3a^^3^^^^^^^^^^^3SE Marine Biological Laboratory Library Woods Hole, Mass. *^^r\s Pre«ented ny McGraw*Hill Book Company N«w York SSE3^^^^^^3^^3^^^^^3a RADIATION BIOLOGY VOLUME II COLLABORATING EDITORS Farrington Danikls, Professor of Physical C'hemistry, University of Wisconsin, Madison Alexander Hollaender, Director, Biology Division, Oak Ridge National Laboratory, Oak Ridge John R. Loofbourow, Professor and Executive Officer, Biology Depart- ment, Massachusetts Institute of Technology, Cambridge Arthur W. Pollister, Professor of Zoology, Columbia University, New York Lewis J. Stabler, Professor of Field Crops, University of Missouri, and Agent, U.S. Department of Agriculture, Columbia ^ / RADIATION BIOLOGY Volume II: Ultraviolet and Related Radiations Edited by ALEXANDER HOLLAENDER Director of Biology Division Oak Ridge National Laboratory With the cooperation of Farrington Daniels Arthur W. Pollister John R. Loofbourow Lewis J. Stadler Prepared under the Auspices of the Committee on Radiation Biology, Division of Biology and Agriculture National Research Council National Academy of Sciences Washington, D.C. New York Toronto London McGRAW-HILL BOOK COMPANY, INC. 1955 RADIATION BIOLOGY, VOLUME II Copyright, 1955, by the McGraw-Hill Book Company, Inc. Printed in the I'nited States of America. All rights reserved. This book, or parts thereof, may not be reproduced in any form without permission of the publishers. Library of Congress Catalog Card Number 5;i-()042 TlIK .\1.\1'LE PRESS COMl'A.N'Y, YORK, FA. This volume is dedicated to the memory of two members of the Volume II Editorial Committee Dr. Lewis J. Stadler Professor of Field Crops, University of Missouri Agent, U.S. Department of Agriculture, Columbia, Missouri and Dr. John R. Loofbourow Professor and Executive Officer, Biology Department Massachusetts Institute of Technology PREFACE This second volume of "Radiation Biology" covers the field of ultraviolet radiation. It also contains considerable material on high-energy radiation. Since both parts of the spectrum induce similar biological effects, no clear line can be drawn between the two. Microbiology, for example, is dis- cussed in both volumes but occupies a more prominent place in the second. Actually, Volume I and Volume II compose a unit and should be read as such. Alexandek Hollaender vu 14 71 CONTENTS Preface vii 1. Photochemistry ] Robert Livingston, Professor of Physical Chemistry, University of Minne- sota, Minneapolis 2. Practical Applications and Sources of Ultraviolet Energy ... 41 L. ./. Bvitolph, Engineer, General Electric Company, Lamp Division, Cleveland, Ohio 3. Sunlight as a Source of Radiation 95 ./. .4. Sanderson, Superintendent Optics Division, Xaval Research Labor- atory, Washington, D.C. Edward O. Hulhurt, Director of Research, Xaval Research Laboratory, Washington, D.C. 4. Technique of Study of Biological Effects of Ultraviolet Radiation 119 Jesse F. Scott, Associate in Biophysics, Massachusetts General Hospital, Boston; and Research Associate, Department of Biology, Massachusetts Institute of Technology, Cambridge Robert L. Sinsheimer, Associate Professor of Biophysics, Physics Depart- ment, Iowa State College, Ames 5. Ultraviolet Absorption Spectra 165 Robert L. Sinsheimer (see Chap. 4) 6. A Critique of Cytochemical Methods 203 A. W. Pollister, Professor of Zoology, Columbia ITniversity, New York City 7. The Effect of Ultraviolet Radiation on the Genes and Chromo- somes OF Higher Organisms 249 C. P. Swanson, Professor of Botany, Johns Hopkins University, Balti- more, Maryland L. J. Stadler, Professor of Field Crops, University of Missouri, and Agent, U.S. Department of Agriculture, Columbia 8. The Effects of Radiation on Protozoa and the Eggs of Inverte- brates Other than Insects 285 Richard F. Kimball, Senior Biologist, Biology Division, Oak Ridge National Laboratory, Oak Ridge 9. Radiation and Viruses 333 S. E. Luria, Professor of Bacteriology, University of Illinois, Urbana IX 69919 X CONTKNTS 10. Effects of Radiation on Bactkria 365 M . h'. Zilh . I'rolrssor of liactoriology, Cornell University, Ithaca, N.Y. Alexander HoUnnuler. Miophysieist, BioloRy Division, Oak Ridge National Laboratory, Oak Hidni' 11. R.\i)iATioN Stiidies ON Fungi l-^l Seymour I'omper, Hioloni-^t. Riolop;y Division, ();ik Hidge National Laboratory. Oak Ridge Kinihiill ('. Alirood, Senior Biologist, Biology Division. Oak Hidge Na- tional Laboratory, Oak Ridge 12. I'lIOTOREACTrVATION 456 Renato Dulbecco. Associate Professor of Biology, California Institute of Technology. Pasadena 13. Sunburn 487 Harold F. Blum, Physiologist, National Cancer Institute, and \'isiting Lecturer, Princeton University, Princeton, N..J. 14. Ultraviolet Radiation and Cancer 529 Harold F. Blum (see Chap. 13) Name I.ndex 561 SiTBJECT Index 577 RADIATION BIOLOGY Volume I J CHAPTER 1 Photochemistry Robert Livingston School of Chemistry, University of Minnesota, Minneapolis Introduction. Primary steps: Absorption — Franck-Condon principle — Direct optical dissociation — Half life of the excited state — Predissociation — Internal conversion — Phos- phorescence and long-lived fluorescence — Long-lived energetic states — Quenching of excited states — Transfer of excitation — Solvent effects — Cage effect — Photochemical transfer of electrons or protons to the solvent. Secondary steps: Bimolecular steps — Unimolecular steps — Tennolecular steps — Diffusion-controlled processes. Mechanism of complex reactions: General problem — Steady-state approximation. Examples of the principal types of photochemical reactions: Decomposition reactions — Reactions of molecular oxygen — Polymerization and dimerization — Intramolecular changes — Sensitized reac- tions. References. INTRODUCTION The development of photochemistry as a quantitative science was made possible by the discovery of the Einstein photochemical equivalence law and by the accumulation of spectroscopic knowledge. The equivalence law can be stated as follows: a photon can induce a photochemical reac- tion only by being absorbed and, on being absorbed, will activate one and only one molecule. The spectroscopic behavior of practically all (stable) diatomic molecules is well understood. In studying the optical proper- ties of complex molecules, it is usually necessary to be guided by general principles and qualitative analogies. In principle, there is little difference between photochemical reactions utilizing visible light and those produced by ultraviolet radiation. The observed change in a photochemical reaction, as in a thermal reac- tion, is the result of the concurrence of a number of simple reaction steps. A set of reaction steps, which is consistent with all available information about a reaction, is called the "mechanism of the reaction." It is con- venient to divide the steps which constitute the mechanism into two groups, called "primary" and "secondary" steps. The primary steps are those chemical or physical processes which are the direct consequence of the absorption of the photon and which involve only the absorbing species (and possibly the solvent). Reactive molecules, radicals, or atoms are produced by the primary steps. These reactive entities can 1 2 RADIATION HIOLOGY (hen uiulergc) (or initiate) a series of siinplc thermal reactions, the second- ary steps. Secondary reaction steps arc imiinolecular, himolecular, oi- termolecuhir reactions. ( )1 these, himolecuhir reactions, which involve a radical or atom and a stable molecule, are probably the most important. Bimolecular reactions also occur between two radicals or atoms. In the gas phase, recombination of two atoms always occurs as the result of a three-body collision. This is very probably the only important type of termolecuhir reaction step. Unimolecular reaction steps are limited to the "spontaneous" decomposition or rearrangement of comple.x molecules. PRIMARY STEPS ABSORPTION Each photon is absorbed by a single molecule,' and, under all ordinary conditions, multiple e.xcitation of a molecule by successive capture of two or more photons is of negligible importance. By capture of a photon the molecule is raised from the ground state to a higher electronic state. With few exceptions (notably molecular oxygen and "odd" molecules; Herzberg, 1950), the ground state of a stable molecule is a singlet one, and the molecule will be excited preferentially to a higher singlet level. The selection rule, which "forbids" transitions betw^een states of different multiplicities, is called the "intercombination rule." For ordinary mole- cules the probability of a transition between energy levels of unlike multi- plicity can be 10^-fold less than that for an otherwise similar transition which does not violate the rule. This is one of the few selection rules which apply to complex molecules as well as to atoms and diatomic mole- cules. For certain cases, such as those involving heavy atoms, e.g., mer- cury, the rule applies less strictly, and the probability of transition is reduced by a factor of only about 10'*. FRANCK-CONDON PRINCIPLE The Franck-Condon principle is probably of greater importance than the selection rules in determining the photochemical behavior of mole- cules. This principle states that electronic transitions which involve appreciable changes in the positions or momenta of the constituent nuclei have relatively low probabilities.- At ordinary temperatures, molecules which exist in thermal e(|uilibrium with their surroundings are in their lowest oscillational states. Accordingly, the Franck-Condon principle and the potential-energy curves determine the oscillational ' In this disrussion, certain typos of crystals and crystalline micelles are to be regarded as single molecules. ^ This discussion of the Franck-Condon principle is based on its original classical formulation. For a discussion of the modifications introduced by quantum theory see Herzberg (1950). PHOTOCHEMISTRY U A^B* A+B r [a) states of the excited electroiiic state that can be reached directly by the absorption of a photon. This is illustrated for a diatomic molecule in Fig. 1-1, in which the ordinate r represents the distance between the nuclei and U is the potential energy of the molecule. In Fig. 1-la, the eciuilibrium separation for the nuclei is about the same in the excited state as it is in the ground state. The probable transitions from the ground state can be represented by vertical arrows lying between a and 6 since they corre- spond to small changes of position or momentum. In this way, a simple application of the Franck-Condon principle demonstrates that direct optical dissociation is improbable for a molecule whose potential-energy curves are of the type illustrated in Fig. 1-la. In the gas phase at low pressure, where the life expectancy of an excited molecule is short com- pared to the time interval between collisions, such a molecule would emit one quantum for each one ab- sorbed. As is suggested by the vari- ations in the length of the downward arrows, the fluorescence or emission spectrum is much more complex than the corresponding absorption spec- trum. In a condensed phase or in a gas at higher pressure, there is a high probability of the excited molecule losing its excess oscillational energy by collisions of the second kind (Franck, 1922) during the lifetime of the excited state. As a result, prac- tically all the transitions, corresponding to the fluorescence, will start from the lowest vibrational level of the excited state. Stokes's law holds under these conditions, and the fluorescence spectrum is shifted to the red side of the absorption spectrum. Freciuently the fluorescence spectrum is approximately a mirror image of the absorption spectrum (Lewschin, 1931). u A+B 'B r (b) Fig. 1-1. Potential-energy diagrams for a diatomic molecule. DIRECT OPTICAL DISSOCIATION In the case illustrated by Fig. 1-1 b, the molecule is nuich less stable in its excited than in its ground state and, correspondingly, the equilibrium separation of the nuclei is increased. It follows, from the Franck-Condon \ RADIATION BIOLOGY priiuiplt', I hut the range of probable transitions (starting from the ground state) Hes between the vertical arrows a and c. Transitions whose arrows lie between arrows b and c are similar to those discussed in connecti(jn with Fig. 1-1 a. Transitions arising from points to the left of arrow 6 result in the formation of an excited molecule whose vibrational energy is greater than that required to dissociate it into atoms, one normal and one excited. Accordingly, direct optical dissociation is a probable event. If the gas is irradiated with photons of energy greater than that indicated by the length of arrow h and less than that of arrow a, the radiation will be strongly absorl)ed. For each such absorption act, a molecule will be dis- sociated (within the half period of a single vibration), and there will be no fluorescent emission. Since the end state is not quantized, the absorption spectrum will be contiiuious in this region. HALP^ LIFE OF THJ: KXCITED STATE If no chemical process, such as optical dissociation, can take place, the half life (ti,,,) of the excited state of a molecule is greater than 10~^ sec, and the molecule will lose all or part of its energy of excitation by emitting a photon. Since the restrictions which limit the probability of a transition apply equally to absorption and emission, a high absorption coefficient corresponds to a short half life of the excited state. For example, the direct optical excitation of a normal molecule from its ground singlet state to an excited triplet state (with a half life of lO"'' sec) would be so improb- able that it would not be observed in the absorption spectrum under ordi- nary conditions. These conditions were put in quantitative form by Ein- stein in 1917 (Herzberg, 1950, pp. 20, 381). If the effect of possible degeneracy of the levels is neglected, the Einstein relation can be put in the following form: NaC In 2 _ 1 aT\., — Stt X 10» v^ X 4 (1-1) where Na is Avogadro's number, c is the velocity of light, and v is the freciuency of the radiation absorbed or emitted. The Beer's law extinc- tion coefficient, off is defined by the following equation, in which in is the concentration of the absorbent in moles per liter, / is the length of the light path in centimeters and /n and Iir are the intensities, respectively, of the incident and transmitted light: tr — InC It should be noted that the product ary, is inversely proportional to the square of the fre(}uency. While Eq. (1-1) applies only to monochromatic light (and therefore approximately to atomic spectra), it can be modified to apply to the broad-band absorption and emission of a molecule. For the latter application, a must be known as an empirical function of u, and hi — ^jiNn, Ifi _ kfi _ T labs kfi + kc To PHOTOCHEMISTRY 5 the expression must be integrated over all frequencies which correspond to the electronic transition under consideration. Equation (1-1) is based on the assumption that an excited molecule can lose its energy of excitation only by emitting a photon. If the energy can be lost in any other way, either spontaneous or induced, the lifetime of the excited state will be correspondingly reduced. When the system is illu- minated with light of constant intensity, a steady-state condition will pre- vail, and the concentration A^'.v of molecules in the excited state will be constant. If "intensities" are expressed in photons absorbed per cubic centimeter per second and the rate Vc of the nonradiative disap- pearance of excited molecules in corresponding units, then labs = Ifi + ?'c = {kfi + k,)NN. The coefficient k^ can be a function of added substances, but, for any given solution, kfi -\- k^ is a constant and is equal to the reciprocal of the mean life r of the excited state under these special conditions. Since it follows that (1-2) where ro is the natural mean life or the life which the excited state would have if the emission of fluorescent light was the only possible degradative process. The ratio of Ifi/ labs is called thfe fluorescence yield. PREDI8S0CIATI0N The fluorescence yield, and correspondingly the mean life, of an excited molecule may be reduced by a process known as "predissociation." This process is possible when a stable vibrational level of an electronically excited state overlaps a dissociation region of another state. For a dia- tomic molecule this corresponds to the crossing of the potential-energy curves of two excited states. Under these conditions, when both the energy and the nuclear configuration are the same in the two states, the molecules can cross over from the stable, quantized state into the unstable, nonquantized state. While the energy of the primary excited state must be greater than the thermochemical energy of dissociation, it may be much less than that required for direct optical dissociation. The probability of such a transition may have any value from unity to prac- tically zero. It is determined by the Franck-Condon principle and by certain selection rules. The chemical detection of the resultant atoms or radicals is the most sensitive test for the occurrence of predissociation. The weakening of the fluorescence intensity is a direct measure of the probability of predissociation. The disappearance of the rotational structure of an absorption band indicates that the probability of the cross- fi KADIATION HIOLOGY oNtT Iroiu OIK' state to llic otlicr lias hccoMK- so j;rcat that the mean life of the excited molecule has been reduced to a value comparable to its period of rotation. Some niolcculcs which cxhil)it a fluorescence yield of unity at low pres- sures dissociate when they are illuminated at high pressures. A collision of the excited molecule with a normal molecule of its own kind or of an added gas induces its dissociation. A process of this type, which is called "induced predissociation." was first observed for I2 (lierzberg, 1950). INTERNAL CONVERSION In addition to fluorescence, direct optical dissociation, and predissocia- tion, excited complex molecules can undergo a process called "internal conversion" (Franck and Sponer, 1949). This process consists in a radia- tionless transition from a low oscillational level of a higher electronic state to a high oscillational level of a lower electronic state. The difference in energy between the two electronic states appears as an increase in the oscillational energy of the molecule after the transition. In internal con- version, both the initial and final states are (juantized; in this respect internal conversion differs from predissociation. Like predissociation, it can occur only when the molecule is in a specific nuclear configuration for which the total energy and the nuclear configuration of the molecule are the same in the two electronic states. Since a complex molecule has many generalized oscillational degrees of freedom, the time required for the molecule to reach the required configuration may be relatively long. Under experimentally realizable conditions, the time between collisions appears to be much less than the average time required for the molecule in the lower electronic state to return to the crossing point and thus to have a chance of coming back to the original state. Collisions between sur- rounding molecules and the vibrationally excited molecule quickly reduce the vibrational energy of the latter and so make the reverse transition impossible. In this way, internal conversion followed l)y a number of col- lisions of the second kind can lead to the complete degradation of the energy of excitation into thermal energy of the system. This is very probably the explanation of why many molecules which absorb strongly in the visible or near ultraviolet are nonfluorescent and do not react photochemically. Immediately after the act of internal conversion, the molecule has a large amount of energy in its oscillational degrees of freedom; in other words, it is a " hot " molecule. As such it can undergo pyrolytic reactions such as decarboxylation or the elimination of a hydrogen molecule. It is diflRcult to conceive of any other simple explanation of the direct photo- chemical dissociation of a complex molecule into two stable molecules, a j)rocess which requires the simultaneous breaking of several bonds and the formation of new bonds. Depending on the nature of the molecule and PHOTOCHEMISTRY 7 the amount of energy available, internal convn'rsion may lead to the dis- sociation of a molecule either into two stable molecules or into radicals. PHOSPHORESCENCE AND LONG-LIVED FLUORESCENCE Metastable states appear to play an important part in the photo- chemistry of complex molecules. The existence of these states has been demonstrated indirectly by the analysis of photochemical data (Shpol'skii and Sheremet'ev, 1936) and directly by a study of the "phosphorescence" and "long-lived fluorescence" of these molecules (Pringsheim, 1949; Forster, 1951). Practically all complex molecules (at least those which contain a double bond) are either fluorescent or phosphorescent (or both) when they are dissolved in glassy media or adsorbed on suitable solids. One of the first examples to be studied quantitatively was the dye trypa- flavin adsorbed on siUca gel (Pringsheim and Vogels, 1936). At ordinary temperatures this system emits a strong fluorescent green band and a separate weak orange band. The green band is made up of ordinary short-lived fluorescence and a relatively long-lived emission, having the same wave-length distribution. The half life Ty^ corresponding to the latter process is an exponential function, ry, = ke^'^'^, of temperature. There is no short-lived fluorescence corresponding to the orange band. The half life corresponding to this latter transition is independent of tem- perature and equal to 1.2 sec. As the temperature is reduced, the life corresponding to the green phosphorescence eventually becomes longer than that pertaining to the orange, long-lived fluorescence, and the slow emission becomes predominantly orange. This general behavior is exhibited by a wide variet}^ of substances (Kasha, 1947). Many measure- ments of this type (Lewis and Kasha, 1944, 1945) have been made with absorbing substance dissolved in a solvent, such as a mixture of ether, pentane, and alcohol, which is fluid at ordinary temperatures and becomes rigid at low temperatures. Under these conditions, only ordinary fluorescence is observed in the fluid solvent, the temperature-dependent phosphorescence appears when the solvent becomes very viscous, and the temperature-independent, long-lived fluorescence becomes noticeable at still lower temperatures. A reasonable explanation of these phenomena, which was first proposed by Jablonski (1935), is illustrated by the simplified energy diagram of Fig. 1-2. The several electronic-energy levels are represented by horizontal lines, capped by a bundle of horizontal lines which indicate the overlap- ping generalized oscillational levels. For an ordinary stable molecule, the ground level N and the two excited levels F and F' are singlet states. The metastable level M is, presumably, a triplet level. The transitions which correspond to arrows 1 and 2 represent the absorption of photons, which raise the molecule into its first or second excited (singlet) state. In its initial state the molecule will be in thermal equilibrium with its surround- 8 RADIATION HIOLOGY ings, and its oscillational enerf^y will Ix' at or near its zero-point value. As determined l»\ the Franck-Condon principle, the electronically excited molecuh^ will usually have an excess of oscillational eiierj^y. In a con- densed medium or in a gas at moderate pressure, the molecule will (|uickly lose this excess of vibrational energy by successive collisions of the second kind. As a result the fluorescent light, transition 3, will usiuilly have a larger wave length than the corresponding absorption. In addition to these permitted transitions, radiationless transitions, 4, 5, and (>, are pos- sible. Each of these acts corresponds to a process of internal conversion. The occurrence of step 4 is proved by the fact that fluorescence cor- responding to transitions from /'" to A'^ or from F' to /'' is never observed Fk',. 1-2. Schematic potential-energy diagram for a complex molecule. with complex molecules such as dyes. Illumination with light of shorter wave length, which raises the molecule to the second (or a higher excited) level F', results only in the long-w'ave-length fluorescence (transition 3). Phosphorescence and long-lived fluorescence involve the metastable state M . In order to reach this state the molecule must undergo an act of internal conversion, step 5. Once in state M , the molecule can return to state F, by way of step 5, only if it receives thermal energy e(|ual to or greater than e, the difference between the energies of levels M and F. This sequence of events corresponds to the temperature-dependent phos- phorescence. Step 8 is forbidden, by the intercombination rule, and will occur only if there is no other probable mode of escape from M. The relativ^e importance of this emission, the long-lived fluorescence, increases as the temperature is lowered. Internal-conversion steps, 6 or 7, con- tribute to the inefficiency of photochemical and fluorescence processes, (^ne offect of adsorbing the molecule or dissol\iiig it in a rigid medium is to PHOTOCHEMISTRY 9 reduce its number of oscillational degrees of freedom and thereby to lower the probability of internal conversion. It seems reasonable to assume that this effect of binding the molecule to its surroundings has a relatively small effect on the initial transition from F to M (or F' toF), since the molecule and its surroundings momentarily have available an energy excess equal to the difference between the energy of the photon and the energy difference betw'een levels F and N. If the preceding simple explanation is correct, the difference between the energ}^ levels F and .1/ can be determined in two independent ways, which should yield identical results. The difference in potential energy between levels F and .V is measured approximately by the quantity hp.i, corresponding to the long-wave-length limit of the normal fluorescence. Similarly, the energy difference between M and N is approximately equal to hvs for the long-wave-length limit of the long-lived fluorescence. The energy difference between F and AI is therefore equal to hv^ — hva. This same energy difference should be obtainable from measurements, at two or more temperatures, of the half life of the phosphorescence. In terms of the simple Jablonski mechanism, e in the following equation should be equal to Aj^s — hv^: For all cases for which the experimental evidence is available, this is approximately true. Although there are minor and understandable dis- crepancies (Pringsheim, 1949, p. 441), the available data support this interpretation. The measured values (Kasha, 1947) of the M-F energy difference lie in the range 5-40 kcal/mole. The lower values belong to dyes, and the higher ones to simpler molecules, such as the aromatic hydrocarbons. Since e~'^*^ will be a very small fraction for the higher values of e, phosphorescence will be very inefficient and probably unde- tectable in these cases. LONG-LIVED ENERGETIC STATES It Avas formerly maintained by many photochemists that fluorescence and photochemical action are strictly complementary actions. How- ever, a large amount of information, chiefly qualitative but in part quan- titative, which is definitely incompatible with this belief, has accumulated. Efficient photochemical reactions are known which involve compounds whose fluorescence yield is small, even in dilute solutions in inert solvents. Particularly in some dye-sensitized photooxidations involving weakly fluorescent sensitizers (Shpol'skii and Sheremet'ev, 1936; Franck and Livingston, 1941), the absorbed photon has too small an energy to dis- sociate the absorbing molecule. At least in these cases a long-lived excited state must be an intermediate in the photochemical reaction. To demonstrate this, let us consider a particular example: the auto- 10 RADIATION BIOLOGY oxidutioii of allyltliiouicii pliotosensitizod \>y cliloiopliyll ((lulTroii, 1927). It seems safe to assume that the reaction involves an interaction between an energy-rich chlorophyll molecule and either an oxygon or an allyl- thiourea molecule. It iiotinal chloropiiyll is represented by G, its fluo- rescent state by G*, some long-lived excited state by G', and the reacting molecule, either oxygen or the reducing agent. I)y B, a generalized (and simplified) mechanism for the reaction may be written as follows: (1) G -\- hu ^ G* ' (absorption), (2) G* -^G -\- hvf (fluorescence), (3) G* —> G' (internal conversion), (4) G'-^G (degradation), (5) B + G* — >-^ G + products (chemical reactions). Expressed in appropriate units (einsteins per liter per second), the rate of step (1) is i'l = lab.- The rates of the four subsequent steps are, respec- tively, V, = /.-,[. I *],/•:, - A-4.l*],r4 = k,[A'], and V, = k,[B][G*]. As long as the measurements of the fluorescence and of the chemical reaction are made under the same conditions, it is immaterial whether ks is a function of the nature of the solvent, i.e., whether the solvent quenches the fluo- rescence. Under steady-state conditions, djA*) „ , d{A') „ -^^0 and ^^-0, and thus Vi = i>2 + Vs + /'o, and v^ = Vi may be written. Substituting for the several Vi, the following values for the fluorescence yield ;as as readily as in a condensed system. When such reactions are exothermic, their heats of acti\ation are usually low, ratifying from 0 to 10 kcal. It does not follow that any such reaction which can be pos- tulateil will occur in practice. For example, the reaction D + C'M4-^CH3l) + H apparently does not take place under ordinary conditions, and theoretical calculations indicate that its heat of activation may be as high as 40 kcal. The reaction between atomic sodium and cyaiingen, Na + C2N2 -^ NaC^X + CN, is slow o\>ing to the snialiness of its probability factor, although its energy of activ'ation is approximately zero. When sufficient energy is available an atom may react with a molecule to form two radicals. A step of this kind (Lewis and von Elbe, 1938), H + U^^ OH + H. undoubtedly plays an important role in the explosive combination of hydrogen and oxygen. Combination of two atoms results in the formation of a molecule pcjs- sessing more oscillational energy than is necessary to dissociate it. As a result, the life of the quasi molecule is equal to the time of a single oscilla- tion (about 10~^'^ sec), and at ordinary pressures it dissociates before it has a chance to make a stabilizing collision with a normal molecule. In other words, combination of atoms in the gas phase as a bimolecular reac- tion cannot occur. However, two radicals, or an atom and a moderately large radical, can combine to form a molecule whose energy can be dis- tributed among several degrees of freedom and whose mean life will there- fore be comparable to the time between collisions in an ordinary gas. Examples of this type are H + C2H4 -> C2H5 and 2C2H.^ — > ( '4H11). There is, of course, no restriction on the combination of atoms in a con- densed system, since the colliding atoms will be continuously in collision with the surrounding solvent molecules. Disproportionation and probably metathelical reactions occur and influence the mechanisms of reactions. The reaction 2C2H:,-. C2H4 + r>\u is detectable under suitable conditions but is apparently less probable than the simph? combination of the ra(li<'als to form l)utane. PHOTOCHEMISTRY 19 Secondary steps are, of course, not restricted to reactions involving radicals; the reactive species may be a relatively unstable molecule or an excited atom or molecule. Tn some cases the excited molecule in a sing- let, fluorescent state may enter into the reaction, as is illustrated by Hg*(6^Pi) + H2^HgH-^H, "" ^'cH^ + 02^ peroxide. Most photochemical reactions of complex molecules, such as the autooxi- dation of rubrene, which take place by way of an excited state appear to involve a long-lived (possibly triplet) excited state. UNIMOLECULAR STEPS True unimolecular reaction steps are limited to complex radicals or molecules. They can result in the formation of (1) two radicals, (2) two stable molecules, or (3) a stable molecule and a radical: (1) Hg(C2H5)2^HgC2H5 + C.>H,, (2) CH3OH -^ HCHO + H2, (3) CH.CO -> CH3 + CO. Internal rearrangements of complex molecules can be the result of uni- molecular reactions. Examples of this type, which have been studied, include cis-trans isomerizations, racemizations (probably), as well as reac- tions of the following type: CHo CH2 CHo — > CH:{ — CH = CH2. Many first-order reactions which occur in solution involve a molecule of the solvent and are therefore bimolecular rather than unimolecular reactions. TERMOLECUI.AR STEPS With few possible exceptions, termolecular gas-phase reactions are recombinations of atoms (or radicals), occurring as three-body collisions iiu'olving some other molecule or radical, i.e., 2Br + No^ Br2 + N2. In solution, termolecular reactions involving one or more molecules of the solvent are probably of much more frecjuent occurrence. DIFFUSION-CONTROLLED PROCESSES The rates of some reaction steps which take place in condensed systems or in gases at moderate or high pressures are controlled by diffusion proc- esses. Most of these reactions involve heterogeneous or microhetero- geneous media. There is an important class of such reactions which occur 20 U\ni\TI<>\ lUOLOGY ill homogeneous, coruhMised phases. Steps of this type have very low heats of activation and steric faetors of the ordcf of magnitude of unity; accordingly, practically every collision results in reaction. Examples of this type are the combination of atoms or small radicals, the quenching of the fluorescem-e of dyes by efficient (juenchers, and enzymatic reactions at low concentrations of the substrate. These reactions occur at every encounter of the reactant molecules. The frequency of encounters, unlike that of collisions, is determined by the rate of diffusion, which in turn is dependent on the viscosity of the solution. Under the normal conditions in which gas-phase reactions are com- monly studied, recombination of atoms frequently is a wall-catalyzed process, whose rate is fixed by the rate of diffusion of the atoms to the wall of the vessel. The rate of atomic recombination 2 A = A2, occurring both by three-body collisions and by wall catalysis, can be represented by an equation of the following form (Kassel, 1932, pp. 170-180): ^ = kPcW + ^^ [A]. The factors Pc and P« are linear functions of the partial pressures of the components of the gas. The coefficient k' is influenced by the geometry of the vessel, becoming greater as the surface-to-volume ratio of the vessel increases. In a photochemical steady state the homogeneous recombination (whose rate is proportional to the square of the concentra- tion of atoms) is favored by an increase in the intensity of the absorbed light. If atoms or radicals are formed in a liquid-phase photochemical reac- tion, the observed quantum yield is likely to be influenced by the rate of stirring of the solution since, in an unstirred solution, the steady-state con- centration of atoms will, in general, be spatially nonuniform. This effect is especially important if a large fraction of the actinic light is absorbed in a thin film near the window through which the light enters. MECHANISM OF COMPLEX REACTION GENERAL PROBLEM Few, if any, chemical reactions are kinetically simple in the sense that they involve only one reaction step which is of simple order and which is identical with the stoichiometric reaction. The observable course of a photochemical reaction is the result of the simultaneous occurrence of a number of reaction steps. A set of reaction steps, which is consistent with the stoichiometry and kinetics of a reaction, constitutes the mecha- nism of the reaction. The kinetic equation for the over-all reaction can be obtained by combining the rate equations for the several steps in such a way that the concentrations of the reaction intermediates are eliminated. PHOTOCHEMISTRY 21 For relatively simple reactions (Kassel, 1932; Laidler, 1950) this can be done by solving simultaneously the differential rate equations for the individual steps. In general, the resulting solution cannot be obtained in terms of simple functions, and the analysis demands a mathematical skill which the average photochemist does not possess. As a result it has become conventional among students of kinetics to use approx- imate methods to deduce the over-all rate equation from a postulated mechanism. STEADY-STATE APPROXIMATION The most generally applicable of these simple methods is the so-called "steady-state approximation." A steady state may be defined as a con- dition in which the rates of change of the concentrations of the several intermediates are very small compared to the rates of change of the con- centrations of the reactants and products. This condition is realizable whenever the ratio of the concentrations of the intermediates to the con- centrations of the reactants is very much less than unity. When this condition is not attained, the method is not applicable; however, it should not then be necessary since the (larger) concentrations of the intermedi- ates could be measured by experimental means. In no reaction is the steady state attained instantaneously. However, the time required for its attainment is usually a negligibly small fraction of the half time of the reaction. The steady-state approximation consists in setting the rates of change of each of the intermediates equal to zero and in solving simul- taneously the resulting algebraic equations. This process can be explained most easily by outlining the details of two well-known examples. Examples. The decomposition of hydrogen iodide is a classic example (Warburg, 1916; Bonhoeffer and Farkas, 1928) of a carefully studied and thoroughly understood photochemical reaction. Although this reaction is so simple that it is not necessary to use the steady-state approximation method in its analysis, it will serve to introduce the fundamentals of this procedure. Gaseous hydrogen iodide strongly absorbs light of wave length 3000 A or shorter. It is nonfluorescent, and its absorption spec- trum is continuous, showing no vibrational or rotational structure. It follows from these observations that the primary act is one of direct optical dissociation, HI 4- /iv ^ H + I. The secondary steps must be reactions of hydrogen and of iodine atoms. They may combine to form hydrogen iodide or molecular hydrogen and iodine, or more probably they may react with hydrogen iodide: I + HI^l2+ H, H 4- HI-^ H2 + I. The first of these tw^o reactions can be ruled out since it is strongly endo- 22 RADIATION HIOLOGY thermic (A// = 34 kcal). Tlu* .socoikI, l)oiiit>; an cxijlhc'iiuic reaction hetwocn an atom and a diatomic molecule, .sliould have a .small heal of activation and a steric factor not dirt'eriiig {greatly from unity. The two possible alternative reactions that a hydrogen atom can undergo, M + H + I-^ III + M and M + 11 + II -^ U, + M, can occur only by three-body collisions with any molecule, M, or by diffusion to the wall. The relativ^e importance of these atomic combina- tions is further reduced by the fact that the steady-state concentrations of the atoms must be much smaller than the concentration of the reactant, hydrogen iodide. Since the hydrogen atom concentration is kept very small by its efficient reaction with hydrogen iodide, the only reaction which iodine atoms can enter into, appreciably, is association to form molecular iodine. Accordinglj^, the mechanism for the reaction may be written as follows: (1) HI + /;i^-^ H + T* (primary step), (2) H + HI ^ Ho + I I (3) M + 21 -^ lo + M ) (secondary steps). Expressing the "intensit.y " lahs of the absorbed light in the photochemical units of einsteins per liter per second and the rates of the chemical reac- tions in moles per liter per second, the rate equations for the three steps of the mechanism may be written Ih = labs, Vo = k.j[Hl][Ul ih = k,Pc[l]-. The rate of decomposition of hydrogen iodide is the sum of the rates of steps (1) and (2), - '-^ =^ V, + Vo = La,. + A-.[HIlfin. Introducing the steady-state assumption then dt -^' lab. = A-2[HI][H] may be written. Therefore dt ~ *'"''• PHOTOCHEMISTRY 23 The (luaiitum yield ^ expressed in terms of molecules of hydrogen iodide decomposed is d[lil]/d( , (f = J = 2. ^ abs This is in excellent agreement with the experimentally determined quan- tum yield of 2.00, the value which has been observed over a wide range of conditions, including pressures down to 0.008 mm Hg. There can be no reasonable doubt of the correctness of this mechanism. The thermal formation of hydrogen bromide was perhaps the first reac- tion to which the steady-state approximation method was applied (Chris- tiansen, 1919; Herzfeld, 1919; Polanyi, 1920). At temperatures in the range from 150° to 200°C, a photochemical formation of hydrogen bro- mide can be observed which is relatively free from disturbance by the ther- mal reaction. The quantum yield of this reaction is the following func- tion of concentrations and intensity (Bodenstein and Lutkemeyer, 1924): dlHBiydt Av[H2] I'^s rH K I [HBr] Absorption of blue or near-ultraviolet light leads to optical dissociation of bromine. The primary step of this reaction is therefore (1) Br. -\- hu^ 2Br. The bromine atoms might be expected to react with either molecular hydrogen or hydrogen bromide: (2) Br + H. ^ HBr + H {AH = 16.7 kcal), (2') Br + HBr ^ Br + H {AH ^ 40.6 kcal). Both these reactions are endothermic, with the heats of reaction indicated. Since the steric factors for these two reactions are very probably of the same order of magnitude, the rate of the second reaction should be smaller than that of the first bv a factor of about g— 40,600/2r g— 16,200/2r g— 24,400/2r In the temperature range under consideration this is a very small number, and the second reaction may be justifiably dropped from consideration. The hydrogen atoms, formed in step (2), can undergo similar reactions, (3) H + Ih:, -^ HBr + Br and (4) 11 + HHr^ U,-\- Br, 12 1 RADIATION HIOLOGY l)()th of which are exothermic. Xoiie of these reactions reduce the total immhcr of atoms present. Therefore the formation of atoms in step (1) must be balanced by their disappearance in atomic combination reactions: (5) M+2H-^Br2 + M, (6) M + H + Br -^ HBr + M, (7) M + 2H -^ H2 + M. Since no hydroj^eii atoms arc formed directly and since, furthermore, steps (3) and (4) are much more efficient than the endothermic step (2), the concentration of hydrogen atoms must be much less than that of bromine atoms. Accordingly, the rates of step (6) and (7) must be small com- pared to the rate of step (5). As a working hypothesis, let us assume that only steps (1) through (5) affect the course of the reaction. The corre- sponding mechanism may be written as follows, where the expression for each rate follows its chemical equation: (1) Br, + /)i'-^2Br Vi = Us, (2) Br + H.2 -^ HBr + H v^ ^ /,-,fH,][Br]. (3) H + Br., -^ HBr + Br v^ = A-4Br,][H], (4) H + HBr^ H., + Br v^ - A-4[HBr][H], (5) M + 2Br-^ Br,, -f- M v, = k,Pc[Br]- = /.{[Br]-. Introducing the steady-state assumptions ^[Br] _ m] _ dt ~^ ^'^"^ dt ""' then 2^1 + v-.i + ''4 = ''2 -f 2?'-, and V-2 = Vs + ?'4 may be written. Therefore Vl ^ Vr, or Similarly. and [Br] = (J^'- /;,[H.,][Br] = {A-.[Br,l + ^•4[HBr]![H] k,[BT,] + A-4[HBr] \ /,-.; / In terms of the mechanism the rate of formation of hydrogen bromide is given by d[HBr] -r. — = V2 + V:i — Vi dt = A-2fH.,l[Br] -f lUBv,] - A^4[HBrl}[H]. PHOTOCHEMISTRY 25 Introducing the values for the concentrations of the intermediates and simplifying, d( ~ A-4[HBr] ■ A-.[Br2] The corresponding equation for the quantum yield, ?t^Hl f/[HBr]A// A-f ^ '^

H, + HS j 2HS -> HoS + S (secondarj^ steps) . The photolysis of a variety of aldehydes and ketones has been investi- gated extensively and in detail (Xoyes and Leighton, 1941). The results PHOTOCHEMISTRY 27 of these studies are far from simple. It appears to be fairly definite that two different kinds of primary acts can occur. The excited molecule can dissociate either into radicals or into two such stable molecules as a hydro- carbon and carbon monoxide. The formation of the radicals is probably a process of predissociation, and the production of molecules, the result of internal conversion. Croton aldehyde, at temperatures below 150°C, is photochemically stable in spite of the facts that it is nonfluorescent and that its absorption is continuous. Although this failure to react is con- ceivably the result of rapid recombination of radicals formed in the pri- mary act, it seems much more likely that the energy of excitation is lost by way of an act of internal conversion, followed by degradative colli- sions, i.e., collisions of the second kind, with surrounding molecules. The photochemical decomposition of formaldehyde is probably as sim- ple a reaction of this type as has been studied. Although the results of the several investigations of this reaction (Steacie, 1946) do not agree in all particulars, the broad outline of the mechanism appears to have been reasonably well established. The products of the reaction are carbon monoxide and molecular hydrogen. At 110°C the ciuantum yield is approximately unity for wave lengths from 2600 to 3500 A. At the longer wave lengths the absorption spectrum shows fine structure but corresponds to a region of predissociation in the shorter wave-length range. The yield increases with increasing temperature, reaching a value of about 100 at 350°C. Hydrogen atoms can be detected under all experimental conditions, but there is some evidence that there is an appre- ciable direct formation of molecular hydrogen when the gas is illuminated with light in the longer wave-length region. The following mechanism seems to be compatible with the published results: (1) HCHO + hv— CHO + H (chief primary step), (2) HCHO + hv -^ [HCHO] -^ CO + H, (primary step at long wave lengths), (3) HCO ^ H + CO (chain-carrying secondary step), (4) H + HCHO ^ H2 + CHO (chain-carrying secondary step), (5) M + 2H -^ H2 + AI (chain-breaking secondary step) . Steps (3) and (5) can occur either in the gas phase or by diffusion to the wall. For the gas-phase reaction the heat of activation of step (3) is about 13 kcal. There are, of course, a number of other possible steps, but these five are sufficient to explain the available data. REACTIONS OF MOLECULAR OXYGEN In biological systems those photooxidatix'e reactions which involve molecular oxygen are by far the commonest. The primary step of such a 28 RADIATION BIOLOGY reaction may be either the optical dissociation of the oxygen molecule or the photoexcitation or dissociation of a molecule of the reducing agent. Since oxygen absorbs chiefly at wave lengths less than 1800 A, reactions of the first type are limited to relatively transparent substrates such as hydrogen or carbon monoxide (Noyes and Leighton, 1941, pp. 246-254). In those cases where the reducing agent absorbs the light, the initially reactive species may be an excited singlet (i.e., fluorescent) state, a long- lived (triplet) excited state, or a pair of radicals, produced by some type of photodissociation. Solutions of aryl hydrocarbons in hexane or similar solvents are fluores- cent. In the presence of o.xygen, their fluorescence is quenched and peroxides are formed (Bowen and Williams, 1939). With few exceptions the sum of the fluorescent yield and the peroxide quantum yield is dis- tinctly less than unity, in some cases being as small as 0.1. None of the data for the 14 hydrocarbons investigated by Bowen and Williams are consistent with the view that the only effect of o.xygen is to quench the fluorescence by reacting with the excited molecule (in its singlet, fluores- cent state) to form a peroxide. Apparently oxygen can quench the fluorescence of these molecules without forming any detectable product. In five cases (benzene, w-xylene, fluorene, acenaphthene, and triphenyl- methane) the evidence is compatible with the postulate that only the singlet, fluorescent state is involved in the peroxide formation. For the others (especially hexamethyl benzene, anthracene, naphthacene, toluene, and p-.xylene) the experimental results strongly indicate that some or all of the peroxide is formed by a reaction between an oxygen molecule and an energy-rich nonfluorescent (triplet?) state of the hydrocarbon. This is particularly obvious for hexamethylbenzene, where a quantum yield of peroxide formation almost fivefold greater than the maximum fluores- cent yield was observed. The preceding conclusions are based on the assumption that only the 10 following reaction steps occur. In these ecjuations, A* stands for the singlet, excited state and A' for the triplet state of the hydrocarbon molecule, A. (1) \ + hu-^ A*, (6) (), + A* ^ AO2, (2) A*^ A + ItPf, (7) 0, + A' ^ AO2, (3) A* -^ A, (8) 0-2 + A* ^ A + O2, (4) A* -^ A', (9) O2 + A* -^ A' + (),, (5) A' -^ A, (10) ().. -f A'^ A + ()... Oxidations which are initiated by the photochemical dissociation of the reductant frequently exhibit the characteristics of chain reactions. Their quantum yields are functions of the temperature, of the concentrations of reactants and products, and sometimes of the intensity of the absorbed light. Often the products are complex, and the relative amounts of the several compounds formed vary with the conditions. The detailed PHOTOCHEMISTRY 29 mechanism of the reaction for such a case has not been estabhshed with reasonable certainty. The photochemical oxidation of formaldehyde (Style and Summers, 1946) is a good example of this type of reaction. It has been studied in the temperature range of 100° to about 275°C and at a variety of pressures and compositions. Its principal products are CO, CO2, HCOOH, Ho, and H2O. Their several yields vary from values of less than 1 to 30 or 40, depending in a coniplex way on the conditions. It is well established that H, CHO, and HO2 are reaction intermediates. The (chief) primary process is HCHO + hp^U + CHO. Although the detailed mechanism is not known, the experimental evidence is compatible with the postulate that the following reactions serve as (some of) the secondary steps of the reaction: H + HCHO ^ H. + CHO, CHO + HCHO -^ Ho + CHO + HCHO, 2CH0 ^ CO + HCHO, M + H + O2 ^ H62 + CO, CHO + 02^ H62 + CO, HO2 + HCHO ^^ [CO + CO2 + HCOOH + H], 2HO2 -^-^ H2O + 3.2O2. Organic peroxides are the principal products of some reactions of this general type. Peroxides may also serve as photochemical sensitizers. For example, the chief product of the photochemical oxidation of cyclo- hexene is the corresponding peroxide. As the concentration of the perox- ide builds up in an illuminated solution containing oxygen and cyclo- hexene, the peroxide absorbs an increasing amount of the incident light, and the reaction is accelerated (Bateman and Gee, 1948). These observations are consistent with the postulate that the primary process in the absence of the peroxide is RH (cyclohexene) + /( j^ -^ R + H, and in the presence of the peroxide is predominantly ROOH + hv^ ROO + H. At temperatures at which the thermal reaction can be neglected, the over- all process is a short chain reaction. It seems very probable that R, ROO, H, and HO2 are important intermediates in this process. Since the quantum yield of the reaction is inversely proportional to the square root of the intensity of the absorbed light, the chain-breaking step must be a bimolecular reaction between chain carriers (i.e., intermediates) leading to the production of stable molecules. 30 KADIAIION lUoLOfJV POLYiMKHIZATION AM) 1)1 MKRIZATION There are two general types of polymerization: .simj)l(' reactions leading to the formation of definite molecules, such as dimers or t rimers, and chain reactions, whose products are macromoleciilcs of indefinite molecu- lar weight. One of the first polymerizations to be studied with reason- able care (Luther and Weigert, 1905) is the dimerization of anthracene. At moderately elevated temperatures (80°-200°C') the dimer reaches a measurable sleady-state concentration in a dilute solution of anthracene illuminated with ultraviolet light (wave length 3GG0 or 3130 A). Over the range of intensities and concentrations studied, the steady-state con- centration is directly proportional to the intensity of the absorbed radia- tion. The quantum yield of dimerization increases with increasing anthracene concentration, approaching a limiting yield of about 0.5 (Weigert, 1927). The available measurements are insufficient to deter- mine the mechanism of the process. It appears very probable that the first five steps which were proposed in the discussion of the oxidation of aromatic hydrocarbons occur in the polymerization reaction. However, any of the three following alternative reactions may be responsible for the formation of the dimer: A* + A-> Ao, A' + A -^ Ao, 2A' -^ A,. The last of these is consistent with the observed limiting yield of 3-^. This interesting reaction is certainly worthy of further study. The gas-phase polymerization of cyanogen (Hogness and Ts'ai, 1932) is at least superficially simple. The absorption of this compound is fairly strong in the wave-length region 2150-2250 A. The absorption bands are diffuse, and the gas is nonfluorescent. Under the conditions of measurement the ciuantum yield is 3.0. The product is a brownish solid. The authors propose the following mechanism: C2N2 -\- hv —* 2CN (primary process), CN + C2N2^ (CN)3 (secondary process). This is probably an oversimplification, since it is difficult to understand why the (CN):i molecules would react with themselves to form para- cyanogen but would not r&act with the remaining cyanogen. The formation of large polymer molecules (Mark and Raff, 1941 ; Bawn, 1948) ma}^ occur either by successive condensation or by addition of sim- ple molecules. Addition polymerization is essentially a chain reaction and, as such, can be studied effectively by photochemical methods. Determinations of the chain lengths of thermal reactions can be made only indirectly, usually by the use of inhibitors (Alyea and Biickstrom, 1929). In a photochemical reaction the ratio of the over-all (juantum PHOTOCHEMISTRY 31 yield to the yield of the primary process (which is commonly close to unity) is a direct measure of the average chain length. Furthermore, knowledge of the nature of the primary product is frecjuently very helpful in the prediction of the secondary steps. The primar}^ act in association polymerization is the formation of two radicals or a diradical. Each radical or diradical can then add to a monomer molecule, forming a new radical of greater molecular weight. Large polymers are built up by the successive addition of monomer mole- cules to the growing radical. In most cases studied, the addition of monomer to the radical rec^uires a heat of activation of a few kilocalories. The specific rate of addition is only slightly influenced by the size of the radical. In the absence of inhibitors the chain is, in the great majority of cases, terminated by a reaction between two radicals. This chain- stopping step eliminates two radicals either by their disproportionation or by their addition. Free radicals may be formed by the photochemical dissociation of the monomer or of an added sensitizer such as acetone (Jones and Melville, 1946). Since the chains are broken by bimolecular reactions between growing radicals, the rate of polymerization is proportional to the square root of the intensity of the absorbed light. A determination of the ratio of the rate constants for the chain-propa- gating and chain-terminating steps may be made by analyzing the kinetics of a polymerization reaction. This analysis is made by the usual steady-state approximate method. Special methods are required to evaluate either of these individual constants. Melville (1947) has shown that these individual constants can be obtained if the polymerization occurs under intermittent illumination. This technic^ue, which has proved very useful in the study of polymerization kinetics, is a relatively old one in photochemistry, having been used by Berthoud and Bellenot in 1924 and subjected to a thorough theoretical analysis by Dickinson (Noyes and Leighton, 1941, pp. 202-209). INTRAMOLECULAR CHANGES Relatively few photochemical isomerizations have been studied ciuanti- tatively. One group of reactions which has received some attention is the cis-trans isomerizations. For reasons of experimental convenience, most of the kinetic measurements have been made with substituted ethylenes. However, knowledge of their spectroscopic properties is limited to the simpler compounds. Figure 1-3, which is taken from the work of Mulliken (1942), is a schematic representation of the electronic energy levels of ethylene. In addition to the ground level A'^, two excited singlet levels, V and R, are shown. Absorption bands, corresponding to transition from N to either V or R, are strong. According to the Franck- Condon prijiciple, the angle between the hydrogens cannot change appre- 32 RADIATION HIOI.OGY V R \ c'iably tluriiifi the electron triinsitioii, and therefore tlie energies corre- sponding to transitions N —^ R and N —* V will overlap. Transitions from iV to R result in sharp bands, and from A'' to F in diffuse general absorption. The optical and photochemical properties of cis- and trans-atiWyvAiv. were carefully investigated by Lewis ct at. (1940). 7Vo/is-stilbene is fluorescent (wave length 3300 to 4400 A), and its absorption spectrum (wave length 2000 to 3400 A) shows distinct "oscillational" structure; as-stilbene is nonfluorescent and its absorption spectrum is apparently structureless. Irradiation of either pure compound with radiation of wave length 2537 A produces partial stereoisomerization. Since cz's-stilbene undergoes a photochemical side reac- tion to an unknown product, quantum yields had to be based on measurements of the initial rates. Starting with the pure fzs-compound, the quantum yield of /raws-stilbene formation is 0.26 and of the side reaction is 0.10. The cor- responding quantum yield for the forma- tion of cis- from ^rans-stilbene is 0.35. The interpretation of these facts is ren- dered uncertain by the lack of knowl- edge of the potential-energy diagram for stilbene. The steric interference between the phenyl groups, which is responsible for the relative instabil- ity of the cts-form, undoubtedly renders the potential-energy curve for the ground state unsymmetrical and probably has a similar effect on the curves for the excited states. Conjugation between the benzene rings and the ethylenic link must also affect the energy levels. For lack of other information, let us assume that the potential-energy curves for stilbene, although asymmetric, are otherwise essentially similar to those for ethylene. A molecule excited to the state V will quickly lose its extra energy of oscillation by successive impacts with solvent molecules and will end up in the (approximately) 90° trough of the electronic state. The sul)se(iuent transitions of the molecule are, of course, independent of whether it was originally a normal cis- or /raw. s-stil bene molecule. Since transitions between states A'' and V are permitted, it might be expected that the excited mole('ule could emit a (luantum and return to the ground state of either the cis- or trans-iorm, depending on the relative asymmetries of states A'^ and V. If this were the mechanism of the process, the quantum yields of fluorescence and isomerization would not be complementary, and the limit of the sum of these yields would be 2 rather than 1. That this mechanism does not apply to this case is shown by the nonfluorescence of cis-stilbene. The strong fluorescence of the AT 0° 90° 180 Fig. 1-3. Schematic potential-energy diagram for ethylene. PHOTOCHEMISTRY 33 trans compound demonstrates that some (or all) of these excited mole- cules are in a state which is peculiar to the /rans-configuration, possibly- state R. If czs-stilbene is excited to state R it must go, by internal con- version, to state 1' (or possibly to the ground state with a high excess of oscillational energy) in a time much less than the natural half life of the excited state. The fluorescent yield of /rans-stilbene was not measured. Lewis and his coworkers (1940) assumed that the ratio of the nonradia- tive return to the cis- and /ra/ts-forms was independent of whether the excited state was formed by the irradiation of normal cis- or trans-siW- bene. This assumption leads to a value of about 0.5 for the fluorescent yields. Although this latter assumption is consistent with the available data, it is by no means the only reasonable interpretation. Olson's con- clusion (1931) that the compound formed from the excited state will be predominantly the isomer of lower stability should not be expected to apply to a reaction which takes place by way of internal conversion. The probability of such a process will depend on the relative forms of the several potential-energy surfaces and on their points of intersection and not merely on relative times spent in the two rotational configurations. Photochemical reactions of this type deserve much more attention than they have received. They are intrinsically interesting, and an under- standing of them should prove helpful in the interpretation of more com- plex photochemical processes (Pinckard et al., 1948; Stearns, 1942). The photoisomerization of o-nitrobenzaldehyde to o-nitrosobenzoic acid involves the breaking of two bonds and the formation of two new ones, but the reaction appears to be strictly an intramolecular process. The course of the reaction is independent of whether the compound is present as crystals, is in solution in a solvent, e.g., acetone, or is in the vapor phase. No detectable oxygen is liberated. In the condensed systems (Leighton and Lucy, 1934) at room temperature the quantum yield is 0.50 ± 0.03. In the vapor phase at 90°C, where the vapor pres- sure is about 4 mm of Hg, the quantum yield is 0.70 + 0.05, but the yield is reduced by the addition of molecular nitrogen, reaching a value of about 0.5 at a nitrogen pressure of 700 mm of Hg (Ktichler and Patat, 1936). The yield in solution for the corresponding reaction of 2,4-dini- trobenzaldehyde is also about 0.5 but is approximately 0.7 for 2,4,G-tri- nitrobenzaldehyde. It is plausible that the reaction involves the hydro- gen-bonded quasi six-membered ring and that it takes place by way of an act of internal conversion. Why the yield reaches a limiting value of 0.5 in condensed systems or in the presence of an inert gas is not obvious. The theoretical predictions of Leighton and Lucy are incompatible with the subsecjuent experiments of Ktichler and Patat, and therefore this detailed theory apparently must be rejected. The photochemical denaturation of proteins and inactivation of enzymes can be classed, somewhat arbitrarily, as rearrangements of the 34 RADIATION BIOLOGY hydrated protein molecules. These reatrtions have been studied exten- sively in recent years (McLaren, 1!)M)), and some empirical generaliza- tions can lie deduced from the results of these studies. The (juantum yields of the reactions an^ in the range from 1()~- to 10~'. Radiation of wave lengths shorter than 3100 A is reciuircd to produce the reactions. Photochemical denaturation is irreversible. The primary jjliotochemical product remains in solution at low temperatures (e.g., 4°Cj, but a precipi- tate forms rapidly when the previously irradiated solution is heated to 40°C. The (juantum yield of the primary process is practically inde- pendent of temperature over the narrow range availal)le. Photochemical denaturation results from the irradiation of "dry" proteins as well as proteins in dilute aqueous solution. The (juantum yield is not a function of the intensity of the absorbed light. If the quantum yield is strictly independent of the intensity of the light absorbed by the native protein, the primary act involves the interaction of one photon with each molecule; i.e., it is a "single-hit" process, in which there is no cooperative action between two or more photons either suc- cessively or simultaneously. It should be realized, however, that no simple mechanism predicts that the yield is independent of the product of incident intensity and time of irradiation since the (dissolved) dena- tured protein must act as an efficient internal filter. The "one-hit" kinetics have been interpreted in terms of a primary act in which one peptide linkage is broken by a single absorl)ed photon. The low (juantum yields indicate that this primary process is very inefficient; most of the absorbed quanta are degraded to heat. One plausible explanation for the observed inefficiency is that the light, which is absorbed by aromatic nuclei in the molecule, becomes available for chemical action by an act of internal conversion. A few facts which support this tentative explana- tion are (1) the yield increases with increasing frequency (i.e., energy) of the photon, (2) the yield decreases with increasing size of the molecule, and (3) the yield is greater for adsorbed films of proteins than it is for solutions. SENSITIZED REACTIONS In sensitized reactions the substance which absorbs the light does not undergo any permanent chemical change. This absorbing substance, called the "sensitizer," catalyzes the photochemical reaction. The simplest known example of this type is the xenon-sensitized photochemical dissociation of hydrogen (Calvert, 1932). The resonance radiation of xenon has a wave length of 1409 A, which corresponds to an energy of 193 kcal/mole. Molecular hydrogen, whose dissociation energy is 103 kcal/mole, does not absorb radiation of wa\'e length longer than 849 A. If a mixture of xenon and hydrogen is illuminated with a xenon arc, hydro- gen atoms are formed as was demonstrated by their color reaction with PHOTOCHEMISTRY 35 solid tiHigstic oxide. If pure hydrogen is substituted for the mixture of gases, there is no reaction. The reaction steps are as follows: Xe + hv, - ->Xe* (absorption), Xe*- -^ Xe + hv. (fluorescence). H., + Xe* - ^ Xe + 2H (collision of the second kind) Mel-cury vapor is a l)etter-kno\vn sensitizer for the dissociation of hydro- gen (Noyes and Leighton, 1941). The first resonance radiation of mer- cury, wave length 2537 A, corresponds to an energy of 112 kcal/einstein, which is only slightly more than is necessary to dissociate molecular hydrogen. It should be expected that the reaction H., + Hg*(()^Pi) -^ Hg((3'Po) + 2H should be very efficient. Although the interaction between an excited mercury atom and a hydrogen molecule is indeed very probable, HgH appears to be one of the products: H2 + Hg* ^ HgH + H. If other reactant gases, such as carbon monoxide or ethylene, are present, the hydrogen atoms initiate a series of reaction steps leading to a variety of products. Many such mercury-sensitized reactions have been studied. Photochemical cis-trans isomerizations are sensitized by iodine (Ber- thoud and Urech, 1930; Dickinson et al, 1949). The sensitized reaction is a short chain process with an appreciable heat of activation. The pri- mary act is the photodissociation of molecular iodine. Iodine atoms can add, with an appreciable heat of activation, to carbon atoms adjacent to the double bond. This opens the double bond, permitting rotation of the groups. Subsecjuently the iodine atom can split off. This mechanism is summarized in the following equations, in which C and T stand, respec- tively, for the CIS and trans form of the molecule: I2 + hv-^2\, i+ T^fi, i + c ^ CI, CI ^ fi, 2\ -^ I2. In some systems an absorbing compound, undergoing a permanent photochemical reaction with a yield of about 1, simultaneously induces a chain reaction between other reactants. The chain reaction so over- shadows the inducing reaction, that the whole process may be thought of, loosely, as a sensitized photochemical reaction. Examples of this type are the oxidation of hydrogen (Farkas et al, 1930) and the polymerization 36 KADIATIOX BIOLOGY of elliyk'iu' (^'I'uylor and Enioliius, li)31j induced by tlie prcdisfjuciatiou of ammonia: NHs + hv^ XH2 + ft. When a mixture of hydrogen, oxygen, and ammonia, at a moderate!}' elevated temperature, is illuminated with light of wave length 2200 A or shorter, ammonia is decomposed and water is formed. The cjuan^um yield for the formation of water increases from about 25 at 290°C to approximateh^ 380 at 405°C. At 420°C, irradiation of the system results in an explosion. The kinetics are complex (Lewis and von Elbe, 1938) and probably involve the amide radical as well as the hydrogen atom. If a mixture of carbon monoxide and chlorine is illuminated with light which is absorbed by the chlorine, a chain reaction ensues, the product of which is phosgene. The kinetics of the reaction are complex, but the primary act is certainly the dissociation of chlorine and the radicals CI and COCl are involved in the secondary reactions. If an excess of oxy- gen is added to the system, the formation of phosgene is suppressed, and the predominant process becomes the sensitized formation of carbon dioxide (Rollefson and Burton, 1939, pp. 313-319). The quantum yield of carbon dioxide formation is large and is a complex function of tem- perature and the partial pressures of the reactants. A numl)cr of reac- tions of this general type have been studied, but the mechanism of none of them is completely understood. The photolysis of ethyl iodide is sensitized (West and Miller, 1940; West, 1941) by naphthalene and a number of its derivatives. The direct photolysis of ethyl iodide occurs both in the gas phase and in solution. In hexane solutions the quantum yields corresponding to wave lengths 3130 and 2537 A are about 0.30 and 0.40, respectively. The quantum yield of the naphthalene-sensitized process is about 0.30 for cither wave length. The maximum fluorescence efficiency of naphthalene in hexane solutions is approximately 0.15. As was clearly stated by West (1941), this demonstrates that ethyl iodide can interact with a nonfluorescent excited state as well as with the fluorescent excited state of the naphtha- lene molecule. The yield of the sensitized reaction is independent of the naphthalene concentration but falls off to small values when the e{\\y\ iodide concentration is decreased much below 10~'- M. These results, as well as observations on the effect of changing the \iscosity of the solvent, show that the sensitization is a coUisional process, that the efficiency of such collisions in producing the reaction is high (probably greater than 0.1), and that the collisions in the condensed system occur in bursts (p. 15). All these data are consistent with the following mechanism, which is strikingly similar to the mechanism here offered as an explanation (p. 28) for the photoautooxidation of aromatic hydro- carbons (Howen and Williams, 1939): PHOTOCHEMISTRY 37 A* -^ A + hvf, A* -^ A', A' -> A, A* + Co^sl -^ A + C2H5 + I, A' + C2H5I -^ A + C2H5 + I. Photoaiitooxidations of reactive reducing agents are sensitized by a wide variety of dyes and pigments (Hurd and Livingston, 1940). These reactions occur in aqueous solutions or in organic solvents, such as methanol or acetone. In cases where the kinetics have been studied in detail, they appear to be complex, and in no case has a completely satis- factory mechanism been proposed. These reactions are produced by either visible light or ultraviolet radiation, depending chiefly on the absorption spectra of the sensitizers. Sensitized photochemical redox reactions are of great importance in biology, the outstanding example being photosynthesis by green plants (Rabinowitch, 1945). Photodynamic action and certain pathological skin reactions (Blum, 1941) are also of interest. The oxidative inactiva- tion of enzymes is sensitized by riboflavin (Galston and Baker, 1949) as well as by certain dyes. REFERENCES Alyea, H. N., and H. L. J. Backstrom (1929) The inhibitive action of alcohols on the oxidation of sodium sulfite. J. Am. Chem. Soc, 51: 90-109. Bamford, C. H., and R. G. W. 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(1937) Die lange Lebensdauer angeregter organischer Molekiil(> erlautert am Beispiel der Rubrenoxydation. Z. physik. Chem., B37: 437-46!. Galston, A. W., and R. S. Baker (1949) Inactivation of enzymes by visible light in the presence of riboflavin. Science, 109: 485-486. Herzberg, G. (1950) Spectra of diatomic molecules. 2d ed., D. Van Nostrand Com- pany, Inc., New York. Herzfeld, K. F. (1919) Zur Theorie der Reaktionsgeschwindigkeiten in Gasen. Z. Elektrochem., 25: 301-304. Hogness, T. R., and L.-S. Ts'ai (1932) The iihotochemical polymerization of cyanogen. J. Am. Chem. Soc, 54: 123-129. Hurd, F., and R. Livingston (1940) The quantum yields of some dye-sensitized photooxidations. J. Phys. Chem., 44: 865-873. Jablonski, A. (1935) Weitere Versuche i'lber die negative Polarization der Phos- phoreszenz. Acta Phys. Polon., 4: 311-324. PHOTOCHEMISTRY 3<) Jones, T. T., and H. W. Melville (1946) The free radical polymerization of the vapours of certain vinyl derivatives, l^roc. Roy. Soc. 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Quantum efficiency of photo-oxidation. J. Am. Chem. Soc, 65: 2419-2423. Lewis, G. N., M. Calvin, and M. Kasha (1949) Photomagnetism. Determination of the paramagnetic susceptibility of a dye in its phosphorescent state. J. Chem. Phys., 17: 804-812. Lewis, G. N., and M. Kasha (1944) Phosphorescence and the triplet state. J. Am. Chem. Soc, 66: 2100-2116. (1945) Phosphorescence in fluid media and the reverse process of singlet- triplet absorption. J. Am. Chem. Soc, 67: 994-1003. Lewis, G. N., T. T. Magel, and D. Lipkin (1940) The absorption and re-emission of light by CIS- and /ra«s-stilbenes and the efficiency of their photochemical isomer- ization. J. Am. Chem. Soc, 62: 2973-2980. Lewschin, W. (1931) Das Gesetz der Spiegelkorrespondenz der Absorptions und Fluoreszenzspektren. Z. Physik, 72: 368-381. (1935) Correspondence between absorption and luminescence of dilute solu- tions of dyes. Acta Physicochim. U.R.S.S., 2: 221-238. Livingston, R., and C.-L. Ke (1950) Quenching of the fluorescence of chlorophyll a solutions. J. Am. Chem. Soc, 72: 909-915. Luther, R., and F. Weigert (1905) Uber umkehrbare photochemische Reaktionen im homogenen System. Anthracen und Dianthracen I. Z. physik. Chem., 51: 297-328. McCIure, D. S. (1949) Triplet-singlet transitions in organic molecules. Lifetime measurements of the triplet state. J. Chem. Phys., 17: 905-913. McLaren, A. D. (1949) Photochemistry of enzymes, proteins and viruses. Advances in Enzymol., 9: 75-170. Mark, H. F., and F. A. V. Raff (1941) High polymeric reactions. Interscience Publishers, Inc., New York. Moelwyn-Hughes, E. A. (1947) The kinetics of reactions in solution. Oxford University Press, London. Mott, N. F., and R. W. Gurney (1940) Electronic processes in ionic crystals. Oxford University Press, London. Mulliken, R. (1942) Structure and ultraviolet spectrum of ethylene, butadiene, and their alkyl derivatives. Revs. Mod. Phj^s., 14: 265-274. * Noyes, W. A., and P. A. Leighton (1941) The photochemistry of gases. Reinhold Publishing Corporation, New York. Olson, A. R. (1931) The studj-^ of chemical reactions from potential-energy diagrams. Trans. Faraday Soc, 27: 69-76. 40 RADIATION lUOI.OOY Pinckard. .1. 11.. B. Wille, and L. Zochinoistor (UM8) A coniparativo study of tho throostc'iooisonu'iic 1, l-diplii'iiylhutiidinics. J. Am. Chcm. Sof., 70: Hi38 I'.IM. I'olanyi, M. (H»20) lleaktionsisocliore uiid Roaktion.sgcscliwiiidigkcit voin Staiid- ])unkto der Statistik. Z. Elcktrochcm., 26: 49-54. (1932) Atomic reactions. Williams and Norgato, Ltd., London. Pringshoim, P. (1949) Fluorescence ami plio.sphorcsconco. Intcrsciciicc l'ul)lislicrs, Inc., New York. Piingslioim, P., and H. Vogels (1936) Phosphorescence et fluorescence a longue dur(5e des colorants organiques. J. chim. phys., 33: 345-355. I'rins, J. A. (1934) Spectrum of chlorophyll. Nature, 134: 457-458. Rahinowitch, E. (1945) Photosynthesis, VoL I. Intersciencc Publi.shers, Inc., New- York. Uahinowitch, 1-:., and L. F. Ep.stein (1941) Polymerization of dyestuffs m solution. Thionine and methylene blue. J. Am. Chem. Soc, 63: 69-78. Rollefson, G. K., and M. Burton (1939) Photochemistry and the mechanism of chemical reactions. Prentice-Hall, Inc., New York. Rollefson, G. K., and R. W. Stoughton (1941) The qvienching of fluorescence in solution. III. The nature of the quenching process. J. Am. Chem. Soc, 63: 1517-1520. Shpol'skil, E., and G. Sheremet'ev (1936) Quenching of the fluorescence and photo- chemical sensitization in .solution. Investigation of the photochemical sensitiza- tion in solutions. J. Phys. Chem. U.S.S.R., 8: 640-652. Steacie, E. \V. R. (1946) Atomic and free radical reactions. Reinhold Publishing Corporation, New York. Stearns, E. I. (1942) Phototropic dyes. J. Opt. Soc. Amer., 32: 282-284. Style, D. W. G., and D. Summers (1946). The photochemical reaction between formaldehyde and oxygen. Trans. Faraday Soc, 42: 388-395. Taylor, H. S., and H. J. Emelius (1931) Photochemical interaction of ethylene and ammonia. J. Am. Chem. Soc, 53: 562-574. Terenin, A. (1947) Basic problems of photobiochemistry. Bull. Acad. Sci. U.R.S.S., Ser'. biol., 369-376. Terenin, A., and A. Kariakin (1947) Proton transfer between organic molecules caused by light. Nature, 159: 881-882. Terent'ev, A. P. (1949) Reversible changes of the pH of solutions of some organic compounds on illumination. Doklady Akad. Nauk S.S.S.R., 68: 537-539. Vavilov, S. I. (1943) The theory of the influence of concentration on th(> flviore.scence of solutions. J. Phys. U.S.S.R., 7: 141-152. Warburg, E. (1916) Ueber den Energieumsatz bei photochemischen Vorgangen m Gasen. Sitzber. kgl. preuss. Akad. Wiss., 1916: 314-329. Watson, W. F., and R. Livingston (1950) Self-quenching and sensitization ol fluorescence of chlorophyll .solutions. J. Chem. Phys., 18: 802-809. Weigert, F. (1927) Uber den Mechanismus der photochemischen Polymcri/.at ion d(>s Anthracens. Naturwissenschaften, 15: 124-126. ^\'ei.ssman, S. I. (1942) Intramolecular (>nergy transfer. The fluorescence of com- plexes of europium. J. Chem. Phys., 10: 214-217. West, W. (1941) Primary processes in fluorescence and photo.sensitization with par- ticular reference to simple aromatic compounds. Ann. N.Y. Acad. Sci., 41: 203-230. West, W., and W. E. Miller (1940) Photosensitization and fluorescence by aromatic hydrocarbons. J. Chem. Phys., 8: 849-860. Zifnmerman, G. (1949) Photochemistry of permanganate ions. Ph.D. thesis. University of Chicago. Manuscript received by the editor Mar. 19, 1951 CHAPTER 2 Practical Applications and Sources of Ultraviolet Energy L. J. BUTTOLPH General Electric Company, Lamp Division, Cleveland, Ohio Inlroduclion. Germicidal-aclion curves: Action curve tentative at shorter wave lengths- Action curve approximate at longer wave lengths—The unique 2537 .4 mercury line Susceptibility to ultraviolet: Injury, mutation, and kill—Comparison of susceptibility to ultraviolet and to other lethal agents-Logarithmic nature of kill— Unit kill— Reactivation by heat and light— Germicidal action of ultraviolet of wave lengths greater than 2800 A— Composite of killing factors. Killing exposures: Reciprocity of time and intensity. Erythemal action of wave lengths 2537 and 2967 A: American Medical Association tolerance— Face and eye protection and treatment. Commercial sources of ultraviolet- High- and low-pressure mercury arcs— Ultraviolet of wave length 2537 A— Conversion factors— Intensity— Ozone formation— Photochemical effects of 2537 and 1849 A energy- Temperature and ventilation— Depreciation. Ultraviolet disinfection: Air disinfection— f luid disinfection- Disinfection of surfaces of granular materials. Ultraviolet-induced m utantsfor new fungi. Protection and processing of products: Mold, antibiotics, and par- enteral fluids-Blood plasma— Syrup, fruit-juice, and wine storage— Meat storage Higher pressure mercury sources of ultraviolet: Intensity and variations with distance- Individual line intensities-Starting and restarting times-Life and depreciation- Research determination of output and intensity— Mercury-amalgam and other metal arcs — S unlamps. References. INTRODUCTION Innumerable applications of ultraviolet energy are suggested in a volu- minous amount of old literature, in which there is little of practical value because of the failure to specify the ultraviolet wave lengths, the inten- sities, and the exposure times used. This is equally true of the many chemical, the indefinite therapeutic, and the few biological effects of the ultraviolet. Ellis et al. (1941) have comprehensively reviewed the chemical and biological applications of the ultraviolet; Laurens (1933) has done the same for the physiological effects. Meyer and Seitz (1949) and Roller (1952) have excellently reviewed the sources, measurement, and various applications of the ultraviolet. Lea (1946) has contrasted the excitation effects of ultraviolet with the ionization effects of X ray and shorter wave length radiations in a practical discussion of the theoretical bjises of both effects, 41 42 RADIATION HIOLOGY The practical biological applications of ultraviolet are those utilizing its erythenial effects, its ergoslerol activation, and its inactivating and mutational effects on bacteria, fungi, and viruses. The action spectra describing all these effects as functions of wave length are subjects of other chapters; this chapter is concerned with one outstandingly practical biological application, the germicidal effect. There arc included dis- cussions of commercially available sources of ultraviolet for this effect and for research on this and other effects. GERMICIDAL-ACTION CURVES If bacteria are irradiated with ultraviolet of various wave lengths and with an identical exposure (intensity times time) for each wave length, 1.0 0.9 0.8 0.7 0,6 0.5 0.4 0.3 0.2 0.1 S i ' \ \ w i 1 ^ \ ri 1 f \ > Q: 1 I ^ „'/ \ s s \ \ if / \ \ \ 1 . \ \ 1 y \ \ \ \ 1 \ \ \ \ / t < 1^' \ \ J \ c \ \ x y v^ / ' N ^ 2000 2200 2400 2600 2800 WAVE LENGTH, A Fig. 2-1. Bactericidal- and orytluMiial-action curves. Illumination, Berlin, 1985.) 3000 3200 3400 {International Commission on which is sufficient to give a convenient unit of killing, oO, 63.2 (lethe; see p. 49), or 90 per cent, at the optimum wave length, the data may be plotted as a germicidal-action curve. Such a curve has not been stand- ardized as have the curves for luminosity and the erythemal action, but Gates (1929-30), Hollaender et al. (1940), Jones ct al. (1940), and others have studied the action of specific wave lengths on specific organisms. Caspersson (1931, 1937) associates the germicidal-action curve with the ultraviolet-absorption curve of the nuclear protein. For most bacteria and fungi and for some viruses, the optimum killing w^ave length is at ^2650 A. The relative effects at longer and shorter wave lengths are so similar that a single tentative action curve for the average germicidal effect on various bacteria and fungi and on many viruses is shown in Fig. 2-1 along with an erythemal-action curve standardized by the Inter- APPLICATIONS AND SOURCES OF ULTRAVIOLET 43 o o o 01 o IT) o o W (X) 5 > < \, N ^ N . ^^^CV^ 'v. --\ ^^. <4 > \ S \ \ \ 1- UJ I P- liJ \ \ v \ \ \ \ \ \ S, \ N \ \ ^ ' \. '> V N, V, \ N V s •v. \ 1 \ s. ^ \ \ \ \ s \ y \, s \, \ s. \ \ -t 6 y ^. \ V \ \ \ \ \ ^ s \ \ -> h N \ \ N \ s. \ V V \ -^/ N \, \ \ > k \ \ 'o.. \ >., \ ^ N \ \ \ s ^. N, > s \^ V \ \. ■h ^. \ k|. \ \ \ V ^v s. (y. *> 1, \ ' N. ->> \ > s V \ > V \ % \ \ ^ > i 1 \ L \ \ O'. S. °^ V \ \ \ s \ NJ \ > i \ \ \ \ s Iw, 4\ ^^ ^ \ \ \ L \ \ s \ \ K •■'o k \ \ \ V \ ^ \ N 1 ^ ^ \ ^ ^ \ \ \ \ \ S k ■^ \ \ s Si ^ \ \ ^ \ \ \ s. s. '-i! s, i>. s V \ \ ^ V ^ N s. ^ s. \ L \ \ \ ' \ ^\ > ■^ s. \ > .> ) V '4 < V s 4 s. \ \ \ \ '\ V !>> > \ \ ^^^: \, s rv \, \ \ s. s. \ ^" kv > V 1 V \ >^^ s V V, N^ ^^ V \ \ c '^^ s N \ ^ \ ^ +/, N s s > V \ 1 S s^ ^i s^ \ \ \ \ N \ -^ k \ ^ \ U —1 be X \-- LiJ _J J_ \ \ r ^ \ \ \ 1- 3 1 \ K \ \ 1 o o ^ o C) fO o o OJ CO s .2 'S O u n y o 00 r/) O -"l (U O o CO C« o fc > ^ ^ %-i o n ro 5 bO =t. ,~ M in 73 7 0) III a> t— ? bi) 0) ■ri \- Ul EQ -J 1 o o > < 03 1- > _l -) Lh O 7' <« < T a; K fl «: 1 1 73 OJ o .!5 3 73 Z a - S CD c X ICl- o 7! UJ ;-i o 3 in to O 6 D, T X o ro (N o i d OJ M O ta o o o o (\jO O O o O o 01 CO f^ ro'O in T rO CVJ ~ IC iniX 39WJ.N30a3d 44 RADIATION BIOLOGY national Commission on lllnmination (ICI), Berlin (11)35). Both curves are plotted with the relative effect at the optimum wave length as 100 per cent. The relative 100 per cent bactericidal elTcctiveness of 2V)'){) A and the 85 per cent relative effectiveness of 2537 A energy shown in Fig. 2-1 should not be confused with the possibility of a 100 per cent absolute kill by 2537 A energy shown in Figs. 2-2 and 3. The ICI factors for the erythemal-action curve and tentative factors for the bactericidal-action curve proposed in Fig. 2-1 are shown for indixidual mercury lines in Table 2-1. These factors are useful for calculating the relative effectiveness and Table 2-1. I^kytiikmal- and Bactericidal-action Factors Mercury ICI Tentative spectrum erythemal bactericidal lines, A factors factors 2353 0.55 0.35 2446 0.57 0.58 2482 0.57 0.70 2537 0.55 0.85 2576 0.49 0.94 (2650) 1.00 2654 0.25 0.99 2675 0.20 0.98 2700 0.14 0.95 2753 0.07 0.81 2804 0 06 0.68 2857 0.10 0 . 55 2894 0.25 0 . 46 2925 0.70 0.38 2967 1.00 0.27 3022 0.55 0.13 3130 0.03 0.01 efficiency of mercury sources whose relative line intensities are known (see Tables 2-3 and G). ACTION CURVE TENTATIVE AT SHORTIHl WAVE LENGTHS The germicidal-actioii i-nvvc for wave lengths less than 2500 A is still tentative since theory and some research suggest that the action continues to increase at shorter wave lengths (greater frequencies and greater energy content of the quanta). A rather rapid drop in the curve at wave lengths less than 2500 A is, on the other hand, characteristic of the absorption curve of nuclear protein and very representative of practical germicidal effects in which nearly all the liquid and gaseous elements in the environ- ment of an organism absorb the ultraviolet of shorter wave lengths and APPLICATIONS AND SOURCES OF ULTRAVIOLET 45 thus protect the organism itself. Perhaps the decreasing germicidal action at wave lengths less than 2500 A found by some workers may represent the absorption curves of the culture media rather than the absolute susceptibility of naked bacteria to ultraviolet killing. 8 ' o'i^^gth '^ J^ 9 !^Q A o ~ £\j O Fig. 2-3. Composite of Figs. 2-1 and 2. ACTION CURVE APPROXIMATE AT LONGER WAVE LENGTHS The germicidal action of radiant energy extends even into the visible spectrum, the action decreasing rapidly with increasing wave length (decreasing frecjuency and energy content of quanta). The action is of the order of magnitude in the near ultraviolet and visible estimated by Luckiesh and Taylor (1946; see also Hollaender and Claus, 1935-36) in Fig. 2-4 where a logarithmic ordinate is used to extend the killing action to lower effectiveness levels. The reactivating effects of wave lengths 3600-4400 A reported by Kelner (1949) suggest that, for all practical pur- poses, the curve of Fig. 2-4 might well end at 3600 A. In any case, since •46 RADIATION BIOLOGY ^ 0.00002 ^ 0 00001 7000 "2000 3000 4000 5000 6000 WAVE LENGTH, A Fig. 2-4. Relative bactericidal action extended to the near-ultraviolet and visible regions for E. colt on agar. (Luckiesh et al., 1947 ; Hollacndcr and Claus, 1935-36.) no effects on fungi are reported, the curve probably applies only to the more susceptible organisms, at wave lengths greater than 3600 A. THE UNIQUE 2537 A MERCURY LINE Sixty per cent of the electrical input to a low-pressure mercury arc is converted directly into radiation of wave length 2537 A. Wave length 2537 A produces 85 per cent of the maximum germicidal effect on most bacteria, fungi, and viruses which is possil)le at ^2650 A. This efficient production of ultraviolet of nearly optimum germicidal wave length is one of the more unusual coincidences in biophysics. The shape ot the germicidal-action curve is su(^h that the effectiveness of 2537 A ultraviolet is only 10-20 per cent less than the maximum effectiveness possible at 2650 A, an uncertainty well within the variations in the action curves for various organisms and within the experimental errors inherent in the determination of the curves. For these reasons the low-pressure mercury arc has been selected as the practical source of ultraviolet for germicidal effects. About the only practical interest in the germicidal-action curve is to appraise the relative inefficiency of high-pressure mercury arcs in fused (luartz glass, the only artificial sources of ultraviolet that are at all comparable with the low-pressure arcs. In Fig. 2-5 the action curves are superposed on block diagrams of the relative line intensities of typical high- and low-pressure mercury arcs. The block diagrams are calculated on the basis of etjual amounts of power (in watts) into th(> two types of arcs in order to show graphically their relative ultraviolet efficiencies as well as their germicidal and erythemal effectiveness. AMPLICATIONS AND SOURCES OF ULTRAVIOLET 47 1 0 T 1 09 .0^2 ^ I— ^T pv -^ . \I^- \ 08 .--^ '/^ > ^ \ r«; t ^ V 07 ^/^ / -J < Q _ / -I < \ \ T 06 / o tr UJ — o < — [ < - 5 UJ I . \- V 1^ _ UJ / UJ ) V / \ 05 z o- / 1 I u - O - \ / _i < \ 0.4 rs] O '^ / / 1- o - X . \ / CO > \ 03 \ 1/ a \ J ^ — N V m 0? 7 / M in \ s. / 10~ tn c in \ §01 / 'j rOS. CM \ V Cvj CM CM fr K, "- ^— 1 ^ ^ — ID rO 1 ^ o > / — N \ fri 1.0 20 ^ ^^c. M V 1 rrn \,^ / [\ s — '- \\ u) 09 00 w n , SBC \ M 32 oc 36 / 00 " S40 00 44 00 48 00 52 7 < 56 00 6000 6400 UJ > 0.8 .^ \ 1 / \ /J —^ \ 1 — 307 "=06 / o o < \ (b) r \ 1 I luj < _ 5 UJ X 1- 1 s ~ UJ V / -^ k 0.5 UJ o — \ ICD / X o \ / < \ 0.4 O 1 \. -JV 1 UJ / H O T \ / > T 03 ( 1 i/ Q. \ / / — \ \ 0.2 1 -f — 1^ m \ K / \ 01 01 / CM v3V^ v\ \ in \ / » 1 / \ V. r A ^V^ \J^ ^ ^ 1 I8C )0 22 00 26( 30 30( )0 34 00 38 WAVf oo : LE 42 NGT oo H, A 46 00 50 OO 54( oo 5800 6200 660 Fig. 2-5. Relative energy distribution of (a) high- and (6) low-pressure mercury arcs, transmission of arc tube glasses, and action curves. SUSCEPTIBILITY TO ULTRAVIOLET INJURY, MUTATION, AND KILL An understanding of the biophysical nature of cell injury, mutation, and kill is not essential for the practical applications of the ultraviolet! In this discussion, for practical purposes, an organism is considered dead when it is unable to reproduce. The possibility of this being a condition far short of complete destruction, and the logarithmic nature of ultra- violet killing, leave considerable uncertainty as to just when all the organ- isms in one group are dead. In most of the practical ultraviolet applica- tions a complete kill is not necessary, but whenever it is necessary it can be pro\'ided by adequate factors of certainty in exposure. There is ample evidence from practical experience that the growth of fuiigi can be prevented by exposures of the order of those effective for bac- teria killing and by exposure of only a fraction ( a tenth to a hundredth) of those required for the killing of bacterial spores. The wilting and death of common plants such as ivy and tomato under such exposures sug- 48 KADI ATION lUOLOr.Y gest suppression of tlio mycdia without serious damage to the spores, buf the mechanism should tx' ;iii interesting subject for research. COMPARISON OF SUSCKl'l I HlLl'l^ TO TLTRAVIOLET AK\) TO OTHIOR ij/niAi, a(;kxts The intrinsic susceptil)ility of \arious species of bacteria, fungi, and viruses to killing by ultraviolet of 2537 A, or any other wave length, varies over an exposure range of as much as 1-3. The extrinsic susceptibility, determined by acquired tolerance and age, may also \'ary over a range of 1-3. In contrast with liacteria and viruses, for any arbitrary percentage kill of various mold spores, the exposure may be 15-300 times that required for the same kill of dry air-borne Escherichia coli, with a difTer- ence as great as 1 to 1000 between the most susceptible bacteria and the most resistant mold spores. The exposure necessary to kill any one kind of organism may vary considerably, depending on its environment, tem- perature, illumination, and physical condition, illustrated in Pig. 2-2 by humid water-borne E. coli which requires four times the exposures of dry air-borne E. coli for comparable killings. Although the effects of radiation of shorter wave lengths on various microorganisms seem to be very similar to those of the ultraviolet, there is little similarity between the effects of ultraviolet and of other lethal agents such as heat, dryness, or chemicals except in the logarithmic nature of the kill. Markedly thermoduric organisms, for example, are readily killed by ultraviolet energy. LOGARITHMIC NATURE OF KILL The practical ultraviolet killing of nearly all microorganisms is more or less logarithmic in nature. Wyckoff (1932) and Rahn (1932, 1945) have discussed this rule and its exceptions in detail. The logarithmic nature of kill is in accord with the general exponential attenuation law, ,V = AV-", (2-1) where iVo = the initial concentration of organisms, ,V = concentration of organisms after an exposure I'or tinu- / to an ultra- violet intensity, / = the ultraviolet intensity, and e = the base of the natural logarithms (about 2.718). For a unit exposure, 7/ = 1, the concentration of survivors .V becomes N = Noe-' = 0.3(58 No, (2-2) and the kill, .Vo — N, becomes No- N = 0.632 .Vn. (2-3) I Al'l'LICATIONS AND SOURCES OF ULTRAVIOLET 49 The same attenuation law also covers the dilution of fluid-borne con- tamination by the admixture of a neutral and sterile fluid when the unit volume of diluent is substituted for / in the exponent. UNIT KILL This relation suggests 63.2 per cent as a basic unit of sanitation for which the term "lethe" has been suggested. With air sanitation in mind, Wells (1940) has also used the term for a unit ultraviolet exposure pro- ducing a 63.2 per cent kill of a standard organism (E. coli) under elabo- rately specified conditions. A lethal exposure then becomes equivalent /xw/cm^ TO KILL IN I min 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MINUTES TO KILL WITH I //watt/cm^ Fig. 2-6. Typical data from Fig. 2-2 shown on a logarithmic scale of percentage of survivors and uniform scale of exposures. in effect to the air change of mechanical ventilation, as discussed later. The lethe unit of kill is indicated in Figs. 2-2 and 6. The exponential form of the curve of killing as a function of exposure has also led to the suggestion of a unit kill of 50 per cent, by analogy with the half-life rating of radioactive materials. The logarithmic nature of the ultraviolet germicidal efi^ect is illustrated in Fig. 2-6 where the data on dry and wet E. coli are plotted on a loga- rithmic scale of survivors and on an arithmetic scale of exposures. Most of the published data plot as straight lines on such scales within the experimental errors of the measurements [see Lea (1946) for a discussion of methods of plotting such data]. An arithmetic or linear scale of kill is used in Fig. 2-2, along with a logarithmic scale to e.xpand the range of .-,0 RADIATION BIOLOGY lesser exposures and yet cover a thousandfold exposure range. The sig- moid form of the curves is inherent in the scales used. REACTIVATION BY HEAT AM) I.KIIIT The j)ractical signilicaiu'e of the reactivating effect of time, heat, and light on ultraviolet-injured bacteria and molds was overemphasized by the manner in which the experimental data were first presented by Kelner (1949). For example, a definite 20 per cent revival of the total initial number of irradiated bacteria per experimental unit volume was presented as a 3000 per cent increase in the number of viable organisms from the indefinite few left after a theoretical killing of 99.99994 per cent. By this method of presentation, if the "killing" had been complete, the per- centage increase in viable organisms would have been infinite regardless of the actual revival. The 20-25 per cent revivals of bacteria resulted from light exposures of the order of 5-8000 ft-c-hr, exposures provided only by 2 3 days of the highest levels of practical indoor illumination. The eciuivalent of a 2- to 3-hr exposure to 100 ft-c provided less than a 0. 1 per cent revival, and few practical germicidal applications involve exposures of bacteria to as many foot-candle-hours. This suggests that the reactivating effect of light is of little or no significance indoors but may somewhat reduce the apparent susceptibility of organisms to the ultraviolet of the sun. GERMICIDAL ACTION OF ULTRAVIOLET OF WAVE LENGTHS GREATER THAN 2800 A Buchbinder ct al. (1941) have shown that sunlight, direct and through window glass, as well as the ultraviolet from common artificial light sources, has measurable germicidal effects on bacteria exposed to common illumination intensities for a day or two (Fig. 2-4). The daylight inten- sities and exposure times may have been somewhat comparable with those used in the reactivation experiments, in which case the killing must have been the difference between the germicidal action of wave lengths greater than 3000-3200 A and the reviving or protective action of wave lengths greater than 3600 A. COMPOSITE OF KILLING FACTORS Only a three-dimensional model would completely represent the rela- tions of wave length and exposure to killing. However, the outstanding practicality of wave length 2537 A suggested making its plane representa- tive of the wave length and plotting on it a fourth indeterminate variable, the .susceptibility of organi.sms to killing by that wave length, as typical of the killing by other wave lengths. The result was a consolidation of Figs. 2-1 and 2 as Fig. 2-3. ■ APPLICATIONS AND SOURCES OF ULTRAVIOLET 51 KILLING EXPOSURES RECIPROCITY OF TIME AND INTENSITY Like the photographic effects of Hght, the germicidal effect of ultra- violet results from an exposure (intensity times time). The basic factors in an exposure are the incident power, the time, and the irradiated area. The erg, often used in biophysical work, is a unit of energy only and must be referred to time in order to define power. Ergs per second become units of power, and ergs per second per square centimeter become units of uitensity. Ergs per second per square centimeter-second, or, more usually, ergs per square centimeter, become units of dose or exposure. In practical work there are advantages in basing the intensity unit directly on the watt since it is also used to define the power output of ultraviolet sources. The microwatt, equal to 10 ergs/sec, becomes a con- venient unit of power, and the microwatt per square centimeter becomes a convenient unit of intensity. For practical purposes the microwatts per square centimeter equal the milliwatts per square foot, the multiply- ing factor being 0.9290. The microwatt per square centimeter-minute, often written as microwatt-minute per square centimeter, becomes a prac- tical unit of exposure equal to 600 ergs/cml The microwatts per square centimeter-minute emphasizes the reciprocity of exposure intensities and times which may be adjusted over a very wide range to obtain a specified exposure under various conditions. Theoretically, an e.xposure of 25 MW-min/cm-' or 1500 ergs/cm^, for example, may be obtained either in a long time (1 day) with a low inten- sity (0.018 ultraviolet /xw/cm^) or in a short time (0.001 min) with a high intensity (25,000 ultraviolet /xw/cm^). In practice, the exposure time IS usually determined by the nature of the job to be done and ranges from a fraction of a second for the disinfection of rapidly moving air or products to 1-10 min for air disinfection in relatively quiet places. The exposure intensity must then be adjusted to obtain an adequate exposure. ' Such intensities may range from a few ultraviolet microwatts per square centimeter for bacterial air disinfection to several ultraviolet milliwatts per square centimeter for product disinfection from molds, as suggested by Fig. 2-7a,b and in greater detail for the short exposures in air ducts by Fig. 2-8. The reciprocity of time and intensity is also illustrated by the upper and lower legends on the otherwise identical scales of exposure of Fig. 2-2. ERYTHEMAL ACTION OF WAVE LENGTHS 2537 AND 2967 A As shown graphically in Figs. 2-1 and 5, the germicidal ultraviolet is also erythemal in action, the effect at 2537 A being about half as great as at the optimum wave length of 2967 A. As indicated in Figs. 2-1 and 7a 52 RADIATION lfI()L(JGY 0.000 001 0.001 0.01 0.1 I 10 100 1,000 EXPOSURE TIME, min 100,000 I U II 1,000 7 hr I hr EXPOSURE TIME, min Fifi. 2-7. Reciprocity of tiinc and intensity for various exposures and the kill of typical inioroorganisius. APPLICATIONS AND SOURCES OF ULTRAVIOLET 53 erythemal exposures are about 10 times the germicidal and are comparable with the fungicidal exposures, so that the time element becomes important in practical applications. Intensities, which are germicidal within the few seconds and minutes reciuired, in many cases become erythemal with 10-15 times longer exposures of the face and eyes, thus making some form of protection usually necessary. As suggested in Fig. 2-76, an exposure to 1,000 500 80 250 LINEAR AIR SPEED, ft/mm 100 50 25 to 5 10,000 8,000 6,000 5,000 4,000 3,000 2,000 80,000 60,000 50,000 40,000 30.000 20.000 q: UJ < O < O o a: o t,000 800 < u. CO o o S o > < 0.001 0.0O2 0.005 OOI 002 0.05 01 0.2 EXPOSURE TIME IN AIR DUCTS, min/ft Fig. 2-8. Reciprocity of time and intensity for short-time exposures in air ducts. 2537 A energy of 450,000 ergs or 750 MW-min/cm- will produce a minimum perceptible erythema of many skins (see Chap. 13, this volume). AMERICAN MEDICAL ASSOCIATION TOLERANCE The American Medical Association (1948) specifies 0.5 M^/cm"^ as the maximum permissible ultraviolet intensity for 7-hr day exposures, an exposure of 126,000 ergs, or 210 MW-min/cm-, but they reduce this to 144 MW-min/cmVday (an intensity of 0.1 fiw/cm^) for contimious exposure and acknowledge that the specification is based on experience with wave lengths 2800-3200 A rather than wave length 2537 A. 54 RADIATION BIOLOGY Altlit)ut»;h this exposure toleranee mi{i;ht seem to supply an unnecessary factor of safety, it provides for some unusual skin sensiti\ities bordering on the pathologic. Experience in hospitals and in inchistria' applications indicates, for example, that some adult face skins are more sensitive to ultraviolet irritation than the face skin of the average infant . The Amer- ican Medical Association has not had occasion to specify a corresponding sui^erythemal exposure for '29()7 A ultraviolet, but its unit exposure for a minimum perceptible erythema on average untanned skin is 300 nw- min/cm-, as indicated in Fig. 2-76. It should be noted here that there is no theory or evidence that the erj'thema produced by 2537 A energy differs at all from that produced by 29G7 A except that the former is more superficial and transient. Expo- sures to comparable erythemas which result in skin peeling after 2957 A produce only scaling after 2537 A. This difference in effect, shown in the erythemal-action curve of Fig. 2-1, may be due entirely to the slightly deeper penetration of 29G7 A into the skin (see Chap. 13, this volume). Useful germicidal intensities of 2537 A ultraviolet range from 5 to 6000 Mw/cm-, and the corresponding suberythemal exposure times are from less than 40 min to 2 sec. The intensity of ultraviolet reflected from sur- faces and walls of a minimum reflectance of 5-10 per cent may range from 5 /iw downward. From this it is obvious that there is always a problem of face and eye protection in practical applications of ultraviolet energy. Table 2-2 extrapolates the American INIedical Association's permissible exposure through practical ranges of time and intensity. Table 2-2. M.\ximum Permissible Daily F^cposures Exposure time Intensity on Exposure time Intensitj- on per 24 hr, hr faces, /xw/cm^ per 24 hr faces, Mw/cm* 24 0.1" 2hr 1.8 18 0.2" 1 hr'' 3.6 12 0.3 30 min 7.2- 9 0.4 10 min 21.6' 6 0.6 1 min** 216.0 4 0.9 30 sec 432.0 3 1.2 5 sec 2()00.0 "Permissible intensity in hospital infant wanis; onc-liftieth or one one-hundredth that recommended for hospital air disinfection. * Exposures (time times intensity) of 3.6 juw-hr/cm'. 'Intensity recommended for hospital upper-air disinfection; tolerated only 10-30 min if on the faces of personnel. '' Exposures (time times intensity) of 216 /j\v-min/cm^. FACE AND EYE PROTECTION AND TREATMENT Commercially available sun glasses and face shields designed to cover the eyes from the sides and the ears completely provide adequate protec- tion. Hands and arms may be protected by plastic or rubber or very APPLICATIONS AND SOURCES OF ULTRAVIOLKT 55 closely woven textile gloves, but these gloves should be tested before long- time use. The discomfort from ultraviolet-irritated eyes may be relieved by exposing them for 15-20 min to as high an intensity of heat as can com- fortably be borne from a heat lamp or from an ordinary 50- to 60- watt incandescent lamp held close to the eyes; the treatment is effective through closed eyelids (author's personal experience). In extreme cases, a doctor should be consulted, but when this is impracticable, the usual first-aid treatment is the application of ice packs. In any case, the irri- tation produced by the ultraviolet may disappear within a day or two and much more quickly than a corresponding degree of irritation from a longer wave ultraviolet source. A severe conjunctivitis may, however, make the eyes susceptible to secondary infection until the lesions are healed. COMMERCIAL SOURCES OF ULTRAVIOLET Mercury-vapor sources of ultraviolet for practical and experimental uses may be grouped as (1) commercially available low-pressure (0.004- 0.02 mm of Hg) germicidal lamps, (2) high-pressure (400-60,000 mm of Hg or 0.5-75 atm) photochemical, therapeutic, and filtered sunlamps, and (3) special experimental lamps of limited availability. The character- istics of all but the low-pressure lamps are discussed later in this chapter. Arc lamps with rare-earth cored carbons provide powerful sources of energy for many photochemical, photographic, and photocopying appli- cations of the ultraviolet. For various reasons their biological applica- tions have been rather limited and are not discussed in this chapter. HIGH- AND LOW-PRESSURE MERCURY ARCS As indicated graphically in Fig. 2-5, low-pressure mercury arcs are 5- 10 times more efficient in germicidal action than high-pressure arcs in envelopes of the same transmission. It should be noted that high-pres- sure quartz-mercury arcs may be of practical use, regardless of efficiency, in places where it is impossible to provide the essential ultraviolet inten- .sities from the much more bulky low-pressure lamps. For example, the germicidal effect per unit of total volume of a 360-watt high-pressure arc in quartz is 5-10 times that from low-pressure arcs, but the germicidal efficiency of the high-pressure arc is one-fifth to one-tenth that of the low. For another example, the germicidal effectiveness of the radiating part of the UA-3 and UA-11 high-pressure mercury arcs of Table 2-6 can be duplicated only by 8-12 times the radiating length of the more efficient low-pressure lamps. ULTRAVIOLET OF WAVE LENGTH 2537 A INHERENTLY LOW EMISSION INTENSITY OF SOURCES OF 2537 A The possibility of a source of 2537 A ultraviolet with the high power output per unit of source area, or radiant-flux density, of the high-pressure 56 RADIATION niOLOGY a z W E- a o % O 03 H O « O o ^ < ^ I (N 3 a. 3 O CC ^ w ^ P5 3 > 03 ? -3 ^ :s <; o H o CO o E-i to H U « j3 •- j3 ■^ D, d a, 2 £ E c a O o CO £ ^ r 1^ I t; P t. >< o is OJ C 3 a o _ -»J o c 3 ^ C -r "c S « c 83 S3 §^ 3 -^ CO c« c •- X c r -i^ . =3 = £ ^.S ^ s c3 ~ X o .:s -^^ '^ - ^. ^ P t- fc- a. t- SJ OJ Q 5 ::j -a a c C3 ^ a> C 3 O |2 ace 03 03 c > 5 §i 3 t^ Ch a.S ^ d o d ?- ?5 h o < •« -e t: Q O O O o o c o 2 o o o ic 'O lO lO lO .-: ic o lO Tt c>^ 01 CO o 00 ^ 0) Ph iC t^ iC (M or ■^ ro 1- _ 00 1 X t^ c -r 05 CO >c 00 1 o •— ( c^ (N 1 t^ ■* 00 ir : o ^ re a a ■•• ■-^ ^ •-. •-s V V a 3 00 ■^ iC h - H H H ir 3 O ■* 00 H ro O a c 5 C ' 1 g 0 c 0 c o 0 c ♦r 'T (N C lO 'C iC uo -r oi oj Cq C^ IM t- (M IM (N (N — ' — —, o c o o C^ C^ C^ CC — c<\ yt f lit o c »o O O lO ic cc Lt c o c o • • ■ o o o o o c o O C C O O O C lO lO iM C^ (M (>) 0^*0 o — lo -,o c; C-. r: cs (M (N ?C CO — C^ r: O) (M (M (N >o lO lO »0 l> lO (N Oq r^ cc t^ lO '* C5 Tt- ■* CO o -^ ■* -^ '^ (N CQ CO CO -H CO CO CO CO CO lO o o O O (M CO 1 1 t 1 O CO CO o 1 1 1 1 -* t- X iC o o o N CO ■* iC CO t^ X — cr. -^ c. M c IK = -3 -^ 2 O tH S £ M C 3 ^ C ~ S o ° Q « « a, n o lO c _> -5 £ '^ "S ^^ o -^^ ^ ;- S X -f^ d 3 ^ t3 .2 '^ 2 CM .2 -^ ^ « -^ 5 ■^ -^ C i; C »-* — . H. -5 c c p o .2 .2 5 -"- t^ Ou '- o ■*^ c o o 3 T3 O O o X CM X 1^ " u 3 -2 3 g-oo c a, CM O . *^ 3 t- •— ' =5 > +-' O 3.S N CO ^ ■£ o S o C3 O C •2 5 -3 ->^ £ ^ ;= . * 3 --.1 -^ tri — as ■^ -3 ^ •t; 3 3 1^ h-5 ^ 3 O C ^ 3 •? ^ .^ 3 P ^ .3 M SS o c O O . > 3 X _- = '-S '7 "^ 3 t: !, t. t. O 03 X Q o M m < s ►S > > a — =^ S^ 72 <; I— I 58 RADIATION BIOLOGY mercury arcs lias long taiitali/od exporimoutcrs. From the well-known absorption of the 25.37 A resonance line by mercury vapor and from an erroneous association of efficient 2r)37 A production solely with low mercury-vapor pressure, the experimenters have inferred that water or air cooling of a lamp should permit a great increase in the electric power input and the 2537 A emission. In the search for the optimum con- ditions for 2537 A production, rather definite optima of vapor pressure and power input, corresponding to lamp-tube temperatures of 40°-60°C, have been found. Radical decreases in mercury pressure by cooling or increases in power input, either separately or concurrently, produce rela- tively small changes in 2537 A output but produce radical changes in efficiency. The output ratings of commercial sources are the maxima consistent with good efficiency and life. Users of 2537 A sources who may be willing to sacrifice both life and efficiency for higher output power density must now be reconciled to a maximum emission of the order of 30-50 ultraviolet mw/cm- of source surface provided by about 0.1-0.15 watt of electrical power input per square centimeter of tube surface. This is about twice the output of commercial sources. The difference between power input and emission (3 to 1) results from the inefficiency of the conversion of electrical power to radiant power in the lamps and a subsequent absorption of about 20 per cent in the glass tube. Tenfold increases in power input, which are possible by water or air cooling, pro- vide but slight increases in the 2537 A output per unit of source area. These generalizations have little or no bearing on the radiation character- istics of higher pressure mercury arcs discussed later. SOURCES OF 2537 A ULTRAVIOLET Table 2-3, based partially on the lES Lighting Handbook, 2d ed. (1952), presents the physical, electrical, and radiation characteristics of most of the commercially available sources of 2537 A energy. In each ease the amount of electrical input (in watts) to the arc, the length of the radiating source, and the total radiated 2537 A energy in watts, here- after called "ultraviolet watts," and the ultraviolet watts per square centimeter at a distance of 1 meter are closely associated. This permits calculation of the efficiency of the sources, of their input and output per unit of source length and area, and of the ultraviolet intensity provided by them at various distances. Division of the intensity in ultraviolet microwatts per sc^uare centimeter at 1 meter by 10,000 provides a useful practical rating in ultraviolet watts per square foot at 10 ft. Multipli- cation of ultraviolet microwatts per square centimeter at 0.9290 converts to ultraviolet milliwatts per square foot, but for practical purposes they are equivalent. Multiplication of the 10-ft rating by 100 and division by the distance squared provides intensity in the same units for other distances greater than the length of the radiating source. APPLICATIONS AND SOURCES OF ULTRAVIOLET 59 CONVERSION FACTORS POWER, INTENSITY AND DISTANCE, AND WORK AND ENERGY The use of metric and U.S. units, separately and together, named units, and various time units, with little standardization of practice, requires frequent use of conversion factors. In Table 2-4 are listed various con- Table 2-4. Conversion Factors From Multiply by To Power Output and Intensity UV watts output, total UV watts output, total UV pw/cm^ UV output /steradian UV output/steradian UV output/steradian UV intensity, unit area, 1 meter , UV intensity /cm^ at 1 meter. . . . ergs /sec joules /sec 10.0" 0.001 0 . 929 10.0" 0.01 0.0001 0.176 10 X 103 0.1 1.0 UV Mw/cm^ at 1 meter UV juw/sq ft at 10 ft UV mw/sq ft UV output total UV output/cm^ at 10 cm UV output /cm 2 at 1 meter UV intensity, unit area, 10 ft UV intensity /sq ft at 10 ft UV MW UV watts Work and Energy* joules joules ergs ergs g-cal Btu r (roentgens) in air Photons or quanta at 2536 A. 1.0 0.01665 X 10« 0.1 0.011665 69.77 X 10' 17.4 X 106 0.00018315 0.0131 X 10 '2 UV watt-sec UV /iw-min UV MW-sec UV MW-min UV /Ltw-miu UV /iw-min UV MW-min UV MW-min Exposures joules /cm 2. ergs /cm 2. . g-cal /cm 2 . r/cm^ 0.01665 X 106 0.001665 69.77 X 103 0.00018315 UV MW-min/cm^ UV nw-min/cm^ UV MW-min/cm^ UV MW-min/cm^ " Approximate. ^ When on equal areas — exposures. versions to the centimeter-microwatt-minute units used in this chapter. The relation between ultraviolet output and maximum intensity refers only to essentially linear sources, to distances greater than the length of the source, and to directions of maximum intensity perpendicular to the center of the source. Energy per steradian refers only to the steradian in that maximum intensity direction and, literally, only to a very small part of the solid angle represented by the steradian. For this reason, energy 60 HADIATION HIOI-OC.Y per steradiiui is uii unsatisfactory description of the output of linear sources. 2050 A i:cn IVALKVCE Since 25;i7 A (Micrj>;y has only about 85 per cent of the hactcricidal action of 2050 A energy (Table 2-1), the ultraviolet watts output and ultraviolet microwatts of Table 2-3 must be multiplied by 0.85 for the 2050 A bactericidally equivalent ultraviolet watts of Table 2-0. INTENSITY RELATIVE ENERGY DISTRIBUTION The inherent spectra of all low-pressure mercury arcs are dominated by the 2537 and 1849 A lines. Other lines are so relatively weak and from such low-intensity sources that they are of little practical value (Table 2-5 and Fig. 2-56). The output of the 1849 A line is determined Table 2-5. Relative Energy in Various Spectral Lines or Groups of Typical Low-pressure Lamps Wave Length, A Relative luiergy, % 2537 100 2652 0.14 2753-2893 0.12 2967 0 . 37 3022 0.17 3126-3132 1.43 3650-3()63 1 . 30 3906-4077 1 . 60 4339-4358 3 . 40 5461 2.25 5770-5791 0.60 over a wide range by the fused cjuartz and the special glasses, in various thicknesses, whose transmissions are shown in Fig. 2-5a. Since the 1849 A energy is rapidly absorbed by air (about 50 per cent in 1 in.) and since it penetrates licjuids and cellular proteins much less effectively than the 2537 A, little practical bactericidal application has been found for energy of this wave length. Since 1849 A energy is only slightl}^ absorbed by nitrogen but is readily absorbed by oxygen, it provides ozone in air, relatively uncontaminated by oxides of nitrogen, and some practical application of this energy for this purpose is being made. Commercially available sources in thin glass provide 1849 A energy to an extent 1-2 per cent that of 2537 A energy. Greater 1849 A energy output is possible through thin fused-fjuartz glass. RESEARCH SPECIFICATIONS OF ULTRAVIOLET INTENSITY The ultraviolet power output and intensity ratings of Table 2-3 are average values for new sources. The variation and service depreciation i APPLICATIONS AND SOURCES OF ULTRAVIOLET Gl of all commercial sources are such that the output ratings should be used or approxmiations only. A description of a lamp type and of its elec- trical characteristics is an essential guide to the mechanics of a laboratory research but is. of little value as a means of specifying the radiation nitensities provided. The effective intensities obtained fn any „h should be measured at the irradiated surface or throughout the irradiated volume and should be specified in general terms entirely independent of the source, the usual laboratory unit being the microwatt per square centimeter, and the corresponding engineering unit, the milliwatt per square foot. ^ INTENSITY VARIATIONS WITH DISTANCE For distances greater than the length of the radiating source the intensity varies inversely as the square of the distance. For distances ess than about one-third the radiating length of these linear type sources he intensity varies inversely as the distance. The variation at the transition distances can be measured directly or estimated from the ac-tual mtensities produced by such typical sources as are shown in HIGH INTENSITIES AT CLOSE RANGE The maximum hitensity prcn-ided by a single tubular source is at its own surfac-e. This is a useful point at which to start a study of the variation of intensity at short distances from the tube. Distances are measured from the center of the tube although it radiates ultraviolet r' r^ 1 T r^ ""^ ^^' *"^' '''^'^ *^^ «^^^^^- The effective emitting ng h of the l-in.G30T8 tube of Fig. 2-9 is 32 in., and its circumferenc^ •s 314 m. From this surface area of about 100 sq in., 7 ultraviolet watts 0 ^00 ' "">' T""'T '""^'''"'^^ "^ ^-^^ ultraviolet watt/sq in. or il^800 Mw/cm-. A surface or a material in contact with the tube would therefore be irradiated at that intensity. Similarly, a cylindrical surface 2 in. in diameter would have double the tube area^ J* .would intercept practically all the radiated 7 ultraviolet watts with half the intensity. Similarly, the intensity on a 3-in cylin- drical surface would be one-third as great, and on a 4-in. cvlinder one- ourth as o^reat On cylinders of these diameters, small compared with heir eng h, the ultraviolet intensity is uniformly distributed, except tor a length at each end equal to about the radius of the tube Th,> intensity on the surfaces of such irradiated cylinders varies with their diameters or inversely as the distance from the tube axis to the irradiated suitace. This relation is true out to distances of the order of one-third the effective length of the source, as shown in Fig. 2-9. 62 RADIATION mOLOGY ti31N30 3901 kNOtiJ 93HDNI _ _ o o o y31N3D 3901 WOaJ 133J APPLICATIONS AND SOUKCES OF ULTRAVIOLET 63 AVERAGE INTENSITY IX SPACE In the disinfection of fluids, the average ultraviolet intensity, or average radiant-energy density, throughout the concentric cylindrical space sur- rounding the tubular sources becomes a basic factor. It is especially basic in the case of air where there is no absorption to modif}^ the linear decrease in intensity inversely with the distance from the lamp tube. It can be the basic intensity factor in the disinfecting exposure of air where it is possible to provide enough turbulence of flow to expose the air to the full range of intensities, and so to an average intensity, during its travel through an irradiated zone. Since the intensity at less than source-length distances from linear sources varies inversely as the distance and the volumes of successive increments of annular space increase directly as the distance, the products of annular volume increments and their energy density become constant. Thus, within source-length dis- tances, the average intensity occurs at the average distance of one-half the radius of the irradiated zone and is twice the intensity at the outer limits of the zone, as pointed out by Luckiesh and HoUaday (19-l:2a,b). At distances greater than source length, as in directly irradiated rooms or very large plenum chambers of ventilating systems, the intensity throughout the spherical space surrounding a central ultraviolet source varies inversely as the stjuare of the distance, and the volumes of suc- cessive increment shells of space increase directly as the square of the distance, so that the products of successive volume increments and their energy density become constant. Here again the average intensity would occur at the average distance of l/\/3 or 0.577 the radius of the irradiated spherical volume and would be three times the intensity at the outer limits of the volume, as developed by Wells (1940), if it were not for the toroidal rather than spherical spatial distribution of the energy about a linear source. Also, since irradiated rooms are cubical rather than spherical in form, the average intensity occurs at more nearly half the average radial distances to the walls and is again about half the intensity at the outer limits of the spherical or cubical zone. INCREASE OF INTENSITY AND UTILIZATION BY' REFLECTORS Efficient sources of the germicidal ultraviolet are inherently low in intensity compared with high-pressure sources designed for photo- chemical and therapeutic use. A maximum intensity of 10-20 ultra- violet watts/sq ft (10,000-20,000 ultraviolet-/iw/cm'") can be available at a tube surface for experimental work, but, at practical working dis- tances, only about one-fourth this intensity can be obtained as irradiation over an extended area (see Fig. 2-106). The ionized mercury vapor in germicidal tubes almost completely absorbs any 2537 A energy which might otherwise pass through the glass tube itself from an outside source. For this reason, only the thin layer 64 KADIATION HIOLOGY ot incri'ury xapor prtictii-uUy in roiitacl with the luinp tuhe i.s aiiolTcctix-e source of 2537 A energy. Thus, when several such tubes are placed in contact, side by side to form a grid, only about one-third their total ultraviolet output reaches a parallel irradiated surface of an area about one-third the total surface areas of the tubes (Fig. 2-1 Oaj. It is of interest to note that, with such a rectangular or sciuare grid source formed by such an as.sembly, the intensity on the irradiated surface remains constant for distances out to about half the width of the assem- bly. As the distances are increased to the length of the rectangle, there is a transition to a variation inversely with the square of the distance. Single tubes are, in effect, rectangles of so small a width as to have the characteristics of theoretical line sources (Fig. 2-9). (a) (6) Fig. 2-10. Effect of bactericidal-tube spacing on utilization of total ultraviolet output. (a) Close spacing, (b) Open spacing under reflectors. The high intensities possible with germicidal tubes in contact in a grid pattern can be produced more economically with about one-third as many tubes fitted with reflectors. In a tube-and-reflector system the tubes should be spaced on centers three or four times their diameter (Fig. 2-10^). All reflectors for practical uses with germicidal lamps should be of speciallj^ processed, polished aluminum (60-70 per cent reflectance) or polished chromium plate (40-50 per cent reflectance). Luckiesh and Taylor (1946) have shown that no other reflecting materials are of practical value. Special aluminum paint may be used in some places, such as in air ducts, if the service or maintenance is such as to make occasional repainting practical. Such a paint is made of pure aluminum flakes in a vehicle of plastic lacquer of high ultraviolet transmission. Specular aluminum reflectors, designed to intercept about two-thirds of the tube energy, redirect about (55 per cent of the energy to the irradi- ated surface. The efficiency is therefore 65 per cent of two-thirds or 43 per cent plus the 33 per cent directly from the tube or a theoretical total of ^^75 per cent. In practice, commercial etiuipment is only capable of doubling the effective radiation from an equivalent grid of bare tubes. APPLICATIONS AND SOURCES OF ULTRAVIOLKT ()0 The mercury column in the tube absorbs almost all the energj^ redirected to it by the reflector and prevents higher utilization. OZONE FORMATION One-tenth to 5 per cent of the mercury line 1849 A energy is trans- mitted by the glasses used for the tubes of germicidal lamps as indicated in Fig. 2-oa. Energy of this wave length, transmitted only a few inches through air, easily breaks the weak bonds of the oxygen molecule to permit the formation of ozone near the ultraviolet source. Roller (1946) found such ozone to have a half life of 15 hr, from concentrations of several hundred parts per million, in the dry glass containers in which it had been formed. Ewell (1942) had found, however under more prac- tical conditions, that humidity, light, 2537 A ultraviolet, and surface absorptions greatly catalyzed the reversion to oxygen. Under such conditions he found ozone to have a half life of 2-3 min when irradiated with 2537 A ultraviolet and of 6-7 min when not irradiated, in both cases from concentrations of 3-4 ppm. Since this ozone diffuses throughout an irradiated space, its inherent instability is considerably increased by 2537 A energy, and short-lived atomic oxygen occurs in a uniciue manner. Commercial sources permit air disinfection with eciuilibrium ozone concentrations less than the 1 part per 10 million considered permissible by the American Medical Association (1948). Other sources provide the somewhat higher concen- trations traditionally used in certain food-storage applications, where the odor-masking effects of ozone and its concentration by absorption on moist surfaces may be of some value. PHOTOCHEMICAL EFFECTS OF 2537 AND 1849 A ENERGY The photochemical actions of the 2537 and the 1849 A energy are out- side the scope of this chapter except in so far as they are incidental to some of the practical applications in other fields. The 2537 A energy con- siderably increases the normal oxidizing action of oxygen without its obvious ionization. This is greatly increased wherever there is ioniza- tion and ozone formation by 1849 A energy. In all germicidal applica- tions of the ultraviolet the possibility of objectionable chemical changes should be investigated, e.g., formation of phosgene and hydrogen chloride in poorly ventilated dry cleaning rooms where carbon tetrachloride may be used, formation of hydrogen sulfide and mercaptans in egg-drying plants where egg powder may be in the air, and modification of the flavor of irradiated foods such as meat, milk, cheese, and butter. TEMPERATURE AND VENTILATION Like fluorescent lamps, commercial germicidal lamps are designed to operate under average conditions of room temperature and ventilation. 66 RADIATION BIOLOGY Unusual enclosure or extremes of air temperature, such as in refrigerators, ovens, and air ducts, will reduce the ultraviolet (jutput of the germicidal tube to the same extent as the light output of a similar fluorescent lamp. The reduction is about 10 per cent at 50° and 100°F, 20 per cent at 40° and 1 10°F, and 30 per cent at 35° and 120°F. DEPRECIATION In common with fluorescent lamps, bactericidal tubes depreciate rapidly during the first 100 hr of operation. This is considered a part of the manufacturing process, and commercial lamps are given an initial rating as if at \0d hr of normal operation. In Fig. 2-11 the approximate 120 1000 2000 LIFE, hr 3000 4000 1 — I — I — I — I — I — I — I — I — I — I — I — I — \ — I — I — I — I — I — I — I — I — I — r 5000 6000 7000 8000 120 0 10 20 30 40 50 60 70 80 90 100 PERCENTAGE KILL BY UNIFORM EXPOSURES Fig. 2-11. Depreciation, life, and bactericidal-effectiveness curves of typical low- pressure ultraviolet sources. depreciation of low-pressure sources is shown. Those in high-transmis- sion glasses start from 20 to 25 per cent above the 100-hr rating, whereas those in f used-quartz glass or in similar Vycor glass start from only slightly above the 100-hr rating, considered as 100 per cent in Fig. 2-11. The depreciation rate, and therefore the effective life, is affected by the length of the operating periods; the shorter the average operation per start, the faster the depreciation and the shorter the effective life. In general, such lamps fail to start and operate normally at about the end of their effective life, i.e., at about 60 per cent of their 100-hr rating. Lamps with so-called "cold" electrodes provide some exception to these rules, but they still have the basic depreciation limitations of constant opera- tion. They may continue to start and operate normally after their out- put has dropped to ineffective levels. It should be noted that ultraviolet killing is an exponential rather than linear function of ultraviolet intensity. The curve at the right of Fig. 2-1 1 provides a typical illustration of the relatively small amount of change in ultraviolet killing that may result from a large depreciation. APPLICATIONS AND SOURCES OF ULTRAVIOLET 67 ULTRAVIOLET DISINFECTION Practical uses of the lethal action of ultraviolet energy are limited only by the practicality, in each case, of providing an adequate exposure (nitensity times time) on microorganisms. Exposure time is usually defined by mechanical conditions leaving little chance for radical change, and the exposure intensity must be adjusted for an adequate exposure product. Provision of an adequate ultraviolet intensity is conditioned on two basic and independent factors: (1) the variation with distance from the source or sources and (2) the absorption of the ultraviolet by intervening media. The variation with distance has been represented in general terms as insignificant within a few inches from a relatively large assembly of sources and reflectors, as inverse with the distance for a few inches from single tubular sources and reflectors and a few feet from large- area sources, and as inverse with the square of the distance at greater than source-length distances from single tubular sources and reflectors, as well as at considerably greater distances from large areas. The absorption of germicidal ultraviolet by air is entirely negligible even for the irradia- tion distances of large auditoriums. AIR DISINFECTION Ultraviolet air disinfection is commonly accomplished by placing germi- cidal lamps in the rooms or in the air ducts serving such rooms. The two methods well illustrate the definition of exposure times by the mechanics of the problems, with adequate exposure intensities to be provided if pos- sible. As detailed later, an effective exposure for air disinfection is 15,000 ergs/cm^ or 25 ultraviolet /xw-min/cml In the upper air of occupied rooms, practical exposure times may be 1-5 min, and eff'ective intensities may be 25-50 ultraviolet Mw/cml In air ducts the exposure times may be H-}i sec, and the corresponding intensities may be 10,000- 25,000 ultraviolet /xw/cm^. DUCT AIR DISINFECTION The disinfecting exposure of duct air is defined by its transit time through an ultraviolet-filled zone of definite length and by the average ultraviolet intensity during the transit time throughout that zone. In Fig. 2-2, 15 ultraviolet MW-min/cm^ is suggested as a disinfecting exposure for most air-borne microorganisms except fungi. In an air duct with a cross section of 2 sq ft and a rating of 1200 cu ft/min, the linear flow of 600 ft/min through an ultraviolet-filled zone 3 ft long provides an expo- sure time of 0.005 min. The average ultraviolet intensity throughout the zone must then be 3000 ultraviolet yuw/cm^, or about 1800 ultraviolet mw/sq ft for an exposure of 15 ultraviolet ^w-min/cm-. The average intensity throughout a cylindrical zone radially irradiated by a linear 68 RADIATION BIOLOGY source on its axis of a length greater than the diameter of the zone has been shown to be that on a concentric cylindrical surface of one-half the radius. It follows then that the ultraviolet intensity at any given dis- tance from a germicidal lamp, as indicated in Fig. 2-9, will be the average intensity throughout a cylindrical zone of a radius twice that distance. Thus the GSGTO lamp of Fig. 2-9 provides an intensity of 2800 mw/sq ft at a distance of 2'^^ in. and so an average of that intensity throughout a cylindrical space of about the length of the ultraviolet source and a radius of 53^ 2 ii'^-i or a cross-section of 0.95 sq ft. Although two such tubes would provide an effective initial average intensity in the cross section of the duct, three would be specified to increase the minimum intensity at remote parts of the duct and for an effective intensity at the end of tube life. Luckiesh and HoUaday (1942a) have developed the theory of ultraviolet duct-air disinfection in minute detail, and Buttolph (1945, 1951) has given it practical application. Turbulent Flow for Average Exposure. In small ducts that require only one or two germicidal tubes which are of necessity placed parallel with the direction of air flow, there may be a 10-to-l variation in the ultraviolet intensity at distances 1-10 in. from the tube. To ensure that all the air receives an average intensity exposure in its travel through the irradiated zone, either the streamlined flow of the air must be broken into turbulent flow by baffles or more germicidal tubes must be used. In the latter case, as in all cases where many tubes are used, they may be spaced to provide a sufficiently uniform ultraviolet intensity to take care of the streamlined air flow. Increase of Average Intensity and Uniformity by Reflective Duct Walls. Duct walls of pure aluminum of 65-75 per cent reflectance for 2537 A will nearly double the effectiveness of the germicidal tubes by at once nearly doubling the average intensity and by greatly increasing the uni- formity of distribution by multiple reflection. Disinfected Duct Air as Alternative to Make-up Air in Sanitary Ventila- tion. The most that ultraviolet disinfection can do is to make all the air handled by a duct bacteriologically equivalent to make-up air. When- ever the use of enough ultraviolet to provide a theoretical 99 per cent disinfection of the duct air may be impractical, it should be noted that one-half as much ultraviolet will still prox'ide 90 per cent disinfection, and one-fourth as much will provide about 70 per cent disinfection. In such cases, disinfection provides the equivalent of 90 and 70 per cent make-up air in contrast with the 10-20 per cent usually believed to be economically practical in the winter. Outdoor air is usually considered satisfactory for the sanitary ventila- tion of living and assembly (juarters, and its usefulness is limited only by the considerable cost of heating and circulating it in adeciuate quantities. In food and pharmaceutical plants it may, however, carry enough mold APPLICATIONS AND SOURCES OF ULTRAVIOLET 69 and bacterial contaminants to be a continuous hazard to the products. In these instances, ultraviolet air disinfection can perform a job which is not yet possible by available methods of air washing and filtration. ROOM AIR DISINFECTION Ultraviolet disinfection of air is accomplished in occupied rooms by germicidal tubes in cylindrical parabolic refiectors which are designed to project the energy for maximum distances through the air of the room above the head level of the occupants. It is accomplished in vacated rooms or where protection of the occupants can be provided by bare germicidal tubes centrally placed in the rooms or on the ceilings. Because of the greater distances the variations in ultraviolet intensities are much greater through irradiated rooms than in irradiated air ducts. Fortu- nately the convective circulation and the relatively low-intensity long- time exposures practical in irradiated rooms provide an average intensity exposure such as is obtained in air ducts only by induced turbulent flow or by the use of many separate sources of ultraviolet. Unoccupied Rooms or Rooms with Occupants Protected. In the rela- tively simple case of the vacated room or where occupants may be ade- quately protected, effective ultraviolet intensities may be provided by centrally placed bare ultraviolet sources. The effective intensity is then determined entirely by the time available to disinfect the air. Assuming this time to be 5 min, the intensity for an exposure of ultraviolet ^w- min/cm- needs to be only 5 ultraviolet /xw/cm'. This intensity which can be provided by the G36T6 tube of Fig. 2-9 at a distance of 12 ft will disinfect not only the air but also the walls. Bacteria which might be deposited on the walls from the air are thus subjected to only the mini- mum intensity present in a 20- by 20-ft room, such as a hospital room between occupancies or a room in a pharmaceutical factory. In rooms where only air-borne bacteria are a problem the average rather than the minimum intensity becomes the basis of installation, and the same bare, centrally placed tube will provide an average intensity of 5 mw^sq ft throughout a 40- by 40-ft room to disinfect the air in only 5 min. Occupied Rooms. Occupants in a room add three serious complications to ultraviolet air disinfection that compel entirely different approaches to the theory and the practice. In the occupied room the problem is not the simple one of cleaning up the residual contamination but the dynamic one of killing or removing air-borne microorganisms as rapidly as they appear from the noses, throats, and clothing of the occupants. The kill- ing or removal must be in such a way as to reduce to a minimum their air- borne life under an equilibrium condition of origin, of necessity several feet from the place of their killing or removal. In the occupied room the maximum intensities tolerated on more sensitive faces range from 0.1 ultraviolet mw/sq ft for continuous exposure to 0.5 ultraviolet mw/sq ft 70 RADIATION BIOLOGY for 7 lir of exposure per day. Altliough ideally placed, these intensities are not high enough for rapid disinfection. In occupied rooms the value of air disinfection is primarily that of the health value in removal of microorganisms. In the absence of any criteria of the health value or hazard of air-borne organisms, the natural ventilation believed of value becomes a secondary criterion. Conrcrtirc Circulation and Upper-air Irradiation. The (;onvective cir- culation of air, by which the heating of a room from a few localized sources of heat is possible, involves the use of vertical components which provide an interchange of air between the upper and lower parts of a room equivalent to from several air changes per hour to several per minute. 1 n occupied rooms the basic convective ciicul.itioii is increased by the body heat, the breathing, and the movement of the occupants. These factors increase the circulation in proportion to the crowding, the contamination, and the need for air disinfection. Ultraviolet disinfection of the upper third or fourth of a room can provide in these portions of the room a reservoir of air for the dilution of the lower air at rates equivalent to unusual natural or mechanical ventilation. Lacking more direct criteria of value, it becomes convenient to consider ultraviolet air disinfection as equivalent to and a substitute for outdoor air for sanitary ventilation purposes. Upper-air Method of Disinfection. Luckiesh and Holladay (1942b) treat the upper part of a room as a duct containing air in random circula- tion at a velocity (5-10 ft/min) equivalent to about one one-hundredth the linear velocity in wall ducts and room units and irradiated with an average ultraviolet intensity (0.025 ultraviolet watt/sq ft) which is about one one-hundredth that provided in air ducts. The upper part of the room is then treated as a duct serving the lower part. There is, however, no such definite separation between the two parts of the room as this oversimplification suggests, and the following analysis (Buttolph, 1951) is believed to be more realistic. An ultraviolet (2537 A) intensity of 5 ultraviolet mw/sq ft, effective throughout a cubic foot, will kill respiratory and E. coli test organisms at the same rate as they might otherwise be washed or diluted out of the same cubic foot of air by one air change per minute. This is the theo- retical reduction of 62.3 per cent of Fig. 2-2. An additional air change or an additional 5 ultraviolet mw/scj ft can dispose of 62.3 per cent of the remaining 37.7 for a theoretical reduction of 82.5 per cent. The effect is the same whether by 5 mw for 2 min or 10 mw for 1 min, two air changes in successive miimtes or two air changes in 1 min. These rela- tions are plotted on linear and on semilogarithmic scales for comparison in Fig. 2-12. An average ultraviolet intensity of 5 mw/ s(i ft throughout the entire cubage of a room would theoretically provide the disinfection equivalent APPLICATIONS AND SOURCES OF ULTRAVIOLET > Z) to a: < UJ UJ < -, a: -J Q. < h en I < < I- o 2 O liJ _| O Ct UJ 100 90 80 70 60 50 36.8 30 20 10 100 36.8 10 0.1 0.01 - AIR CHANGES PER MINUTE I 23456789 UJ Q: O 70 tiJ I §t P a: z < UJ o (J o cc _; in the room of one air change per minute, such as is thought to be desir- able in very crowded rooms. Only one-tenth that intensity (0.5 ultra- violet mw/sq ft) is, however, tolerated on sensitive faces for 7 hr per day without objectionable "sunburning." So only about one-tenth of an air change eciuivalent per minute is practical by this method. This is, however, equivalent to the six air changes per hour, or 30 cu ft of out- door air per child per minute sometimes specified for school rooms. An average intensity of 5 ultraviolet mw/sq ft throughout a whole room, with only 0.5 permitted in the lower part of the room, can be obtained b}^ an average of 14 mw/sq ft in an upper third of the room or 18.5 in an upper fourth. Since the ultra- \'iolet in the lower part of the room is due mostly to the diffuse reflec- tion from the upper side walls and ceiling, its intensity may vary little throughout the entire area, especi- all}^ under 10- to 12-ft ceilings. However, since the ultraviolet in the upper part of the room is pro- jected through it from a few sources (usually in cylindrical parabolic re- flectors on the walls), the intensity may vary from 2 or 3 ultraviolet mw/sq ft to 2 or 3 ultraviolet watts/sq ft (a thousandfold varia- tion). Such an uneven distribu- tion of the energy in the room is effective only because the convective circulation exposes nearly all the air to nearly the entire range of intensities to provide an integrated lethal exposure, as with turbulent flow in an air duct. Hospital Room Disinfection. The first uses of ultraviolet for air dis- infection in hospitals were to provide the equivalent of local curtains or barriers between the surgeon and his operation (Hart, 1936; Overholt and and Betts, 1940) and across the front of infant cubicles (Sauer et al., 1942; Del Mundo and McKhann, 1941; Robertson et al., 1939, 1943). Ultraviolet intensities ranging from 20 mw at the floor to 200 mw at head level can readily be provided. The 20-mw intensity becomes as effective as the 200-mw intensity because the width of the di\'ergent beam and the distance of the bacterial travel through the beam, and thus the average exposure time, are ten times as great at the floor. These inten- sities may produce erythema at head level in 1 min and at floor level in 10 min, so that such installations are limited to pharmaceutical plants f r I — \ — 1 — 10 - 20 - 30 -\ - 40 \ - 50 1 \ ; l§.2 \ - 70 \ - 80 N. ^99% 90 N. - 63.2^ 90 LlI .1 ^V cc ^ \ ' 3- 2 - _ a: 8 ' - _ _i •^ o 1 1 1 1 1 1 . 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 DISTANCE FROM FIXTURE, ft (6) Fig. 2-13. (a) Spatial distribution of ultraviolet from typical bactericidal tubes and open reflectors, (b) Isointensity lines in milliwatts per square foot in a plane perpen- dicular to the center of the G30T8 tulie of Fig. 2-9. air ducts; the door opening becomes a duct from one room to another in spite of its unconventional cross section compared with its length. The variation in ceiling height and the difference in exposures per day in patient and service rooms of hospitals have led to two distinct types of commercial equipment, an open type for use under high ceilings and where there may be personnel exposure of about 8 hr per day (Fig. 2- 13a) and a louvered tj^pe for use under low ceilings and where there may be continuous exposure (Fig. 2-14a). Figures 2-13a and 14?> suggest a possible way to provide energy intensities of 15-20 m\v/sq ft in the upper third or fourth of a room. APPLICATIONS AND SOURCES OF ULTRAVIOLET IIO" 100° 90° 80° 70° 60° 50° 40" 30° 20° 73 ^ D ^ ^ ^ ^^ O 2.5 ^ ^'0 ? 5 - ^ _ 20 — _^ y -J j/^y^^,y^ ^^...^ * \ 10 S^4 - Z^:^^ 40 ■■^^ ^^^ -^ ^^ g5 - ^^^~ -**v ^ -^^^2^- ^2 - / ----•■'t!!^^^--^^'^!!^^^^--^^ __-- — — "^ ? 1 ^ ^S^eo-N ^^^.^ ^^-^i^:^^;;::^ I^I^^^--^ ' * X ' ~ ^^^ """^ Jxi.,,-'''!^'^^^^^^ r^^^"-""^ ^"^ . C ^^ ^^^-^j^^r^^g^^^* ^1^ jS^^" ^^■^ H^BHB|^^^^^^^^^^^"'^^ *- ^ "- 6 - HJ , It 5 - 3 K . 5 ^ - V d 3 _ O t- 2 _ o: * O o t _ _l ' u. 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 DISTANCE FROM FIXTURE, ft Fk;. 2-14. (a) Spatial distribution of ultraviolet from typical bactericidal tubes and louvered reflectors. (6) Isointensity lines in milliwatts per square foot in a plane perpendicular to the center of the G30T8 tube of Fig. 2-9. Barrier-type units have usually been custom made, but a typical combination unit is described by Fig. 2- 15a and h. HEALTH VALUE OF AIR DISINFECTION Ultraviolet air disinfection is but one of the factors in a complete air sanitation. It is comparable with the removal of dust and noxious vapors. Although air disinfection has been used in industrial applications as such a general sanitary measure, it early came to be thought of as having more specific possible value in preventive medical and public- health applications. Buttolph (1951) has proposed a tentative standard of air sanitation relating ventilation and disinfection to room occupancy. It calls for one air change per miimte where there may be as little as 300 cu ft of room volume per occupant. From that it assumes the need for dilution with fresh air or eciuivalent ultraviolet air disinfection to be proportional to the crowding, to vary dii-ectly with the iuiml)er of occu- pants in a room and inversely with its volume. 74 RADIATION BIOLOGY Hospitals. When new low-pressure sources of 2537 A ultraviolet, with a germicidal ellicieucy fivefold greater than that of previously available sources revived the interest in practical applications, they were first found by Hart (193G) and Overholt and Betts (1940) to improve the air (10" 100° 90° 80° 70° 60 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 DISTANCE FROM FIXTURE, ft (6) Fig 2-15 (a) Spatial distribution of ultraviolet from typical bactericidal tubes and louvered reflectors with a downward barrier reflector, (h) Isointen.s.ty lines ni milli- watts per square foot in a plane perpendicular to the center of the G30T8 tube ot Fig. 2-9. sanitation in operating rooms. This success led to trials by Sauer et al. (1942), Del Mundo and McKhann (1941), and Robertson et al. (1939; 1943) in infant nurseries where an even more definite improvement was found by air sampling as w(^ll as by records of the spread of contagion among infants. On the basis of these tests the use of germicidal lamps APPLICATIONS AND SOURCES OF ULTRAVIOLET 75 is approved for general hospital use by the American Medical Association (1948). Lurie (194G) and Vandivicre ct at. (1949j ha\e shown that tubercle bacilli, either in sputum or air-borne, are readily killed by practical exposures to ultraviolet. Wells and Ratcliffe (1945; Wells et al., 1948) have shown that, in experimental animals, tuberculosis is spread mostly l)y air-borne organisms so small that they remain suspended in air for long periods. These studies suggest a unique value for ultraviolet air sani- tation in tuberculosis hospitals and perhaps even in some homes. Schools. The promising results in hospitals led at once to trials in public schools. Wells et al. (1942) and Wells (1945) in Swarthmore and Wells and Holla (1950) in Pleasantville studied the spread of measles and chicken pox as typical of respiratory diseases in general to find that ultraviolet air disinfection suppressed the epidemic occurrence of these diseases in the sense that their incidence was spread out over longer- than-usual time intervals. This modification of the pattern of epidemic spread was thought worth while in spite of some uncertainty as to a significant long-run reduction in the total cases. Only measles and chicken pox were studied as respiratory diseases, typical in their air-borne manner of spread but atypical in the individual immunity they impart, with the preconception that influenza and the common cold could not be studied directly because of their indefinite diagnosis, their spread in every environment outside the schools, and the almost universal susceptibility to them. As anticipated, air disinfection provided no measurable effect on the incidence of colds and influenza among the school children, and Downs (1950) reported no effect in a surrounding community. The studies by Wells and Holla (1950) showed that measles and chicken pox are too completel}^ typical of respiratory diseases to simplify greatly the study of their epidemiology in schools since two-thirds of their spread occurred outside the school coverage of the ultraviolet installation. In large consolidated schools served by busses from small towns and the surrounding country, Perkins et al. (1947) hoped to study also measles and chicken pox with a minimum spread outside the school environment. There was early indication of some effect on the epidemic spread of the diseases in the schools, but there was also subsequent evidence of their spread in the busses, perhaps enough to blanket the marginal effect of the air disinfection in the school buildings. Ultraviolet air disinfection is justified in school rooms as a supplement to air sanitation by ventilation, especially in northern latitudes. It has a place as a general sanitary measure along with dust suppression, wash- room sanitation, and habits of personal cleanliness which may be taught and practiced in schools regardless of their effectiveness outside the school. 76 RADIATION BIOLOGY Institutions. Many institutions, through their isolation from sur- rounding communities, have provided an opportunity to study the possil)le vakie of a unixcrsal practice of air sanitation in large com- munities. Schneiter et al. (1944) early reported a study in a training school for delin(iuent boys started with such e(|uipment as was then available. DuHuy ct al. (1948) have since reported no effect on the incidence of disease among the boys, and that air sampling showed little air disinfection. If there was no disinfection, no effect should have l)een expected, but the more probable explanation is that the air-sampling method did not properly detect the presence or absence of respiratory- disease organisms, that the obsolete equipment did not pro\'ide an effective use of the ultraviolet energy even though it was supplied in excess, and that any possible effect on the spread of respiratory diseases in the sleeping rooms was nullified by the lack of separation of the boys in the irradiated dormitory from those in the control dormitory during their class, intimate play, and eating periods. Navy Barracks. In contrast with these results, studies in Navy barracks consistently indicated disinfection of the air and a significant reduction in the spread of general respirator}^ diseases. Wheeler et al. (1945) reported a 25 per cent reduction of respiratory illness and a 50 per cent reduction of the relatively highly resistant saprophytic organisms dominating the air contamination. Miller et al. (1948) reported, in a similar but theoretically more effective installation, a 19.2 per cent over- all reduction in total respiratory disease and a 24 per cent reduction in the unusually high streptococcus-disease rates. Willmon et al. (1948), reviewing four years of Navy barracks study, are less certain of the amount of reduction in disease, and Jarrett et al. (1948) reported about 50 per cent reductions in bacteria count but were dissatisfied with the open-plate method which overemphasizes heavier dust-borne contaminants. Conservatism as to Value. The universal appeal of air disinfection as a general sanitary measure and the limited evidence of its specific health value have led committees of the American Public Health Association (1947) and of the National Research Council (1947) to issue warning statements about air disinfection in general and about the ultraviolet method in particular. Both committees point out that, at best, air dis- infection can reduce only that limited part of the spread of respiratory disease which may be air-borne, and they emphasize the difficulties in obtaining effective ultraviolet air disinfection without face and skin irritation of room occupants. There is need for further study of the extent to which air disinfection might supplement the use of face masks, the smothering of the cough and sneeze, and the physical isolation of patients suffering from respiratory disease. APPLICATIONS AND SOURCES OF ULTRAVIOLET 77 Air disinfection would seem to have most of the possibilities and limitations in health value of unusual amounts of ventilation with outdoor air, amounts ordinarily impractical in the cost of moving and heating. Ultraviolet energy in germicidal barriers across openings would seem to provide a bacterial isolation of rooms and people where doors or glass partitions may be impractical, as in some hospital infant wards. Ultra- violet barriers in air ducts can supplement and in some cases serve the purpose of excessive filtration of duct air, as in pharmaceutical factories. FLUID DISINFECTION When intervening media are gases of negligible absorption and licjuids of so great an absorption that the effective penetration distance is negligible, the intensity at the irradiated surface is obviously dominated entirely by the distance through the gas. When, however, there is irradiation through a gas such as air and into the mass of a licjuid of low or intermediate absorption, the intensity at any given point in the licjuid is determined primarily by the total distance from the source to the point, and secondarily by the absorption from the surface of the liciuid to the same point. This absorption can vary over a 10,000-fold range, from water of low iron content which can be disinfected in a duct in much the same way as air, when due allowances are made for its greater absorp- tion and the increased ultraviolet tolerance of wet bacteria, to milk and serum which must be processed in films of thicknesses less than a few thousandths of an inch. WATER DISINFECTION Water was the first liciuid to be disinfected by ultraviolet and with commercial equipment using high-pressure mercury arcs. The method could not compare with chlorine disinfection, economically, and it did not provide any evidence of effective use as does "residual chlorine." Although low-pressure sources of germicidal ultraviolet have greatly reduced the cost, ultraviolet is now used only in instances where chemical methods or boiling cannot be tolerated and where there may be routine bacteriological control, such as in beverage, food, and pharmaceutical processes. Water-borne E. coli recjuire an 8-10 times greater ultraviolet exposure for a given kill than when air-borne, 150-200 ultraviolet /iw-min/cm- for a theoretical 99 per cent kill in distilled water (Luckiesh and Holladay, 1944). The ultraviolet absorption of all water is much greater than that of air and of itself varies more than tenfold from a 90 per cent absorption in 5 in. to the same absorption in 50 in. (Fig. 2-16, from data by Luckiesh ('/ a/., 1947, 1949). This variation is apparentl}^ almost entirely due to dissolved iron salts. Since water readily acciuires sufficient iron for such \ariations in absorption from contact with iron pipes and storage tanks, 78 RADIATION HIOLOGY y / / y / 1 / 1 / f ll ^ 1 I 1 a. ^ ■ ■■i/> •;■': P ^ ,' •' ^ y' in 1 r. ■;*. '-'». fo 1 ii ';■•■ s^ t >p^ >^ m .1 •is i* ^ m^ ^ ti-' i^ MILK / / / .¥ ^ f ^sO^>^ # . r ., vt - ^^ / / /" ^ y -^ --^ -H DOPL vSMfr, SERUM. ^ ^ y ^ -- =: B LU [ ^ lood_ O o o O 01 CO o o o o o o — O en CD h- o o o U> If) ^ - '1 / / / ^1- i ' h / / -J -J Q / c3 1 < 00 Q 1 1 f ^ ^/1 '5/ \ ' I f ^ i 1 J> ^ teifi It.^ 1 ^ ^ ^ ■' e ^^ / > / h^ ' .o^i^ / / n<^ Q^ r / / / ^ / ^ y ^ y ^ ^ -^ 35 M Vlt ^leGAR ^ g -- ^ - - - N ^ O -^ c o _ '^ o -i<: o r;5 a> <> (-) 'Jl o rs o o o ei < n 1- o t^ lli -N-s u. iC 01 u. 1^1 o UJ o CO ^ o .2 c o in (^ o o ro « O '-3 (\J o O ^ - W o o o o o o o o O o o o o o o o o o O O Ol CO 1^ lO in orne organisms would be re(iuired: 200 300 ultraviolet ^iw-min/ cm-. The minimum exposure for the thicker films pro\'ided by gravity devices might i)e increased se\-eralfold to 700-1000 ultraviolet /iw-min cm- such as is retiuired in the disinfection of absorptive water through several inches of depth. Since it is difficult to provide ultraviolet inten- sities greater than 10,000 ultraviolet ^w/cm- (10 ultraviolet watts/scj ft), the exposure times in such devices should not be less than 1-2 sec for the thiimest films to 4-6 sec for the thickest. Although the speed of 500-1000 rpm of the centrifugal devices provides linear film speeds of 200-400 ft/min, this is in a helical, nearly circular path. Only the relatively slow, forward component contributes to the exposure time. The centrifugal force provides this forward component directly only in rotors essentially conical in shape (Fig. 2-1 8C). In a cylindrical rotor the centrifugal force spreads the film in both directions perpendicular to the helical path, l)ut there is effective movement of the film as a whole only in so far as the gravity-pressure equilibrium is dis- turbed by the delivery of liquid from the film and only in so far as the rotor is so inclined as to permit gravity flow. Note also in this connec- tion that the rate of liquid flow through these devices defines the exposure time only through the amount actually and momentarily being processed in the film, an amount usually very difficult to measure accurately. Operating Controls. Where there may be variations in ultraviolet absorption or penetration into an irradiated film such as are shown in Fig. 2-16, there must be provision in commercial devices by which the film thickness and the ultraviolet exposure are completely controlled bj' the licjuid absorption. The ideal control would provide an automatic adjust- ment of the exposure to the absorption with the film thickness held con- stant mechanically. The exposure should, in turn, be based directly on the ultraviolet intensity at the film surface, rather than on the electrical characteristics of the sources, to compensate for their output depreciation. Fast-acting relays and valves should stop the delivery of material instantly upon the shortest power failure and should provide for rejection or reprocessing of material remaining in the device. DISINFECTION OF SURFACES OF GRANULAR MATERIALS There is practically no penetration or reflection of ultraviolet energy in the irradiation of granulated or powdered materials. Only the upper fourth or fifth of the surface of individual particles can be irradiated at any given instant of time. However actively stirred or agitated, the sur- face of such particles is therefore efTectively irradiated only one-fourth to one-fifth of the time. The particles shade each other as soon as there is a layer more than one particle thick, so that there is a "coefficient of APPLICATIONS AND SOURCES OF ULTRAVIOLET 83 .shading" similar to the coefficient of absorption of lifiuid.s which defines the "effective depth of agitation" for granular materials, analogous to the effective depth of penetration of water. Experience indicates that the effective depth of agitation is of the order of ten times the diameter of granulated products. The theory of treating granulated and powdered solids is practically the same as for highly absorptive liquids, with effective depths of agita- tion of the order of the effective depth of penetration into sugar syrups. Although the bacteria on the surface of dry sugar crystals, for example, may be about ten times as susceptible to ultraviolet killing as are those in water, this is offset by the presence of the individual sugar crystals on which the bacteria ride at the surface of the layer for only about a tenth of the time. When the crystals are in the irradiated layer, bac- teria on them are irradiated only about a fifth of the time on the exposed sides of the moving crystals. The result is that granular particles can be disinfected with only about one-fifth the efficiency of water disinfec- tion and that the method is not practical on powdery materials. The disinfection of the surfaces of granular materials is well illustrated by the commercial method used on canner's sugar. SUGAR Thermoduric bacteria survive the vacuum evaporator temperatures of sugar-syrup concentration and, rejected by the sugar crystals during formation, remain in the final film of dilute syrup left on the crystal sur- faces. Ordinarily harmless, they may cause serious spoilage in canned foods and beverages. Such sugar, preferably in coarse crystals in a layer of about 3>-^ in. at rest, is continually vibrated, stirred, or cascaded on a conveyor under closely spaced germicidal lamps (Fig. 2-10) providing of the order of 23,000 ultraviolet mw/scj ft of conveyor surface. The length and speed of the conveyor may be such as to provide a total exposure time of 15-5 sec for an exposure of the order of 500 mw-min/sq ft. GRAINS AND SMOOTH-SKIN FRUIT An ultraviolet method has been reported by Ewest and Leicher (1939) to be effective in reducing the superficial mold contamination of hard grains such as that which develops after storage in the tropics. A similar but simpler method is reported by Matelsky (1950) as effective on smooth-skinned fruit such as cherries. ULTRAVIOLET-INDUCED MUTANTS FOR NEW FUNGI The use of ultraviolet to produce mutants of fungi in a search for new or better commercial characteristics deserves mention because of its 84 KADIATION MIOLOGY iiovoUy rather than its commercial importance. Emmons and FIol- laender (1939) showed that the curve which represents the efficiency of different wa\(> lenf!;ths of ultraviolet producing mutations in fungi par- allels closely the germicidal-action curve. Hollaendcr and l^mmons (1941, 194()) correlated mutants of fungi produced by ultraviolet irradia- tion with the naturally occurring species probably produced by sunlight. From this, techni(iues have been developed for obtaining mutants, for example, for better yields of citric and itaconic acids and of penicillin and the similar antibiotics (see chapters on bacteria and fungi). PROTECTION AND PROCESSING OF PRODUCTS Because of its high germicidal effectiveness compared with its other photochemical, erythemal, and thermal effects, ultraviolet energy, espe- cially of wave length 2537 A, has been used for the protection and disin- fection of many products of so unstable a composition as to prohibit the use of more conventional methods. Such applications have developed in food, pharmaceutical, and beverage processing and storage places. MOLD, ANTIBIOTICS, AND PARENTERAL FLUIDS The mold-derived antibiotics and many parenteral li(}uids are very susceptible to contamination by the normal mold and bacterial content of air. This contamination is often of a chemical nature precluding terminal sterilization by heat. Some serums and antitoxins are developed by the growth of bacteria and viruses which must be finally inactivated by methods which will not at the same time destroy the desirable properties of the preparation. Ultraviolet has served to protect such materials during processing and to provide a final inactivation where controlled bacterial growth has been a part of the process. The germicidal lamps are used in ducts and hoods, over work tables, for upper-air disinfection, and also for thin-film irradiation (Fig. 2-18) l)y the methods previously' discussed. BLOOD PLASMA One of the more unusual applications of ultraviolet is for the dis- infection of blood plasma of the hepatitis virus. Several commercial devices have been developed. All provide for irradiation in films of the order of }/ioi)o in- in thickness or for the violent agitation of somewhat thicker films. It is important to remember that, because of the low penetration of the ultraviolet, changes in film thickness, even of micro- scopic dimensions, may seriously interfere with any of these methods. These methods and devices have already been discussed generally for absorptive liquids (Figs. 2-16 and 18). Preliminary to the use of any APPLICATIONS AND SOURCES OF ULTRAVIOLET 85 of those methods, there must be a complete removal by centrifugiiig or filtration of all clumps down to an empirically determined and specified size. A machine or process should produce a specified degree of sterility in test runs of plasma contaminated with a test organism of an ultra- violet-exposure tolerance comparable with that of the hepatitis virus. Sarcina Infca, whose packet growth habit may simulate the minute clumps remaining in plasma after clarification, is suggested. In operation there should be a continuous record of plasma flow and ultraviolet intensity, similar to the controls used in continuous methods of milk pasteurization. A cadmium photocell (Figs. 2-1 and 5b), described by Taylor and Haynes (1947), and a recording microammeter are suitable for the ultraviolet control. The rate of flow may be controlled by a pump with variable-speed drive. Rapidly operating relays and electric valves should stop or divert the delivery of plasma from an intermediate storage reservoir of a capacity much greater than that of the irradiating device. SYRUP, FRUIT-JUICE, AND WINE STORAGE The sugar content of sugar and fruit syrups is usually such as to prevent fermentation even though a mold scum ma}^ form on exposed sur- faces. Whenever there is condensation of moisture on the sides and tops of syrup-storage tanks, it may dilute the surface layer of the stored syrup enough to permit destructive and otherwise very objectionable fermen- tation in addition to the usual mold formation. Germicidal tubes are being used to prevent such fermentation and mold on the surfaces of tank-stored sugar and fruit-juice syrups used for soft drinks and con- fectionery. Continuous irradiation with an intensity of at least 5 ultra- violet mw/sq ft is required. MEAT STORAGE The most extensive single industrial use of germicidal lamps is to reduce the growth of bacteria and molds on the surface of meat and on shelves, walls, and floors of retail-meat-storage refrigerators operated at 35°-45°F. There is little need for such provisions in cold-storage rooms operated below zero, nor has there been any such need in the larger meat-processing factories where exact control of temperature, humidity, and air movement produces similar results. Proper use of the ultraviolet does not take the place of established periodic sanitary maintenance but does supplement it by a continuous suppression of spoilage and odors. The ultraviolet intensities required are of the order of only the 5 mwsci ft effective for mold suppression in other applications. Ultraviolet is effectively used to suppress surface slime molds on meat stored for 3 days at a temperature of about 60°F for rapid aging or tenderization. Contrary to the impression of some, the ultraviolet has no direct effect on the enzymatic tenderizing. 8() 0 2 H X •y; o o o a. <: > I (« O « D to tc Q O to o EC b: < 6 (M •a . " i" 1) «. as a: 31 — — — c^o f — .lO'O — — ooo o ot m n n ■v -< ec — o«ito , 4) -.2 2 5 ooo o •nc^ioioift O O O lO o c -r i.T o CM cc tc oc ■* o — o -.c CI 3; 3 03 . OOO O 0>0000 rf tfi lO to lOMtOWtO o o o >o O c lO 1(5 'O '»• "O O •M t~ ',C Tl 00 — 0) C 0*0 0 CS CC lO lO IN CM O CC I^ t» t~ CM U5 O O: a> 31 t^ IC -" — < — • — 00'» — ■ r)< ■* IC O -H C i" \flO NJO \C1 \j- vX \^\N ^X N** N ^JX iCkO»0 »C OOCDO^'C CC CMCMfCCC — <—«•—'•—' C^ »C »-0 »C t>- > o o' O-*— < 00 rtOOOlOOOOC tc O C t~ CCOO'^L-cOOCtCOCl^^ CC'HCMiOrl'SlSlCM^TflCtC -< C>l CM -^ M CO « 03 t3 3 a. i>§o CO ■* 31 CO 00 CO to CM 31 CO CM-<-H^OOOO CO CM M CO CO tc CMCOO — tct~3i^tceo3iicotc— ■ — -^COiOt^'lSO CMt~ •^ CO 50 ^co CO CM O CO tc 00 tc 31 31 CM ■* CO C>J iC rj ■* t^ CM CM t~ — O — -HCOt^31COOOCCCO rf ^ c^ 0) o r2 *" 5 6 <: o tc 0) o ■= "^^ "S Si o « 2 3 "n =^ 05 § « 31 CM lO o o o O CO •* o o o o f 00 1^ CM « ~-co O CM o tc t^ o in O tc lO OCOCMOOOOOOO O 31 C OOeOCMt^tCiOOOO -H — t» Ol e<5 CM o o •- «5 O ,CS C^ 00 -^ "^ CM o c ,3 M S ^ ^^CM o K5 -H CO -« O >C CM to O CM CO t^ CO to U5 OOO O "O o — CM O a; — ' X ■ra o o ^ ooo o o o ^ _ o c ^.^ 0 a «^ 00 o o o lO 1^ to o o o ; ; O *-.* ^ a. a M ri -r ^ ■M I^J ^ t^ « c if " ~ " ■- ~ CO . ■if c '-^ '-N •-% *-\ W U .f 0 »»*iVV v^^jwVTs- -q *<*-nV S — is" "O T3 "B 1. -O T: "O 1-1 73 T3 -— If? •«' C t: ^ ^ f^,t'T-^-^iO'>)cc^^c:';c-^^'^a;^- Q a 7'77'77'7<<'7'7'7'7-<<-^'7< 0- a S £-< QD T3 °r- 00 ti -o a X - ^ tiO ^ tc C-i a 0 oi ^^ c J^ c a 0) - X 2 >- a -^ '5 0 **-• cr X a t5 1- m ■S.2 t- •-• a a position, exce H4, which ha\ e bactericidal 0 03 C .= a t- 0 i 0 II t^ '" CI a S «> CT'"" — a 0 a S 2 "*■ 03 > c 0) « c'a *^ '^ o 03 >. '^ T3 3 _ i; .1 ■^5 » a 0) 0) I. 'J a) cd ^ 50 ;5 00 3 — 3 ic -o ^ 00 c E o c .= 3 .ti „ is S 1 ■- >> iL 00 ♦J 5 O ^' 03 _aj n n J3 33 -a u = o « be 1 and "3 3 y '> a Si! »o xO M "— O w — o — •- S'ii^ t- c a X 03 ^ c; dS CI lo O =^jHll as physical mortality. The hurn-out life is usually that at which one-half the lamps of a test fiiroup reach a physical or economic end of life. The latter is very dependent on variable factors of lamp and electric costs and the nature of the ai^plication. For oj)erating intervals of less than 5 hr the life decreases rapidly with the length of the operating interval. For longer operating intervals the total life may increase to several times the 5-hr interval rating. The general form of the depreciation curve is that of Fig. 2-11. An important variable in the depreciation of all higher pressure mercury arcs is the "solarization" effect of the ultraviolet and heat on the trans- mission of the fused (juartz generally used. The effect increases as the wave length decreases and may reduce the output of 2200-2500 A energy to one-third the initial and of 2500-2800 A to one-half the initial in a few hours of operation. This accounts for some of the discrepancies in published data on the shorter ultraviolet output of commercial sources. The effect is relatively small at wave lengths longer than 2800 A. RESEARCH DETERMINATION OF OUTPUT AND INTENSITY As was emphasized for low-pressure sources, although their commercial radiation ratings should be fully specified in all research reports, these ratings can be considered only a first approximation to the energy output and intensity actually effective in any specific research. Whenever direct measurements of energy at the point of application are impractical, calibrated lamps should be used. The intensities that they provide can be calculated for various distances by the methods outlined for low- pressure sources as long as there are no intervening optical systems or filters. In work at wave lengths less than 2800 A, and especially with the higher pressure sources, the source itself should be frequently calibrated for out- put where direct measurements of the irradiated surfaces of material are in practice. MERCURY-AMALGAM AND OTHER MET.\L ARCS Laboratory workers generally fabricate their own electric-discharge metal- vapor arcs, other than mercury, although such lamps have been occasionally imported for sale in the United States. Various commercial types availal)l(' in the i)ast are described by Meyer and Seitz (1949). Their availability is too uncertain and thcii- radiation characteristics are too unstandardized for inclusion in this chapter. It should be noted here, however, that cadmium-amalgam lamps proxiding low-intensity cadmium lines of wa\-e lengths greater than .SOOO A arc commercially APPLICATIONS AND SOURCES OF ULTRAVIOLET ■d o o o o o c ■c o o o o c b- o o o c o 01 C E o ^ C '^ " ^ « O a - = « t^ IN o o c C O 03 ■* o o '-< ec po ^ ■— tit lU 3 S cc »o »o PO ■* Tf 00 c — o 00 ;o lO ic C 10 rt ^ 3 O « — . -H o ■* ■N ■* EC m H ■s S f 5 03 a "r, '^ S e > g lO 00 CO ;0 CC LO "* 00 !© ^ O -^ "O IN CO 00 — c^l > ^ O O ^ S § S 00 »o CO Oi o: ■'t uo M* CO W C^ ^ CO •« t^ C3 lO '^ " 05 .- ^ IN " IN - t; CO 00 o ilO lO o o O "O CO CO ■* o O O O O O — I o ^ is So tH — CO 0) c =s I > o •2 o 03 c o 03 cS 00 t>. CD CO 00 Tf O O O O O (N o o o o o o ;2& CO (N »^ lO lO O O CO IN -H O IN lO > 6-^ Si 00 IN o o o o c d ■w Si - « oj « s *j — o O O O lO o o O O O t>. IN •* ■* — -< IN - 'f CO CC M M GO 02 pH » (iH [X, C3 -t c a -r X X C a a 03 O o 00 w 3 o ■•^ ■B a o 89 C 0 n1 **" > oj a J= n a « u r^ +^ OJ (S 2 3 fi o 7 > S a; - CD D to -M D .- oJ -C C M a o -a -M ID oj >. 0 a ci a c cs a) .. w x: Oi rl ■^^ _c; « 03 X 0 r c3 o S m =3 a 03 B . •p « Ci 2^ § o o o »o C 0 3 --? -»^ -*^ 03 M ca 3 a c -"^ d .5 c ^f! G O F 3 _B "ca o ^ 1- "? 0! C L. 01 S cs jz 01 cS V a. T II 3 - 0 o3 _o o 3 CO a c o ■4^ >> fee r 03 o « m 2^ CO o -♦J '-5 t-r 3 ?^ « •w X 3 7 ^ J3 0 > 1^ lO fc o H H &I c ■o ^ A- -0 in N « ■"* O o 90 KADIATION HIOLOCJY ;i\:iihil)l(' as arc also similar ijotassiuin and sodium lamps. Xoiic ol llicm arc, howcx'cr, sources of ult i'a\'iolct. ol the \va\'c lcnti;llis jicncially desired in biological work. SUN I,. \ MI'S Commercial sunlamps have been used for some biological research. They provide energy down to 2800 A from sources of sufficiently high intrinsic intensity to adapt them to optical methods of isolating narrow spectral regions. They have the general characteristics of high-pressure quartz-mercury arcs with the radiation limited to about 2800 A by the enclosing bulbs or tubes. Laboratory housings for the S-4 lamp have becMi comnKM-cially available. Lamp ('II-3 of Table 2-6 is proving more 2000 2400 2800 3200 3600 4000 4400 4800 5200 5600 6000 6400 WAVE LENGTH, A Fig. 2-19. Relative energy distril)uti()ii of fluorescent and S-4 type .sunlamps. useful in the same devices because it provides essentially the full (|uartz- arc spectrum. Table 2-7 permits comparison with sources of Tables 2-3 and 6. Figure 2-19, which shows the relative energy distribution of sun- lamps, is comparable with Fig. 2-5. The S-1 lamp spectrum is similar to that of the S-4 but with addition of a continuous visible and infrared spectrum from a tungsten filament in multiple with the mercury arc and operating at a temperature of about 2000° K. The RS spectrum has similar visible and infrared components from a tungsten filament in series with the arc. As indicated in Fig. 2-19, tluorescent sunlamps provide a continuous ultraviolet spectrum with a peak at about 3100 A. Mercury-arc lines transmitted by the phosphor make an insignificant addition to it. The generated energy in these lines is comparable with that in the lines of the germicidal lamp and is transmitted to an extent of 60-70 per cent by the phosphor. Although the low emission intensity of these lamps makes them unsuitable for use with optical isolating systems, they are compara- ble with the high-pre.ssure mercury arcs for low-intensity general irradia- tion with energy of wave lengths in the 3000-3200 A range. They are APPLICATIONS AND SOURCES OF ULTRAVIOLET 91 unique sources of energy in the 3200-3400 A region of the ultraviolet spectrum. REFERENCES Ainerican ^lodical Association, Council on Physical Medicine (1948) Acceptance of ultraviolet lamps for disinfecting purposes. J. Am. Med. Assoc, 137: 1600-1603. American Public Health Association, Subcommittee for Evaluation of Methods to Control Air-borne Infections (1947) The present status of the control of air- borne infections. Am. J. Pub. Health, 37: 13-22. Buchbinder, L., M. Solowey, and E. B. Phelps (1941) Studies on microorganisms in the simulated room environments. III. The survival rates of streptococci in the presence of natural, daylight and sunlight, and artificial illumination. J. BacterioL, 42: 353-366. Huttolph, L. J. (1945) Principles of ultraviolet disinfection of enclosed spaces. Heating, Piping Air Conditioning, 17: 282 290. (1951) Ultraviolet air disinfection in room air conditioners. Refrig. Eng., 59: 54-57, 73. Caspersson, T. (1931) Uber den chemischen Aufbau der Strukturen des Zellkernes. Skand. Arch. Physiol., Suppl. 8 {also Referat, Protoplasma, 27: 463-467, 1937). Del Mundo, F., and C. F. McKhann (1941) Effect of ultraviolet irradiation of air on incidence of infections in an infants' hospital. Am. J. Diseases Children, 61: 213-225. Downs, J. (1950) Control of acute respiratory illness by ultraviolet lights. .\m. J. Pub. Health, 40: 1512-1520. DuBuy, H. G., J. E. Dunn, F. S. Brackett, W. C. Dreesen, P. A. Neal, and I. Posner (1948) .\n evahiation of ultraviolet radiation of sleeping quarters as supplement of accepted methods of disea.se control. Am. J. Hyg., 48: 207-226. Ellis, C, A. A. Wells, and F. F. Heyroth (1941) The chemical action of ultraviolet rays. 2d ed., Reinhold Publishing Corporation, New York. Emmons, C. W., and A. HoUaender (1939) The action of ultraviolet radiation on dermatophytes. II. Mutations induced in cultures of dermatophytes b^- expo- sure of spores to monochromatic ultraviolet radiation. Am. J. Botany, 26: 467-475. Ewell, A. W. (19-t2) Production, concentration, and decomposition of ozone by ultraviolet lamps. J. Appl. Phys., 13: 759-767. Ewest, H., and A. Leicher (1939) Getreideentmuffung durch ultraviolette Strahlen. Miihle, 76: 569-570. Gates, F. L. (1929-30) .\ study of the bactericidal action of ultraviolet light. J. Gen. Physiol., 13: 231-260. Hart, D. (1936) Sterilization of air in the operating room by special bactericidal radiant energy — results in thoracoplasties. J. Thoracic Surg., 9: 520. Holiaender, .\., and W. D. Claus (1935-36) The bactericidal effect of ultraviolet radiation on Escherichia coli in liquid suspensions. J. Gen. Physiol., 19: 753-765. Holiaender, A., and C. W. Emmons (1941) Wavelength dependence of mutation production in the ultraviolet with special emphasis on fungi. Cold Spring Harbor Symposia Quant. Biol., 9: 179-186. (1946) Induced mutations and speciation in fungi. Cold Spring Harbor Symposia Quant. Biol. 11: 78-84. Holiaender, A., M. F. Jones, and L. Jacobs (1940) The effects of monochromatic ultra\'iolct radiation on eggs of the nenuitode, Enlirohiux venniciilnris. I. Quan- titative response. J. Parasitol., 26: 421-432. Holiaender, A., and J. W. Oliphant (1946) Experimental inactivation of etiologic 92 RADIATION BIOLOGY agt'iit ill scruiii hy ultraviolet irradiation. U.S. Pub. Health Service, I'ul). Health Kept. tJl: oUS (■>()2. Illuininatiiifi; iMinineeriiif!; Soeiety (l'J52) IKS liniitiiiK handbook. 2d ed., lUuiiii- natiiin iMi^ijiiieerinf!; Soeiety, New York, luteniatioiud (^oniniission on Illuinination, Berlin (UKiS) Compt. rend., •.»: 596-625. Jarrett, K. T., M. l^ Zelle, and .\. HoUaeiuhT (1<)J8) Studies of the control of aeute respiratory disea.se among \aval reeruits. .Vni. J. Hyg., 48: 2'.i'.i-2'.i\) . Jones, M. F., L. Jacobs, and A. Hollaender (1940) The effects of monoclnoin.itic ultraviolet radiation on eggs of the nematode, Enterobius vermicularis. II. Sub- lethal effects. J. Parasitol., 26: 4:^5 445. Kelner, A. (1949) Effect of visit)le liglit on the recovery of Slreptomyces griseua conidia from ultraviolet irradiation injury. Froc. Natl. Acad. Sci. U.S., 35: 73-79. KoUer, L. R. (1946) Ozone production by low-pressure mercury arcs. Gen. Elec. Rev., 49: 50-53. (1952) lUtraviolet radiation. Jolm Wiley & Sons, Inc., New York. Langnniir, A. I)., Iv T. Jarrett, and A. Hollaender (1948) Studies of the control of acute respiratory diseases among Xaval recruits. Am. J. Hyg., 48: 240 250. Laurens, H. (1933) The physiological effects of radiant energy. Chemical Catalog Company, Inc., New York. Lea, D. E. (1946) Actions of radiations on living cells. Cambridge University Press, Cambridge, England (also The Macmillan Company, New York, 1947). Luckiesh, M. (1946) Applications of germicidal, erythemal and infrared energy. D. Van Nostrand Company, Inc., New York. Pp. 110-117. Luckiesh, M., and L. L. Holladay (1942a) Designing installations of germicidal lamps for occupied rooms. Gen. Elec. Rev., 45: 343-349. (1942b) Tests and data on disinfection of air with germicidal lamps. Gen. Elec. Rev., 45: 223-231. (1944) Disinfecting water by means of germicidal lamps. Gen. Elec. Rev., 47: 45-50. Luckiesh, M., and A. H. Taylor (1946) Transmittance and reflectance of germicidal (X2537) energy. J. Opt. Soc. Amer., 36: 227-234. Luckiesh, M., A. H. Taylor, and G. P. Kerr (1944) Germicidal energy — a practical method of measuring transmission and absorption of germicidal energy' by water. Gen. l']lec. Rev., 47: 7-9. Luckiesh, M., A. H. Taylor, T. Knowles, and E. T. Leppelmeier (1947) Killing air- borne respiratory microorganisms with germicidal energy. J. Franklin Inst., 244: 267-290. (1949) Inactivation of molds by germicidal ultraviolet energy. J. Franklin Inst., 248: 311-325. Lurie, M. B. (1946) Control of air-borne contagion of tul)erculosis. Am. J. Nursing, 46: 808-810. Matelsky, I. (1950) Rays curb bacteria, boost fruit quality. Food Ind., 22: 1722- 1723. Meyer, A. E. H., and E. O. Seitz (1949) Ultraviolette Strahlen. 2d ed., Walter de Gruyter & Co., Berlin. Miller, W. R., E. T. Jarrett, T. L. Willmon, A. Hollaender, K. W. Brown, T. Lewan- dowski, and R. S. Stone (1948) Evaluation of ultraviolet radiation and dust control measures in control of respiratory di.sease at a Na\al training center. J. Infectious Diseases, 82: 86-100. National Research Council, Committee on Sanitary lOtigineering (1947) Recent studies on disinfection of air in military estabiisiiinents. Am. .1. P;i()n in the sun in which predominantly contiiuious emission changes rather abruptly into emission of spectral lines. This transition, owing to a rapid decrease in opacity of the solar gases in the region of the photo- sphere, takes place in a few hundred kilometers in a level where the pres- sure is about 10~' atmosphere. The transmission coefficient tx of a homogeneous layer of absorbing gas for light of wave length X is given by h = r-'-x" (3-1) Avhere «x = the absorption coetficient for wave length X and h = the thickness of the absor))ing layer. The sharpness of the plnjtosphere depends on the circumstance that, for visible light, the values of a\h are sufficiently high that a relatively thin layer h of the gases is opaque; that is, the i)roduct a^h is very great. Relative to any comparison wave length, say, in the visible region, radiation emerging from the sun at other wave lengths where ax is greater will come from higher and therefore cooler layers, and the intensity will be lower. At wave lengths where ax is smaller, a thicker layer of the sun is required to be opaque, and the radiations which escape come from a region of higher temperature and are, accordingly, relatively more intense. Thus a part of the visible and near-infrared continuum of the photosphere matches the spectrum of a ()000°K black body rather well, whereas the blue and ultraviolet intensi- ties lie considerably below a (i()00°K source, and there is evidence that the infrared spectrum in the 8- to 13-ju region is fitted better by a 7000° K intensity curve. Although the photosphere is sharp in visible light, it is not uniformly l)right along the diameter, the center of the disk being considerably brighter than the edge. This phenomenon, readily observable with the eye or in photographs of the sun, was examined by Abbott et al. (1922) at seven wave lengths between 3737 and 10.080 A. Considering the inten- sity at the center of the disk to be unity for each wave length, it was found that the intensity decreased toward the edge of the sun for all wave lengths and that the diminution in intensity was more pronounced for short than for long wave lengths. For example, at a distance from the center equal to 0.95 of the photospheric radius, the intensity of wave length 3737 A was 0.4319 of the central intensity, whereas at 10,080 A it was 0.7331 of the central intensity. Thus the limb of the sun is not only less bright but is also redder than the center. The effect is explained by the considerations in the foregoing discussion and is probably due both to absorption and scattering by overlying layers of gases and to the fact SUNLIGHT AS A SOURCE OF UADIATIOX 97 that radiations Avhirh emerge from the edge of the spherical sun come from higher and cooler levels than the radiations from the center of the disk which must pass through minimal thickness of overlying gases in escaping. Since the attenuation by scattering and absorption is greater for short than for long wave lengths, the effect is more pronounced in the blue than in the red end of the spectrum. Although the sun is entirely gaseous, the sharpness of the photosphere, in comparison with more nebulous layers above it, has led to the custom of referring to these latter regions as the atmosphere of the sun. There are three regions of the solar atmosphere distinguishable by the states in Fig. 3-1. Ultraviolet solar spectruiu ol)tained in 1942 at Arosa, Switzerland. {Gotz and Caspar is, 1942.) which matter exists in them and by the radiations which they emit — the reversing layer, the chromosphere, and the corona. The reversing layer is the innermost of these regions and lies just above the photosphere. It extends to a height of 1500 km above the photosphere and is the region in which the transition from continuous to line emission occurs. The temperature is about 4830°K, and the pressure is probably 10"^ to 10~^ atm. In the reversing layer the dark Fraunhofer lines of the solar spec- trum are formed by atomic absorption at discrete wave lengths of the continuous radiation from the underlying hotter photosphere. The ultra- violet spectra of Figs. 3-1 and 2 show many of the large number of Fraunhofer lines in the biologically effective region of the solar spectrum. Babcock et al. (1948) have investigated the ultraviolet solar spectrum between 2935 and 3050 A with a 21 -ft grating spectrograph and have listed 665 absorption lines in this erythemal region. It must not be supposed that the dark lines represent points of zero intensity in the solar spectrum; they appear so in Figs. 3-1 and 2 because 98 UADIATION lUOLOCiY of liij^h phototiiiapliic contrast. Duiiiiji a tcjtal oclipso of tlic .sun when the moon ohscurots the pliotosphcrc, llie reversing layer and regions al)ove it are .seen to emit l)riliiant spectral lines, the so-called "flash spectrum." Although the dark lines reduce the intensity of higher tem- perature radiation from the photosphere, they are (^nly relatively dark and radiate toward the earth with inttMisities appropriate to a lower tem- perature source at about 4800° K. The Fraunhofer absorption is stronger in the ultraviolet than in the visible solar spectrum. Pettit (1940) mapped the spectral energy between the Fraunhofer lines, using a high-dispersion spectrograph and 2900 2800 2700 2600 2500 2400 2300 2200 2100 WAVE LENGTH, A Fig. 3-2. Ultraviolet solar spectrum obtained during a rocket flight of June 14, 1949, by Johnson, Purcell, and Tousey (1952). sensitive photocells, and, from his measurements, estimated the attenua- tion of radiation from the photosphere by the overlying layers of the sun. Comparison of his results for integrated radiation from the sun and for radiation between the Fraunhofer lines indicates that the total intensity between 3200 and 4000 A is about 70 per cent of the radiation which would be emitted by the unobscured photosphere, whereas between 4000 and 7000 A the ratio is about 91 per cent. The chromosphere is a region consisting principally of hydrogen, helium, and calcium, located immediately above the reversing layer; it extends from the top of the reversing layer at about 1500 km above the photo- sphere to a height of 12,000 km which is the greatest height at which H„, the first Balmer line (6563 A), occurs. Other lines of the Balmer series of hydrogen and line spectra of other un-ionized atoms fade out at lower heights. The fla.sh spectrum of the chromosphere as seen during a solar eclip.se contains emission lines of helium and ionized helium together with lines of ionized metals. Menzel points out the existence of ionized helium in the chromosphere and Hoyle gives results obtained from studies of line widths in the flash spectrum as evidence that the temperatures in this region are 20,000-30, 000°K. Prominences originating in the chromo- sphere and sometimes rising several hundred thousand miles also contain matter at these temperatures. The chromosphere also emits continuous radiation, but none of the.se emissions — the line spectra, the spectra of SUNLIGHT AS A SOUKCi: OF KADIATION 99 prominences, or the continuum — are strong. The quantity a\h in Eq. (3-1) is small throughout the chromosphere, and light from the underlying layers is readily transmitted. Since the chromosphere is a weak absorber, it is a weak emitter. The corona is the outermost observable region of the sun, being observa- ble only during a solar eclipse or by use of the coronagraph. The corona begins in the region where the total continuous radiation from the solar atmosphere is about equal in intensity to the total line emission, and this region lies about 12,000 km above the photosphere. The corona extends outward for very great distances. S. P. Langley is said to have observed a coronal streamer extending to 12 solar diameters during the eclipse of 1878. Photographs usually show the corona extending to about 1 solar diameter because of the rapid decrease in its brightness with increasing height. The visible light from the corona consists principally of light from the photosphere scattered by electrons, but emission lines of highly ionized calcium, iron, nickel, and argon are also present, the most intense being the green line at 5303 A due to Fe(XIV), that is, iron with half its 26 elec- trons removed. Temperatures of about one million degrees are required to produce the states of ionization and other effects observed in the corona. Nevertheless, the entire visible light from the corona is about half that of the full moon, or about one one-millionth that of the sun, and its contribution to the visible light and ultraviolet radiations which reach the surface of the earth is inconsequential. The corona and upper chromosphere are, however, of interest as the source of radio waves and of X rays emitted by the sun. Although these emissions are probably too weak to be of biological importance, they deserve a brief description. Radio emissions originating outside the earth, presumably from inter- stellar space, were discovered in 1932 by Jansky (1933) in experiments with 30-meter waves, but emissions from the sun were not known until 1945 when the improved sensitivity of receiving techniques brought about their detection (Hagen, 1951). It was found that the solar radio waves originate in the upper chromosphere and corona in thick regions of the solar atmosphere which center at heights above the photosphere of approximately 8000, 10,000, 13,000, and 18,000 km for wave lengths 0.8, 3, 10, and 50 cm, respectivel3^ For example, during optical totality of the solar eclipse of May 20, 1946, the solar radiations in the respecti^'e wave lengths were reduced to 1, 6, 19, and 33 per cent of their values for the uneclipsed sun. To account for the observed radio-wave energy the temperatures of the regions which were emitting the wave lengths 0.8, 3, 10, and 50 cm were calculated to be 7000, 10,000, 2(),000, and212,000°K. In contrast with the limb darkening of the sun for visible light, when observed with radio waves, the sun brightens at the limb, the l)rightening increasing with the wave length. The solar radio emission is not constant 100 UADIATION Hlol-OGY in intcihsity but varies in an iin'{;iiiai- and unpredictable manner. At the shortest wave length the intensity is most constant. The correlatioii with sunspot area is poor at the shortest wave length and improves at a wave length of about 10 cm. At longer wave lengths the fluctuations in the intensity become more erratic and abrupt. \'ig(>i-()us investigation of all these phenomena is in progress. X rays and short ultraviolet radiations from the sun which had never been detected at sea level were measured by narrow-band photon counters carried aloft in a rocket (Friedman et al., 1951) on Sept. 29, 1949. Ultra- violet radiation in the wave-length band 1150-1350 A was observed above 05 km altitude, and in the band 1425-1700 A above 100 km. Solar X-ray emission was first recorded at 85 km with a counter sensi- tive from 0 to 10 A, which indicated, because of the known absorption of the atmosphere, that the solar emission became undetectable below 1 A. The measured intensities recjuired effective temperatures of the emitting regions, again probably the upper chromosphere and corona, of 4500°, 5000°, and 10«°K for the bands 1425-1700, 1150-1350, and 7-10 A, respectively. It seems nearly certain that X rays longer and softer than 10 A are emitted by the sun, and further rocket experiments have been planned to investigate the subject. SUNLIGHT ON TOP OF THE ATMOSPHERE Two methods have been used to determine the solar spectral energy on top of the atmosphere: (1) by measuring the spectral intensity of the sunlight reaching the surface of the earth and correcting for the trans- mission of the terrestrial atmosphere, and (2) by sending apparatus on rockets to altitudes above most of the atmosphere. Both methods are difficult; the first has been in use for many years but can obviousl}^ give information about only those wave lengths which are detected at the earth's surface; the second is relatively recent and has yielded important new results in the ultraviolet portion of the spectrum. In the first method, unfocused radiation from the entire sun is allowed to fall on the slit of a double monochromator, that is, two monochromators in series in order to reduce contamination, by radiation scattered by the optical parts of the instrument, of the spectrum falling on the exit slit. Back of the exit slit, in a position to intercept the portion of the spectrum emerg- ing from the monochromator is mounted a bolometer, a thermocouple, or a calibrated photoelectric cell with which the intensity may be measured point by point throughout the spectrum or in selected portions of it. Glass or quartz prisms and lenses in the monochromator are used to measure the major portion of sunlight reaching the surface of the earth, which lies between 2900 A and 2.5 ju- Rock-salt pi-isms and diffraction gratings are used to observe the infrared solar spectrum from 2.5 to 25 n. This portion of the spectrum has been of little direct interest in bio- SUNLIGHT AS A SOURCE OF RADIATION 101 physics, although it is of impcjrtaiR-e in the physical state of the atmos- phere and in meteorology. The measurement of the spectral distribution of intensity in sunlight and its correction for atmospheric attenuation has been a major function of the Astrophysical Observatory of the Smithsonian Institution, begin- ning in 1892 under the direction of S. P. Langley who invented the bolom- eter and first measured the spectral distribution of energy in the solar spectrum, and continuing under Abbott and others. Their measure- ments have been made at Mt. Wilson, Mt. Whitney, and Washington, D.C. A convenient summary of their work is to be found in the Smith- sonian Physical Tables (Fowle, 1934b). Many details of method are described in a later publication by Abbott et al. (1942). Independent and, in some spectral regions, improved measurements of solar spectral intensity have been made by other investigators. In nearly every case, however, the measurements were scaled to fit the Smithsonian curves which therefore remain the standards over most of the spectrum. In the ultraviolet and visible portions of the spectrum it is observed that the atmospheric absorption follows an exponential law. Hence i = iV-^T^"'^, (3-2) where i and /n = the intensities of a beam of sunlight at the bottom and top of the atmosphere, respectively, and a, the atmospheric attenuation coefficient, refer to a wave-length interval from X to X + (fX; Z = the zenith angle of the sun; and 7 = a factor which accounts for the curva- ture of the earth, ^^alues for 7 are listed in standard tables; 7 is very close to unity for Z < 80°. In the infrared for certain bands of water vapor and other gases, Eq. (3-2) does not agree with the observed absorp- tions. However, Eq. (3-2) is not wTong; the discrepancy is due to the use of insufficient dispersion to resolve the narrow and complex structure of many of the bands. The air mass M is defined by M = 7 sec Z. (3-3) From Eq. (3-3), .1/ - 1 for Z = 0 and 7 = 1, and therefore .1/ is the amount of atmosphere from the surface to space in a vertical direction. Then (3-4) I = loC -aM To determine io on top of the atmosphere, i is measured for several values of M and is plotted for each wave length on a logarithmic scale against ]\I . The straight line thus obtained is extrapolated to zero air mass, which gives lo when proper account is taken of the transmission of the spectrograph and the spectral response of the accompanying bolome- I ()-i RADIATION mOLOGY Icr or photoelectric cell. The determination reciuire.s that the atmos- pheric attenuation a remain unchanged for several hours as Z changes. Several sets of data obtained hy this method are plotted in Fig. 3-3. They include the data of the Smithsonian Institution (Ahl)ott et al., 1923; Fowle, 1934h) of Pettit (1940) at Mt. Wilson, and of Gotz and Schonmann (1948) at Arosa. The curve of Moon (1940) is an average of all data existing in 1940. The data at an altitude of 55 km were obtained in 1947 i)y Durand ci al. (1949) of the Naval Research Labora- tory with a spectrograph on a rocket; at 55 km the pressure was 2 X 10~' too 90 80 - 70 - t 60 z H ? 50 UJ > 40 30 20 10 r / 1 / / ^ £i SMITHSONIAN PHYSICAL TABLES (FOWLE, 1934b) / I J' o SMITHSONIAN INST, 1920-1922 ( ABBOTT e/ O/, 1922) / r' v\ • , PETTIT, 1940 - hi 1 1 , ^\ A NRL, 65 KM, 1947 (HULBURT, 1947) v\ GOTZ AND SCHONMANN, 1948. h - 1 MOON, AVERAGE TO 1940 6000°K RADIATION q \ r\ A "M ^ • r i \ / • ■^ /*? 0 ^ -/Al >v h> %D / -J ^S„^A - A ^^^.^o o tf' ^~— ~.^___o_ A U 1 1 1 1 1 1 1 1 1 1 0.2 0.4 0.6 0.8 1.0 2.0 2.2 2.4 2.6 1.2 1.4 1.6 1.8 WAVE LENGTH, /Z Fig. 3-3. Solar-spectrum curves on top of tlio atniosphore. atm, and the spectrograph had risen through 4999 5000 of tlie atmosphere and had only 1/5000 al)o\^e it. The solid-line curve of Fig. 3-3 is the spectral intensity of a l)lack body at 6000° K. All the curves of Fig. 3-3 were arbitrarily adjusted to have their maxima at 100. It is seen that the ()000°K curve lies abov^e the solar values in the ultraviolet and also in the red and infrared to 1.4 m- In Figs. 3-1 and 2 are reproduced perhaps the best photographs which have been made of the ultraviolet portions of the solar spectrum. The spectrum of Fig. 3-1 was taken by Giitz and Casparis (1942) at Arosa, Switzerland, using all po.ssible care to reduce the scattered light which always veils the short-wave-length limit of the solar spectrum l)elow the ozone layer. The spectrum of Fig. 3-2 is a composite made from four of a series of spectra obtained by F. S. Johnson, .1. 1). Purcell, and W. SUNLIGHT AS A SOURCE OF HADLATION 103 Toiiscy (1952) of the Naval Research Laboratory with a sportrosraph on a rocket flying above the ozone region from al)()ut 60 to 110 km on June 14, 1949. The spectra of Figs. 3-1 and 2 overlap at the absorption band 2882 A. The curves of Pettit and the NRL (Durand and coworkers, unpub- lished; cited in Hulburt, 1947) shown in Fig. 3-3 in the ultraviolet, are replotted in Fig. 3-4 with the ordinate on an absolute scale and with the 120 no - PETTIT, 1940 NAVAL RESEARCH LABORATORY, 55KM, 1947 STAIR, 1951- 6000°K RADIATION J L 2500 3500 3000 WAVE LENGTH, A Fig. 3-4. Ultraviolet portion of the solar spectrum on top of the atinospliere. scale of the abscissa expanded over that of Fig. 3-3 in order to bring out detail. The data of Stair (1951) of October 1950 are added. The fluc- tuations in the curves indicate certain major absorptions in the solar spectrum but are not fine-grained enough to bring out all the Fraun- hofer lines. Pettit's value of u at 3400 A is taken as standard, and the other two curves are adjusted to it. The ()000°K black-body curve of Fig. 3-3 is continued in Fig. 3-4 to emphasize the fact that the solar intensity continues to fall below it as the wave length is decreased to 2200 A. In the infrared beyond 2.6 n, io decreases rapidly with increasing wave length approximately as for a black body at 7000°K. For example, rela- 101 HADIATION HIOLOGY tive to the inaximuin intensity at KiOO A the intensities at 5, 8, and 14 /x are l.i) X 10 •', 3 X 10-', and 3.7 X l()-'\ respectively, hut the idea that io is approximate to or is less than tiic intensity of a hlack body at ()()00°K must not !)(' pushed too far because, as brought out in the preceding sec- tion, both in the region of X rays and of radio waves the solar energy is such as to indicate the existence of emissive regions of the sun, probably in the corona, which are at temperatures much greater than ()000°K. In Table 3-1 is given the energy in several portions of the solar spec- trum on top of the atmosphere calculated from the areas under the aver- age of the curves of Figs. 3-3 and 4. In obtaining the values in column Table 3-1. Spectral Distribution of Soi.ar Radiation \Vavo-leii}j;th K •action of Flux doiLsity interval total radiation cal/cin^ mill 2200-3150 A 0.014 0 027 3150-4000 A 0.079 0.153 4000-7000 A 0.403 0.78 7000 A-2.6m 0 474 0.92 2.6-14 M 0.03 0.06 3 of the table the solar constant, which is the flux density of total solar radiation on top of the atmosphere at the earth's mean distance from the sun, was taken to be 1.94 cal/cm- sec. It is seen from Table 3-1 that about half the total solar radiation lies in the visible and ultraviolet regions below 7000 A and about half in the infrared. SOLAR ULTRAVIOLET RADIATION AT THE EARTH'S SURFACE The intensity of the solar ultraviolet radiation that reaches a particu- lar point on the surface of the earth depends on the amount of ozone, air haze, and clouds between that point and the sun. Of these materials, ozone is important because of the absorption of its great ultraviolet band which begins at about 3400 A and increases rapidly for shorter wave lengths. Air, haze, and clouds atteiuiate the rays of the sun mainly by scattering with little true absorption; the scattering, of course, causes the sky. For ultraviolet radiation, true absorption of the oxygen of the air does not set in appreciably until the wave length becomes less than about 2700 A. The absorption coefficient a of ozone, observed by Tsi-Ze and Shin- Piaw (1932, 1933) is plotted in curve I, Fig. 3-5, against the wave length; about one-fifth the observations are shown by the dots. The absorption coefficient a is defined by i = z>-«, (3-5) SUNLIGHT AS A SOURCE OF RADIATION 10.3 where io and i are the intensities of a collimatod beam of lij2;ht in the wave-length region from X to X + dX, entering and emerging from a layer of ozone x cm in thickness at normal temperature and pressure (NTP), i.e., at 0°C and a pressure of 1 atm. The long-wave-length region of the ozone absorption from about 3400 to 3100 A has a band structure known as the Huggins bands; the smooth absorption below 3100 A is lOOOrr 2200 2400 2600 2800 3000 WAVE LENGTH, A J200 3400 ''Fig. 3-5. Ultraviolet absorption coefficients of some atmospheric gases. known as the Hartley continuum. The absorption in the Huggins bands varies with the temperature in a complicated way, which is of no interest here; roughly, a decreases between 10 and 30 per cent when the tempera- ture falls from 18° to -50°C (Vigroux, 1950). It is seen from the ozone curve of Fig. 3-5 that, below 3100 A, a rises to very high values; therefore it is not surprising that even the few millimeters of ozone which e.xists in the upper atmosphere is sufficient to prevent the detection of ultra- violet wave lengths from 2915 to 2150 A at the surface of the earth. In the standard atmosphere the pressure is 1.0132 X lO*' dynes /cm-, and the total air in a vertical column of the standard atmosphere from sea level to space amounts to 8 km of air at XTP; 20.75 per cent of this is oxygen. In Fig. 3-5, curve II is the attenuation coefficient a per atnios- ion RADIATION BIOLOGY phore of puii' air calculated from the Rayleigh scattering theory with polarization dofoct. and curve III is a per atmosphere observed by Vassy (1941) for what may be termed a "fairly clear atmosphere at sea level." The observed (Buisson el al., 1930, 1932) absorption coefficient a of the molecular oxygen gas contained in 1 atm, from which the Rayleigh term has been subtracted, is plotted in cur\e IV, Fig. 3-5. There are no avail- able data on the attenuation of ultraviolet radiation passing down through a hazy or cloudy atmosphere. ATMOSPHERIC OZONE Ozone exists in the upper atmosphere of the earth for the most part in the region between 15 and 35 km above sea level; it extends in rapidly T3 Uj" O 3 o I 0. < O UJ o Fig NTP JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC 3-6. Average contours of equal thickness of ozone. Unit, I0~' cm of ozone at (Gotz, 1944.) decreasing concentration as high as 50 km and down to sea Jexel whcMv there are often traces amounting to as much as 0.05 mm of ozone at NTP per kilometer of air (or 5 X 10^'' by volume of air). The total thickness of ozone in a vertical column of air from sea level to space varies from about 1.5 to 5 mm at NTP depending on the latitude and the season. This is brought out in P'ig. 3-6 which gi\-es an axcrage world-wide distribu- tion of ozone derived by Gotz (1944) from a summary of nearly all avail- able observations. Figure 3-(i shows the increase of ozone thickness with increasing latitude up to about (50° foi- all seasons of the year and the spring maximum and autumn minimunv for latitudes greater than 20°. Whether there is a diurnal variation in the ozone thickness above any station is not known with certainty; a few data mentioned by Dobson SUNLIGHT AS A SOURCE OF KADIATION 107 (1930) indicate that a diurnal variation, if it exists, is small. Regarding the change of ozone thickness with sunspots, there are few long-contiiuied series of ozone data. Fowle (1934a, 1935) obtained yearly a\'erage values at north latitude 34° in Arizona and California. Ilis values from 1921 to 1928 varied with the sunspots, but the correspondence was not main- tained from 1928 to 1934. A coamection between ozone and sea-level barometer changes is complicated and has not been clearly established. From the data of six ozone stations in Europe, Dobson concluded that the smallest amount of ozone occurred to the southwest of a barometric high and that the passage of a barometric low over a station was accompanied by an increase in ozone content. In general, since most of the ozone lies between 15 and 35 km, its variations would be expected to be correlated with changes of pressure and winds in the stratosphere rather than in the troposphere, and at this time the relation between stratospheric and tropospheric weather cannot be said to be completely known and understood. CALCULATED ULTRAVIOLET INTENSITY AT THE EARTH'S SURFACE A complete calculation of the solar ultraviolet radiation falling on the surface of the earth from the sun and sky is very complicated and will not be attempted. It is, however, instructive to calculate the ultraviolet intensity of the direct rays of the sun at the surface of the earth for vari- ous zenith angles of the sun. Let in and i be the intensities, respectively, of the rays of the sun outside the atmosphere and at the surface of the earth. Then where a = the absorption coefficient of ozone per centimeter, X = the thickness of the ozone in centimeters at NTP, a„ = the attenuation per atmosphere below the ozone region due to pure air and haze, and Z = the zenith angle of the sun. The symbols i, in, a, and a^ refer to the wave-length region from X to X + r/X. In Fig. 3-7 the curve labeled to is the Naval Research Labora- tory curve of Fig,. 3-4 and is plotted in arbitrary units against the wave length for the ultraviolet region of the solar spectrum where the absorp- tion of ozone becomes important. The values of i for ozone of thickness 4 mm, for no atmospheric attenuation, and for various zenith angles were calculated from Eq. (3-6) with .r = 0.4 cm, «„ = 0, and values of a obtained by drawing a smooth curve through the fluctuations of curve I, Fig. 3-5. The i vs. X curves are plotted in Fig. 3-7 for six values of Z. The total ultraviolet energy E in wave lengths less than 3200 A was 108 RADIATION HIOLOGY calculated I'loni \\\v integral /^' - fr i(i\ (3-7) and the / \'s. X cur\"c.s lor the se\eral xaiues of Z. The resulting values of E are plotted against Z in the 4-mm curve of Fig. 3-8. Families of i vs. X curves similai' to those of Fig. 3-7 were calculated for other ozone thicknesses, and from these curves and Ei\. (3-7) the E vs. / curx'es of 1 1 - SOLAR ENERGY //^\\ 10 - ABOVE ATMOSPHERE ./ //V\^ 9 - '°//^/r^ V) 3 8 - / //// a> > o 1 z ////// 3 2 1 - / 'o'o / / / / / f ^0 / J 1 ^ ^ 1 '0 1 1 1 1 1 1 1 1 ■-'- <\i^^r^i^\-i^-J^\ >< 1 1 1 1 1 t L . 1 1 1 [ 1 1 1 1 1 1 1 L I r ■ 1 2900 3000 3100 3200 3300 3400 WAVE LENGTH, A Fig. 3-7. Observed solar-spectral-energy curve ?n above atmosphere and calculated curves of i after passing through 4 cm of ozone at various zenith angles. Fig. 3-8 for 1, 2, and 3 mm of ozone were calculated. The curves illus- trate the manner in which E decreases with increasing ozone thickness and increasing zenith angle. Curves for integrals to wave lengths less than 3200 A were similar to those of Fig. 3-7 but descended more rapidly to low values. The effect of the atmosphere, in addition to the ozone, is shown by the dotted curve of Fig. 3-8, which refers to a "fairly clear" atmosphere with 1 mm of ozone. It was calculated from Eqs. (3-6) and (3-7) with «„ from curve III, Fig. 3-5. The dotted curve of Fig. 3-8 brings out the almost obvious fact that a relatively small amount of atmospheric haze is more effective in reducing the ultraviolet energy of the direct rays of the sun than, for example, doubling the ozone thickness of the upper atmosphere. However, we must hasten to remark that haze reduces the total ultravio- SUNLIGHT AS A SOURCE OF RADIATION 109 let radiation falling on the earth's surface far less than it reduces the effectiveness of the direct rays of the sun. The reason is that the light scattered out of the direct solar mm Oi rays by haze is not entirely lost, since haze exerts little absorption, but reappears as sk}^ light, which for thin haze is mainly directed downward. A fraction of the sky light is scattered outward to space and is lost to the earth; the fraction in- creases with increasing haze and clouds. As has been mentioned, an exact calculation of all this is com- plicated and would require a com- plete theory of sky brightness and polarization for ultraviolet wave lengths in terms of the ultraviolet optical constants of the atmosphere in all stages of haziness. Such a theory has not been formulated, and such constants have not been determined; therefore only a survey of the observational material is presented in the following section. o o V ^ I mm O3 ^^ + "FAIRLY CLEAR" ^ J I I I 1 L 0 30 60 Z, deqrees Fig. 3-8. Calculated solar ultraviolet energy E for wave length.s loss than 3200 A for several cases. OBSERVED ULTRAVIOLET INTENSITY AT THE EARTH'S SURFACE Many measurements of ultraviolet radiation from the sun and sky were carried out at Washington, D.C., by Coblentz and Stair (1943). The first series of measurements were made with photocells arranged to record En, the radiation received on a plane normal to the rays of the sun, from the sun and a circular region of sky around the sun as a center 22° in diameter. Data were taken during the clearest days over the years 1936 to 1941; for illustration the values of En for 1937 are shown in Fig. 3-9. Two types of photocells were used, one sensitive to wave lengths less than 3200 A and the other sensitive to wave lengths less than 3132 A; the spectral-sensitivity curves of the photocells were not reported. In Fig. 3-9 the abscissas are the air mass M and the zenith angle of the sun Z, and the two scales of ordinates are the En in microwatts per square centimeter for the respective types of photocells. It is seen that they were approximately proportional and that the ultraviolet intensities of wave lengths below 3200 A were roughly 2.5 times the intensities below 3132 A. The data of the other years from 193() 1941 were similar to those of Fig. 3-9 for 1937. For any zenith angle the variation of the ultraviolet intensitv of a factor of about 2, shown in Fig. 3-7, was proba- uo RADIATION TUOI.or.Y bly due largely to haze variations within the (luulitative specification of a "clear" day and, to a lesser extent, to ozone variations. No ultra- violet variation with sunspots appeared, and if any existed, it was obscured l)y the haze variations. The sunspol numbers for the years H)8() 1941 were 80, 114, 110, 89, ()8, and 48, respectively. There was an ill-defined seasonal variation in K„, which is not brought out in Fig. 3-9, partially attributable to the spring-to-fall decrease in ozone (see Fig. 3-6), in that the ultraviolet intensity was often greater in the autumn than in the spring from equal solar zenith angles. Ho\vever, high En values fre- (jucntly occurred in late winter and early spring which may have been 150 - 5 J- 100 o o V ^ 50 60 AIR MASS M Z, degrees Fu;. 3-9. Ultraviolet intensity E,, during clear days in \\ asliiiigtoii. 1937. and Stair, 1943.) {Coblentz due to unusually clear skies or to local ozone variations attributable to stratospheric weather. After 1941 Coblentz and Stair (1944) changed their plan of observation and measured E/,, the ultraviolet energy from the sun and the entire hemisphere of the sky, falling on a horizontal plane, instead of PJ„, the ultraviolet energy from the sun and 22° of the sky, falling on a plane normal to the sunbeams. Values of Eh obtained for clear days in Wash- ington are plotted in Fig. 3-10 as dots, circles, crosses, and triangles for June 4, Sept. 18, and Dec. 21, 1943, and Oct. 17, 1944, respectively. In Fig. 3-10 the two dotted curves outline the spread of value of E„ for clear days in Washington during 193(5 and 1941. Since the points fell in the region between the dotted cur\'es. it was concluded that Eh and A'„were approximately the same; in general, of course, Eh and En cannot be expected to be equal. "N^nlncs of Ei, for .some clear days in high latitudes, obtained (Coblentz el ^ r< , l_ o c; Em - 60.8° 30 60 90 AIR MASS Af Z, degrees Fig. 3-10. Values of Eh and E,, for wave lengths less than .3200 A. The symbols indicate Ei, for some clear days in Washington during 1942 and 1943. The solid-line curve is for Eh for higher latitudes in 1941. The £„ for clear days in Washington 1936-1941 was within the dotted curves. (Coblentz and Stair, 1944; (Coblentz el al., 1942.) least smoky localities; the data for clear and cloudy skies were plotted separately. It is seen in Fig. 3-11 that the curves for the more smoky localities lie below those for the less smoky both for clear and cloudy skies and also that the curves for cloudy skies lie below those for clear skies. Therefore both smoke and clouds decreased the amount of ultraviolet radiation that reached the surface of the earth. Comparison of Fig. 3-10 with the "clear-sky" data of Fig. 3-11 shows good agreement, when it is remembered that Eh for wave lengths less than 3200 A is about 2.5 times Eh for wave lengths less than 3130 A. The conclusion is therefore that, from Fig. 3-10, a rough estimate may be made, correct perhaps within a factor of 2, of Eh in clear weather for all seasons of the year and all times of the day. If a more e.xact value of Eh is required, provision must be made to measure it. To make the rough estimate, an average curve was drawn through the data of Fig. 112 RAnr\TI<)\ ItlOLOGY 3-10 which ti;ave Eh in terms of the zenith nnp}o of the sun. From this, I'Jh was calculated throuj^ihout the day for various latitudes and seasons. The results are plotted in Fijj;. 3-12 for (he twenty-second day of March, June, September, and December for north latitudes 0°, 20°, 40°, ()0°, and 80°. The curves for March and September are the same at all latitudes becaus(» the zenith ;inu;lo of the sini is the same at these epochs; at the — I 1 1 1 1 — 1 1 1 IT 1 ■■■■ i GROUP m \ CLEAR SKY *" \ - \ i 1^ - \ - - \ - - \ - 1 ?\^^ 1 N* 3 4 1 2 3 4 M M Fig. 3-11. Values of Eh for wave lengths less than 3130 A. Group I. for most smoky loealities; Group III. for least smoky localities. {Ives and Gill. 1937.) equator the curves for .June and December are the same for the .same reason. For December at 60° north latitude and for March, September, and December at 80°, the sun does not rise above 10° above the horizon, and Kh i« zero throughout. At 80° the value of Eh in June remains above zero all night, being 3 at midnight because the midnight sun is 14° above the horizon. In the curves of Fig. 3-12, no seasonal adjustments have been made for the fact that the sun is about 3 per cent nearer the earth on December 22 than it is on June 22. Since the values of Eh of Fig. 3-12 are based on the data of Fig. 3-10 obtained on clear days in the United States, they may be expected to be correct for localities outside the United States only if the localities have the same ozone and the same SUNLIGHT AS A SOURCE OF RADIATION 113 clearness of atmosphere and sky as those that occurred in the United States. A few measurements have been made of the ratio of the ultraviolet radiation on a horizontal plane from the total hemisphere of the sky 200 E u o o CM rO V u o /i \\ Nx ^^ '*>.^ ' -7\ - 1 1 1 1 1 1 1 1 1 1. 1 1 1 1111 90° 60° 30° 30° 60° 90° HORIZON ZENITH HORIZON Fig. 3-13. Sky brightness for ultraviolet radiation and visible light. Curve I. for ultraviolet wave lengths less than .■?200 A in California. Curves II and III. for visible light in Brazil and Switzerland. on a meridian through the sun with the sun at an altitude of 50° are shown by the solid-line curves of Fig. 3-13. The values varied con- siderably from day to day because of changes in haze. For comparison two curves of sky brightness for visible light are plotted in Fig. 3-13, one observed in Brazil (Richardson and Hulburt, 1949) and one in Switzer- land (Dorno, 1919, Table Illd). The curves refer to a clear sky, to a meridian through the sun, and to the sun at an altitude of 50° ; the three curves are adjusted to pass through the same point at the zenith. The difference in the two visible-light curves was probably due to the haze conditions in the respective atmospheres. The curves show the well- known lirightness of the sky near the sun and indicate that the sky near the horizon was relatively darker for ultra\iolet radiation than for \'isiblc light. SOLAR INFRARED SPECTRUM AT THE EARTH'S SURFACE In Fig. 3-14 is gix-en the solar spectral-intensity curve /H on top of the atmosphere and the tiansmission cur\'es of th<' important terrestrial al)sorber, water, in liciuid and \apor form as ol)ser\ ed with spectrometers of low resolving power. The transmission curves for litiuid water refer SUNLIGHT AS A SOITRCE OF RADIATION to tliicknossos of 1 and 10 mm of distilled water a1 20°C calculated from the accurate ah.s()ri)tioii coefficients of ('urcio and Petty (1951). The water-vapor curve represents the transmission through 1.85 km of atmos- phere along a horizontal path containing some haze and a total of 17 mm of precipitable water. That is, the water vapor in a column 1 cm square and 1.85 km long would, if condensed to the liquid phase, form a column of licjuid water 1 cm square and 17 mm long. The portion of the water-vapor cur\'e for wave lengths longer than 0.9 /x was measured in 1.00 mm WATER 17 mm WATER VAPOR 0.8 1.0 2.0 22 2.4 2.6 1.2 1.4 1.6 1.8 WAVE LENGTH, /.^ Fig. 3-14. Solar inten.sity 'o and transmis.sion of water and water vapor. 1949 by Gebbie et at. (1950). The short-wave-length portion of the curve below 0.9 yu was from miscellaneous older measurements along horizontal paths in the real atmosphere; the attenuation below 0.6 /x was largely due to haze because water vapor is very transparent in this region. The curves of Fig. 3-14 bring out the well-known differences in the absorption of water in the liquid and vapor phases. For example, beyond 1.4 /x the strength of absorption by liquid water is much stronger than by water vapor. Thus, 10 mm of liquid water and 17 mm of water vapor are opaque at 1.4 m, but, although the transmission of water vapor rises at 1.65 /x to a high value, liquid water remains completely opaciue at longer wave lengths. Also, it is seen that the absorption coefficients of 1 7 mm of water vapor and of 1 mm of liquid water are comparable on the short-wave-length side of the 1.9-m water-vapor band, but, although water vapor regains its transparency at 2 /x, 1 mm of liquid water does not again become transparent. Furthermore, the wave lengths of maxi- 10 RADIATION' laoLOGY imim alisorption do not coiiicidc for the two phases. Approximately the same iiumher of al)sorption hands exist at wave lengths helow 1.9 fi, hut the \apor hands app(Mir at somewhat shoi-ter \va\'e lenf^ths than the liquid bands. Therefore sunlight that reaches the earth through several centimeters of precipitahle water vapor is not entirely' depleted of energy capable of being absorbed !)>■ liiiuid w atcr in organisms or in the seas, and, in particu- lar, this is true in the so-called "atmospheric windows" at 1.65 and 2.2 n, I 17mm WATER VAPOR 17mm WATER VAPOR 0.05 0.04 0.03 0.02 o 0.01 7 8 9 WAVE LENGTH,/^ Fig. 3-15. Solar intensity I'o in tho infrared and transmission of water vapor, ozone, and carbon dioxide. where atmospheric water vapor transmits copiously and relatively small thicknesses of liquid water absorb strongly. The effect of absorption in the infrared is to heat the absorber. In the 2.2-/i region and at longer wave lengths where liquid water is highly absorbing, heating is produced principally at the surface. On the other hand, for shorter wave lengths below 1 /i, the radiation peiietrates to a greater depth before being com- pletely absorbed and produces warming in depth. Figure 3-15 is an extension of the solar-intensity and the atmospheric- transmission curves of Fig. 3-14 to 14 n to show other atmospheric infra- red absorption bands. The absorption at 2.7 m is due to both carbon dioxide and water vapor. The strong absorption at 4.2 fx is due to carbon dioxide, and the great band from 5.2 to 7.5 /x is due to water vapor. Beyond this band to about 14 ^l the lower atmosphere is relatively trans- SUNLIGHT AS A SOURCE OF llADIATION 117 parent except for the complicated but slight absorption of water vapor. The 9.()-M infrared band of ozone of the upper atmosphere is shown. At 14 n, a strong band of carbon dioxide sets in, and thereafter, for longer wave lengths up to about 400 m, or 0.4 mm, water vapor is a strong absorber with the exception of a narrow crevice of transmission at 22 ju. In conclusion, it is apparent that the curves of Figs. 3-14 and 15 can be used to make rough estimates, perhaps correct within a factor of 3, of the solar energy in the infrared at the surface of the earth if the amount of water vapor in the overhead atmosphere is known, but, if greater pre- cision is required, provision must be made to measure the radiation directly. REFERENCES Abbott, C. G., L. B. Aldrich, and W. H. Hoover (1942) Annals of the Astrophysical Observatory of the Smithsonian Institution. Vol. 6, Smithsonian Institution, Washington. Pp. 29-201. Abbott, C. G., F. E. Fowle, and L. B. .AJdrich (1922) Annals of the Astrophysical Observatory of the Smithsonian Institution. Vol. 4, Smithsonian Institution, Washington. Pp. 217-257. — ■ (1923) The distribution of energy in the spectra of the sun and the stars. Smithsonian Inst. Pubs. Misc. Collections, 74 (7): 1-30. Abetti, G. (1938) The sun. Cro.sby Lockwood and Son, Ltd., London. Adel, A. (1939) Atmospheric absorption of infrared solar radiation at the Lowell Observatory. Astrophys. J., 89: 1-9. Babcock, H. D., C. E. Moore, and M. F. Coffeen (1948) The ultraviolet solar spec- trum, XX2935-3060. Astrophys. J., 107: 287-302. Buis.son, H., G. Jausseran, and P. Rouard (1930) Sur la transparence de la basse atmosphere. Compt. rend., 190: 808-810. (1932) Sur la tran.sparence de la basse atmosphere. Compt. rend., 194: 1477-1479. Coblentz, W. W., F. R. Gracely, and R. Stair (1942) Measurements of ultraviolet solar and sky radiation intensities in high latitudes. J. Research Natl. Bur. Standards, 28: 581-591. Coblentz, W. W., and R. Stair (1943) Measurements of ultraviolet solar radiation in Washington 1936 to 1942. J. Research Natl. Bur. Standards, 30: 435-447. (1944) A daily record of ultraviolet solar and sky radiation in Washington 1941-1943. J. Research Natl. Bur. Standards, 33: 21-44. Curcio, J. A., and C. C. Petty (1951) The near infrared absorption spectrum of liquid water. J. Opt. Soc. Amer., 41: 302-304. Dobson, G. M. B. (1930) Ob.servations of the amount of ozone in the earth's atmos- phere, and its relation to other geophysical conditions. Proc. Roy. Soc. London, A129: 411-433. Dorno, C. (1919) Himmelshelligkeit, Himmelsstrahlung und Sonnenintensitat. Veroffentl. preuss. met. Inst., Abhandl. 6. Durand, E., J. J. Oberly, and R. Tousey (1949) Analysis of the first rocket ultra- violet solar spectra. Astrophys. J., 109: 1-16. Fowle, F. E. (1934a) Further ozone measurements and the po.ssible connection of ozone with the sunspot cycle. Trans. Am. Geophys. Union, 160 162. ■ (1934b) Smithsonian Physical Tables. 8th rev. ed., Smithsonian Institution, Washington, Tables 766-769. 1 18 ItADl \'I'I()\ lilOLOOY (1935) Further ozone ineasuros .iiid their connection with snnspot cycle. Trans. \m. (loophy.s. I'nion, l(>l-lt)5. Friedman, H., S. W. Lichtinan, and E. T. Byram (1951) Photon eoiintor nieasure- ments of solar X-rays and (>xtrenie ultraviolet light. Phys. Rev., 83: 1()25-1()3(). CJebbie, H. A., W. R. IlardinR, C. llilsuni, A. W. Pryee, and V. Roberts (1950) Atmospheric transmission in the 1-14 micron region. Proc. Roy. So(r. London, A20t): S7-107. Gotz, F. \V. P., and P. Casparis (1942) Photographie des ultravioletten Sonnespek- tralendes. Z. angew. Phot. Wiss. u. Tech., 4: 05-07. Gotz, F. W. P., and 10. Schonmann (1948) Die spectrale lOnergievertcihrng von Himmels- und Sonnenstrahlung. Helv. Phys. Acta, 21: 151-108. Gotz, P. (1944) Der Stand des Ozone Problem. \'ierteljahrs.schr. naturforsch. Ges. Zurich, 89: 200-204. ilagen, J. P. (1951) A study of the radio-frequency radiation from the sun. Astro- phys.'j., 113: 547-500. Hoyle, F. (1949) Some recent researches in solar physics. Cambridge University Press, Cambridge, England, llulburt, E. O. (1947) The upper atmosphere of the earth. J. Opt. Soc. Am., 37: 405-415. Ives, J. E., and W. A. Gill (1937) Measurements of ultraviolet radiation and illumi- nation in .\merican cities during the years 1931-1933. U.S. Pub. Health Service, Pub. Health Bull. x\o. 233, 1-30. Jansky, K. G. (1933) Electrical disturbances apparently of extraterrestrial origin. Proc. Inst. Radio Engrs., Sec. II, 21: 1387-1398. Johnson, F. S., J. D. Purcell, and R. Tousey (1952) Measurements of atmospheric ozone to 70 kilometers. J. Geophys. Research, 57: 157-170. Luckiesh, M., A. H. Taylor, and G. P. Kerr (1937) Ultraviolet energy in daylight — a two year record. J. Franklin Inst., 223: 099-714. (1939) A four year record of ultraviolet energy in daylight. J. Franklin Inst., 228: 425-431. Menzel, D. H. (1949) Our sun. The lilakiston Company, Philadelphia. Moon, P. (1940) Propo-sed standard solar-radiation curves for engineering use. J. Franklin Inst., 2.30: 583-017. Pettit, E. (1932) Measurements of idtraviolet solar radiation. Astrophys. J., 75: 185-221. (1940) Spectral energy curve of the .sun in the ultraviolet. Astrophys. J., 91: 159-185. Richardson, R. A., and E. O. Hulburt (1949) Sky brightness numsurements near Bocaiuva, Brazil. J. Geophys. Research, 54: 215-227. Stair, R. (1951) Ultraviolet spectral distribution of radiant energy from the sun. J. Research Natl. Bur. Standards, 40: 353-357. Tousey, R., and E. O. Hulburt (1947) Brightness and polarization of the daylight sky at various altitudes above sea level. J. Opt. Soc. Amer., 37: 78-82. Tsi-Ze, X., and C. Shin-Piaw (1932) L'absorption de la lumiere par I'ozone entre 3050 et 3400 A. Compt. rend., 195: 309-311. (1933) L'absorption de la lumiere par I'ozone entre 3050 et 2150 .\. Compt. rend., 190: 910-918. Va.ssy, A., (1941) Sur l'absorption atmosphcrique dans I'ultraviolet. Thesis, Uni- versity of Paris. Vigroux, M. E. (1950) L'absorption de I'ozone dans la region des bandes Huggins. L'influence de la temperature. Compt. rend., 230: 2170 2172. Manuscript received btj the editor Mar. 12, 1951 CHAPTER 4 Technique of Study of Biological Effects of Ultraviolet Radiation Jesse F. Scott* Department of Biologij, Massachusetts Institute of Technology Cambridge, Massachusetts and Massachusetts General Hospital Boston, Massachusetts Robert L. Sinsheimer Department of Physics, Iowa State College Ames, Iowa Introduction. Sources: Classification of light sources — Physical parameters of sources — Choice of a source — Practical aspects. Detectors of ultraviolet radiations: Fluorescent screens — Thermal detectors — Photochemical detectors — Photographic detectors — Photoelec- tric detectors. Methods of spectral isolation: Filters — Dispersing systems. References. INTRODUCTION The technique of the study of the effects of any radiation on Hving systems is divisible, on an operational basis, into (1) the means of produc- ing the radiation, (2) the means of manipulating and estimating the vari- ous parameters of the radiation, and (3) the means of demonstrating and analyzing the effects of the radiation on the biological system under study. This operational outline will be adhered to in a discussion of sources of ultraviolet radiation, detectors of ultraviolet radiation, means of spectral isolation, and accessor}^ optical components. The various means of demonstrating and analyzing the effect of ultraviolet radiation on the biological systems are considered in great detail elsewhere in this volume and will not be taken up here. It must be emphasized that this chapter will not deal with the detailed technique of any particular study but will be concerned with materials for such an investigation. This approach is dictated by the great variety of problems in this field. For example, one investigator may be interested in the abiotic activity of the * A Scholar in Cancer Research of the American Cancer Society. 1H» 1_>() RADIATION lUOLOGY .sunlight as a function of season, altitude, or some other parameter. A second investigator, using the same biological test object, might wish to determine the action sp(M'trum for the lethal effect of ultraviolet radiation in groat detail over a wide range of wave lengths. Each of these workers would draw from the same reservoir of available tools but would combine them in a different fashion for his own particular problem. The function of this chapter is to serve as a guide. SOURCES The source of radiation is of great importance among materials for a study of effects of ultraviolet radiation on biological systems. .V number of excellent chapters have been written containing detailed considera- tions of light sources in general and of tlu; ultraviolet light sources in par- ticular (see, e.g., Ellis d at., 1941; Forsythe, 1937; Harrison etal, 1948; Sawyer and Vincent, 1939; and Roller, 1952). No effort will be made to reproduce these detailed discussions in this chapter but rather to dis- cuss types of light sources and their characteristics, factors to be con- sidered in the choice of a light source, and finally to present a tabular compilation of noncommercial and commercial laboratory light sources which have characteristics making them particularly useful in a study of radiation effects. CLASSIFICATION OF LIGHT SOURCES Sources of radiant energy have been classified in a number of ways among which are: (1) the spectral range of radiation of useful intensity, (2) the method used for exciting the radiation, and (3) the distribution of energy within the spectral range. The ultraviolet spectrum, which is discussed in this volume, covers the range 4000-10 A. This broad range has been subdivided primarily on technical grounds into the near, the far, and the extreme ultraviolet. Sources differ considerably in the fraction of the total energy emitted in each of these ranges. The near ultraviolet extends to 3000 A which is near the short wave-length limit for the sunlight at the earth's surface. The far ultraviolet in biological work extends to about 1900 A. In this vicinity quartz begins to absorb strongly as does atmospheric oxygen (Schneider, 1940; Ladenburg et al., 1932). Because of this increasing atmospheric absorption below 1900 A, the extreme ultraviolet is also known as the vacuum ultraviolet. The lower limit of the extreme ultraviolet is arbitrary. There is considerable overlap in this region between the longer wave lengths of radiation produced by the techniciues employed in the excitation of X radiation and those found by excitation of ultraviolet radiation. Method of Exciting the Radiation. Ultraviolet radiation of any portion of the ultraviolet spectrum may be produced by any one or more of the following means of excitation: (1) incandescent or thermal, (2) spark, ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 121 (3) arc, or (4) discharge. The modern tungsten lamp is an excellent example of a source of the first category. The radiation takes place as a result of heating of the surface of the radiator by some means; in this case it is by the passage of an electric current. For fundamental reasons sources of this category have limited utility, and that only in the near ultraviolet. In most cases the intensity of the emitted radiation falls rapidly between 4000 and 3000 A, approaching zero at the latter figure. Spark sources emit radiation excited by the passage of a high-voltage discharge between electrodes. The material of the electrodes enters the spark stream, contributing the major fraction of the radiation through its excitation by the electrical energy. In the arc also the electrode material evaporates into the arc stream to produce a large portion of the emitting ions in this stream. Arcs are generall}^ low-voltage, high current dis- charges. Radiation is produced in the discharge tube by excitation and ionization of the gas contained at reduced pressure. The radiation is excited by a relatively high potential between electrodes which, them- selves, do not contribute significantly to the ion stream. The distinction between these methods of excitation is not sharp and the reader will find that the foregoing system of classification is not rigidly adhered to in the literature. This is understandable when it is noted that discharge tubes operated at very high current densities may show evidence of evaporation of the electrode material into the ion stream by the appearance of radia- tion characteristic of the electrode material. The heating of the elec- trodes of an arc by ion bombardment ma^^ be sufficient to make the ther- mal radiation from the electrode a significant contribution to the total radiation from the source. A spark operated in air produces radiation which is characteristic of the electrode material, but if operated under water, the radiation produced bears no relation to the electrode material. Spectral Distribution. Of somewhat more practical importance is the classification of light sources with respect to the distribution of the spectral energy emitted. Sources are classified as continuous, line, or band. Continuous spectra generally arise from thermal emittors or from non- quantized energy transitions; line spectra arise from quantized atomic- energy transitions; and band spectra arise from molecular-energy transi- tions or from atomic-energy transitions occurring at high temperatures and pressures. On the basis of the method of production of these various types of spectra it would be expected that many sources would exhibit other than the nominal type of spectrum. Thus, when the hydrogen discharge tube is operated at extremely high current densities in an effort to achieve high brilliance, line spectra are frequently found superimposed upon the typical continuous ultraviolet spectrum of hydrogen. These lines arise from the evaporation and subsequent excitation of electrode material in the ion stream. Mercury discharge tubes, which at low pres- sures and current densities show well-defined line spectra, show increasing 122 J{\1)I ATION 1!1()L(K;Y broadoiiinji; of the lines and the development of iippreciahle contirniou.s l)a('kt>;roun;ht source were mentioned in the precedinj^ discussion of pos- sible classification of sources, namely, the useful spectral range eovercjd by the emitted radiation and the distribution of energy within tliat range (i.e., continuous or discontinuous). A third factor of importance is the amount of radiation emitted at a particular wave length or over a certain band of wave lengths. The amount of radiation may be considered in two ways: first, the total amount of radiation emitted from the whole of the luminous body of the source. Second, the amount of radiation may be considered to be that quantity emitted by a unit area or volume of the source into a unit solid angle. The significance of these two modes of expressing the intensity parameter of a light source will be discussed. Following is a list of the terms which are used to describe the intensity parameter: 1. Radiant Flux: Radiant flux is the rate of flow of radiant energy with respect to time. The quantity is also called "radiance" (P = dU/di) where V is radiant energy and / is time. 2. Radiant Intensity: Radiant intensity is measured hy the energy falling in unit time upon an area subtended by unit solid angle about an}' direction considered and at any distance from the source. This value is also called "steradiance" I ./ = , , ). The solid angle is represented by w. 3. Steradiancy: Steradiancy is the radiant flux per unit solid angle per square centimeter of source w ^ = . , . . ). .4 is the area of the source. It is to be noted that the.se expressions contain no reference to wave length. For studies of the biological effects of radiation it is frequently necessary to know the value of one or more of the preceding intensity expressions w^th respect to wave length. The maimer in which such energy measurements are made will be considered on pages 130 to 142. CHOICE OF .\ SOURCE The choice of a source for a study of the biological elTects of radiation depends on a number of factors which are inherent in the exact nature of the experiment to be conducted. In a general way, decisions must be made as to (1) the size of the area to be irradiated, (2) the range of the ultraviolet spectrum to be covered, (3) the size of the band of energy wdth respect to wave length, (4) the time in which the largest amount of energy is to be delivered to the irradiated area. These factors are to some extent ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 123 independent and yet they are often interdependent in a way which is not always completely understood. This situation can probably be best pre- sented by examples. Assume the practical problem of the sterilization of large volumes of a liquid by ultraviolet radiation. This is to be done by flowing the liquid in a thin film of large area exposed to the total radiation exclusive of the infrared. For practical reasons it is decided to use the mercury-vapor discharge lamp. The amount of ultraviolet energy needed has been established by previous experiments. The mercury discharge lamp has a relatively large fraction of the total ultraviolet output in the biologically potent region around 2600 A but the intrinsic brilliance (steradiancy) is quite low compared with other lamps. This particular problem allows a large area into which the required energy can be delivered. Thus what the mercury discharge tube lacks in steradiancy can be made up by extending the emitting area, which is quite easily accomplished, until the total amount of energy received by a unit volume of liquid during exposure meets the experimental requirement. The broad band of radiation to be used in this experiment is most easily isolated by a filter system which can usually also be extended in area at will (see p. 142). It is quite clear that, in this case, it would have been uneconomical and difficult to have attempted the use of a source which was very bright, i.e., of high steradiancy. Such sources usually attain brilliance by high current densities in small volumes. The total energy output may there- fore be less than a greatly extended source of low steradiancy. Indeed one of the highest rates of total ultraviolet output has been achieved with such a source of low steradiancy. Furthermore, as can be seen in the general references cited, a high current density is often obtained at the expense of simplicity and ease of operation. The steradiancy of a source becomes a matter of importance when for any reason it becomes necessary to use an image of the source for irradia- tion. The whole matter of power transmission through image-forming systems has been considered in detail by Loofbourow (1950) and Blout et al. (1950). The importance of a careful study of these principles may be indicated by the example which follows. Assume that only the cytoplasm of a cell is to be irradiated and a study made of the effects of such irradiation on the nucleus. Such an experiment will require the formation of a reduced image of the source or a portion thereof within the cytoplasm. This would probably be accomplished by use of a reflecting objective as a con- denser. In these experiments also the use of the total emission of a source will be assumed. The following relation, known as Lagrange's Law (see Hardy and Perrin, 1932, p. 43), has been shown to hold by a number of writers including those cited. r_> I RADIATION BIOLOGY i'lie ratio of area in object (source) space A , to that in image space Ao is equal to the ratio of the solid angle of rays forming the image and leaving the source, or expressed in terms of linear dimensions, L.NAi = L,NA,, which says that the product of a linear dimension of the source and the numerical aperture of the rays leaving the source collected by the imaging system is equal to the product of the same dimension of the image of the source and the numerical aperture of the image-forming rays. Let us say that we wish to form an image of the source which is 0.005 mm in diameter with a condenser lens of NA = 0.5. Thus, 0.005 X 0.5 = 0.0025. We assume a field lens for the source which has as high an aperture as the condenser. Then. 0.0025 = LiO.5. Li = 0.005 mm. From this it is clear that only an area of the source of diameter 0.005 mm is contributing to the energy flowing into the cytoplasm. Should a collecting lens of smaller numerical aperture be used, a larger area of the source would contribute, but through a smaller solid angle and, assuming the source to be uniform, the total energy would be the same. In order to increase the amount of energy delivered into the cytoplasm in a given time the product L2NA2 must be increased, the steradiancy of the source must be increased, or both. This fundamental relation has been demonstrated in an example of sim- ple image formation. It has been shown to hold as the limit no matter how many image-forming steps are interposed between the source and the final image used for irradiation. Consider, for example, a possible arrangement of optical components (Fig. 4-1) for the determination of the action spectrum (see p. 384) of the effects of ultraviolet radiation found in the preceding experiment. In this system S is the source focused by a collecting lens on »S'Li, the entrance slit of the monochromator .1/. .S'L,. is the exit slit of the monochromator and / the image formed in the cytoplasm by the condenser. Then, LsNAs = Lsl.NAm = Lsl^NAm = L,NA,. In this case the slits of the monochromator serve as secondary sources. The width of the slits {Lsl,, Lsl,) is determined by the dispersion of the monochromator and the required width of the band of radiation to be iso- lated (p. 148). This may establish a limiting value for the products including these terms because of limitations inherent in available mono- chromators. It is again emphasized that the establishment of a value for any one of the products L^NA^ by experimental re(iuiremeuts or l)y ULTRAVIOLET SPECTROSCOPIC TECHNIQUE V2') instrumental limitations establishes the value for the remaining products and thereby the technical nMjuirements for all other optical components in the system. This treatment has been limited but is intended to indicate the frame- work within which an intelligent choice of a source of radiant energy is made. \ -M ^ / sz, / Fig. 4-1 PRACTICAL ASPECTS The last important group of factors in the choice of a particular source are the practical considerations which include simplicity of construction and operation, ruggedness, useful life, availability, and cost. In the past two decades the commercial availability of many ultraviolet sources together with the power supplies for their operation from domestic mains has increased greatly. Because of the importance of the ready avail- ability of many sources a separate section has been given over to the description of these (see p. 126). To avoid repetition of the descriptive data contained in the general references noted earlier in this section, Table 4-1 has been prepared. This table constitutes a summary of the various types of experimental and laboratory commercial light sources which have been found useful in radiation studies in the ultraviolet range of the spectrum. No effort has been made to summarize all the data on the subject, but rather to give leads to the literature on some of the older sources which have certain useful characteristics and to give more detailed data on the more recent developments. The reader is again advised to consult the following references for a detailed description of the older sources of ultraviolet light: Forsythe, 1937; Ellis ct al., 1941; Harrison et al., 1948; and Roller, 1952. DETECTORS OF ULTRAVIOLET RADIATIONS Ultraviolet radiations may be observed by the use of fluorescent screens, or such radiations may be detected and quantitatively measured by means of the thermal, chemical, or electrical effects they produce. Visual detection is of value in instances where qualitative observations are adequate, as in the alignment of optical systems; thermal and photochem- ical effects may be employed to determine absolute quantities of radia- tion; because of their susceptibility to amplification, some photochemical 1 20 HADIATION BIOLOGY u a Anderson, 1920; For- sythe and Easley. 1931; Sawyer and Becker. 1923 McNicholaa, 1928; Wyneken, 1928; Wrede. 1929 i-» — e - OS ■Sffl Millikan and Sawyer. 1918; Millikan. 1920; Sawyer, 1920; Carter. 1922; Ed- \^n. 1936; Ander- son. 1924 n 4) 0 .2 a; £ E 00 2 « (u » g 2-i'S C O C 0) a ^ 5 i?a3 g •T3 ID .2 3 « •S " « " 5 . = 2 i >. 1 1 -§ s i as a «^ ^*^ 93 2 i a c " _>, - a, •c 4- £ ^ 2 £ £ J! = 3-^0 ■r 0 « ■.- jt- M a; .- •f •" ^ a; =- 'x; 2i T3 .2 ii 0) ^1 a _o 'S _o "a. X V a O a a; C a; CO 03 V o a £ 1 CO 4> O i £ 4) 00 C I. o = o 9 u 7 6.1 IN 2 o «s N -^ ■ ? c u £ a; Oj 1 CD o M, o »; 8"o ■: °- 1 ^ 1 **-■ 6^0 0 O -w -^ e a) o "3 ^ = 5 O > Oj = M CO V o a £ 4) »*- a; u 41 0 m (N IN X2 a S — .2 „ '-5 S 5 o ^ 2 o a — tS 4. X 1 1- <1> 4) 01 ? u £ a 4) c a; ;- a, »^ a.' u~ V 4> I- — O — •| § -^^ f! s c •s g ■^ 2 - — -5: -3 a £ 4) a £ £ 8 ? a £ a CO ■•5 ■^ 5 o CO _ o .= § §2 § GO •So n 3 O 3 _C a o O a 3 0 3 .£ '■*3 a o o a Spectral range (UV)* •a a 3! 03 -a a OS «; Mode of pro- duction of radiation > -a 1-^ - "^ a. fc. c. a; = 3J X I. u T3 a a " 3 o a o a "3 ■♦* ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 127 ff ir ■J. (N J. 1. 4) ., n (N X « m J t— 1 G 3: c < OS S c ■5 :2 "S OS a ;; •5 C oi ii O C8 V c s W CO 4) in 5 cc ■31 03 iJ o .5 rr- 'u (in rT 0) _ ' ' •* 00 n\ t •* Q -3 X B T}< T)< 1- M a> 05 w u ^ 03 w 01 0 QQ O! Oi - .. ^ XI a a «■ 2 D 2: r "^ o <^ S c io ■"■ is OS Oa — 5 - „■ I is 5 I 05 ?. i 2 o = „ < „ 2 0) t^ 5 3 c ^ M --^ 03 -»- oi w 2 S ^ 5 o ^ — T3 C 01 ^ t. S2 0-. e -^ ts: 3 - t3 c ■ o o -o « " « t. 4! r F S .3 - c3 c;. -M tsj 2 = o c •" >:£, o3 C .2^ - ^ S i J .6 :g oS C o O Oi CO » -5 4) O E — P c - '3 i: -^ = > S 2 O S ° 3 j= 03 cc - i' 03 1- C a ^ CO "I re ? SO c <0 <& OQ O O 03 ♦2 08 o ;£ > P = O c — 03 > o o lo t' •a N B CO Bl •^ in d £ (N ? 03 I B CO a; o J, £ ^ Q, B O u T3 ■S i5 ^ " ^ 03 « -a _ 7 ™-. ^ B fi D . _^ B > B =; OJ -5 K j: 03 -d 08 S 13 ■n i: ^ >> B O 03 2. 2 B a: 0) « M ^ o3 bC s -r, -E -3 § 3 E 03 5 T3 3 == ^ j: -^ c « £ :s "^ S 2 -2 2 13 - B © B > ;^3 £ 3 ii E o B 4> O 73 O n. 03 > B O 3 O 3 B O O 5 cc §3 o c8 a: B 03 a, 3; ■d B 03 u 03 t; O) 03 -d B 03 O ■d S3 0! .2 ._- ^ T3 == a e' o a- «- - B S Ph 0 a> 08 X -a 1 (a 0 Ifl u M w » CD = C8 B ~-' 128 o 09 a o as o 02 o % n ■< 4) W 1. 3 - T. g o :3 -^ CO RADIATION HIOLOGY ■a a M •« 2 g OS "* u. ,3 <" 1) t>- O". t~ -o i* C*3 CC ^ o -5 2 2 2k a IN TC3 » ~! O -5 CO C '^ O ♦< .10 3* 00 - Tr* fli . ^^ C3 - e «> C O 2 X ^ CO 1- b£ K - ^ - -0-3 e, -3 -^ Z c u: = a ^ x; «j; t~ -x .V ^ a> ' o •' it *^ - >^ ■■? .i £ a » f J- o (u _^ S. > "c .t^ " ST o •" 03 W 3 •3 O < o O D j: J 3 .5 = -^ j: s ■f^ -3 =3 ^ 2. = M o O ■= c X C c 3 ■* O- — o d <; "^ c o X 5 -o 3 00 03 -0 ^ ^ C. ^ - o £• = o H -3 -fi C C O ^ 2. iJ 1^ •r' a 2 « 2 g S lU -3 -o to T3 |_] Oj C 3 c3 ""'3 o a, -3 ? 2 =^ -a = M ^ 3 5 o. c o 3 — !3 « -3 -^ = c3 'r* «*- o St :5 o i. c o O o -a 0! o -3 ^ i ^-3 c3 u 3 . c o ■" CM tJ 03 O. *" c i: o 5 a " ? OS e 3 o o — C - 0, 5 ■« a -;: « 03 a * 0 OS 0) <«-• i: — ^ ^ XI ^ M a v ■0 1 u *i > 0 d B S nr a> X H -^ 4.' 1? XI •4^ sf 0 c eS .^ > 03 c3 PL, 02 5 5 5 c s i s I 3 O >. M a » a o 3 ^ k. >r ^ - .2 a ^ 3 03 3 K *^ I § o it - "'S .E OS -^ J3 « a o oi o "^ a 03 02 ^ ■a » -5 « »i >^ a o •*- — p-^ 3 - — ■** S g ^1 CQ 3 2 ^ s - . ° . » » O O I, CJ t. 2. — U H -2 J3 M _ 2 2 -^ 4; -< K t: -c > 5 = - 2 I' c -C d t 5 o i :5 I <;• ^. 5 H ^ ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 129 (photographic) effects and the electrical effects make possible the meas- urement of small (quantities of such radiations. FLUORESCENT SCREENS A great variety of substances fluoresce under ultraviolet irradiation (DeMent, 19-15). The properties of certain of these, of especial interest to the lamp industry, have been studied in considerable detail (Kniger, 1948; Fonda and Seitz, 1948). Zinc silicate (willemite) powder can be used to make very satisfactory fluorescent screens over the wave-length region 1000-3000 A (Beese, 1939; Lui, 1945). Maximum excitation is obtained with radiation near 2500 A, for which the quantum efficiency of fluorescence is nearly unity (Fonda, 1939; Schulman, 1946). The emission spectrum of manganese- activated zinc silicate peaks at 5250 A but the emission can be shifted throughout the visible spectrum by addition of beryllium and of various activators (Leverenz and Seitz, 1939). Throughout the region 2200- 3000 A nonabsorbent silicone resins, such as General Electric #9980, may be used as a binder. Magnesium tungstate may also be used as a phos- phor throughout the wave-length region 2200-3000 A with a quantum eflficiency nearly unity (Fonda, 1944; Oszy, 1951). Data on other phos- phors useful on this spectral region are summarized by Thayer and Barnes (1939). For the 3000-4000 A region, sulfide phosphors are quite effective (Klasens et al., 1948; Studer and Larson, 1948; Pringsheim, 1949, pp. 582^., pp. 594^".) with high quantum efficiency about 3650 A (Leverenz and Seitz, 1939). A large number of varicolored pigments are known which respond to radiation in this region (Barnett and Grady, 1949). Special phosphors have been developed to convert ultraviolet radiation at 2537 A to ultraviolet radiation at other wave lengths for particular pur- poses (Froelich, 1947). Thus ultraviolet-sensitive phosphors emitting radiation in the erythemal region (2900-3200 A) (Clapp and Ginther, 1947; Nagy et al., 1950) and the "black-fight" region (ca. 3600 A) (Beegs, 1943; Clapp and Ginther, 1947) have been described. Fluorescent coatings may be employed to extend the usefulness of phototubes to wave-length regions shorter than the transmission limits of their envelopes. Dejardin and Schwegler (1934) used sodium salicylate to extend the effective range of a potassium hydride surface phototube from 3400 to 2200 A. A constant quantum efficiency of fluorescence was obtained over this region. Coatings of salicylate and other materials have been used to extend the sensitivity of photomultiplier tubes to 900 A in the vacuum ultraviolet (Johnson et al., 1951). Again a constant quan- tum efficiency was found with salicylate, independent of the wave length of excitation. It would seem quite feasible to make use of the threshold wave lengths, 130 RADIATION BIOLOGY niul spectral variation of sensitivity of various phosphors, in conjunction with the spectral response of phototubes, to make wave-length-selective detectors (e.g., Luckiesh and Taylor, 1940; Kerr, 1947j. THERMAL DKTECTORS The measurement of radiant energy in absolute units is most com- monly accomplished by the total absorption of such energy in an appro- priate substance, accompanied by a measurement of the increase in tem- perature of the absorber. If the heat capacity of the absorber is known, the energy content of the radiation may then be readily calculated. In practice, the increase in temperature of the absorber may be measured as a resultant change in electrical resistance (bolometer), as an electromotive force (thermocouple), or as a mechanical deformation induced by gas expansion (Golay cell). Radiation detectors based on a measurement of the change in temperature consequent to radiation absorption are called thermal detectors. To absorb totally the radiant energy, the detector must be "black" to all wave lengths represented in the radiation to be measured. It is pos- sible to prepare such "black" surfaces by vacuum deposition, under appropriate circumstances, of such substances as bismuth, zinc, platinum, or gold (Pfund, 1930, 1933, 1937a,b; Harris and McGinnies, 1948). Such surfaces are known to absorb all incident radiation from 0.2 m to beyond 15 ^l. These detectors may be calibrated by the use of a radiation beam of known energy content, thereby avoiding the necessity of a direct measure- ment of their heat capacity. Such a defined beam may be obtained from standard lamps, available from the U.S. Bureau of Standards (Goblentz and Stair, 1933), operated under precisely defined conditions. Bolometer. Measurements of the change in electrical resistance of a detector, consequent to the absorption of radiant energy, are conveniently carried out by using the detecting bolometer as one arm of an initially balanced resistance bridge. The change in resistance leads to unbalance of the bridge with a resultant unbalance voltage which may be amplified to a readily meterable magnitude. A similar bolometer, shielded from the radiation, may be placed in an appropriate arm of the bridge to com- pensate for variations in ambient temperature. If the thermal capacity of the detector is low, it may be used with chopped radiation, with a resultant oscillatory unbalance voltage, which may be amplified by an alternating-current amplifier; in such cases the amplifier may well be .sharply tuned to the chopping frequency to improve the signal-noise ratio. To produce a bolometer detector of high sensitivity, it is desirable to deposit the absorbing coating upon a substance with a high temperature coefficient of resistance. Among the metallic substances, nickel and ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 131 platinum (0.3 and 0.6 per cent per degree Centigrade, respectively) have been used. The semiconductor thermistors have appreciably larger, negative coefficients of resistance (to —5 per cent per degree Centigrade) (Becker et al., 1940) and are widely used (Dodd, 1951). Both thermistor and metallic bolometers hav-e been made with a limiting sensitivity of about 10~* watt, with a response time of a few milliseconds (Baker and Robb, 1943; Jones, 1946; Billings, Barr, and Hyde, 1947; Billings, Hyde, and Barr, 1947; Schlesman and Brockman, 1945), or increasing sensi- tivity to 10"^" watt with response time of a few seconds (Jones, 1949). Thermocouple. By placing the absorbing surface in good thermal con- tact with a bimetallic junction, a change in the temperature of the absorber may be measured as a change in the potential difference across the junction. To minimize the effects of ambient temperature changes on such a thermocouple detector, this potential is usually measured with reference to the potential across a similar junction, in thermal contact with a second absorbing surface, adjacent to the first, but not exposed to the radiation beam. If several such pairs of junctions are connected in series to produce a larger total change in potential difference, the device is called a thermopile. The potential difference thus developed may be amplified in a direct- current amplifier, or it may be mechanically interrupted in a "breaker" amplifier and thereby converted into an oscillatory signal to be amplified in an alternating-current amplifier (Liston et al, 1946). Alternatively, if the absorber has a small heat capacity, the radiation beam may be mechanically chopped to provide a cyclic voltage. This latter method has the additional advantage that it minimizes the effects of slow "drifts" between the potentials of the measuring and reference junctions. The rate of change of potential difference across such a junction with change of temperature is the thermoelectric power of the junction. The highest thermoelectric powers are obtained with junctions between bis- muth-antimony and bismuth-tin alloys (Pfund, 1937a; Hornig and O'Keefe, 1947); with such junctions, powers of 120 mv/°C may be obtained. However, because of the fragility of bismuth alloy junctions, other metallic couples such as constantan-chromel (77 mv/°C) are occa- sionally used (Launer, 1940). With such bimetallic couples and with careful design, it is possible to measure a rise in temperature of the absorber of the order of 10~*°C. Such radiation detectors can provide a sensitivity of 50 mv/mw or greater (Schwarz, 1949; Jones, 1949). For use in photochemical experiments a thermopile with a large receiv- ing area may be desired (Crane and Blacet, 1950). Golay Detector. The Golay radiation detector is characterized by both a high sensitivity and a relatively rapid response time (Zahl and Golay, 1946; Golay, 1947a, b). The radiation is absorbed in a blackened surface 132 UADIATION IJIOLOGY of low Ileal capacity ininuT.scd in an atmosphere of xenon in a .small cham- ber. The rear wall of the chamber is a thin collodion film, .silvered on the outside surface. When exposed to radiation, the absorbed heat is rapidly transferred to the gas, which expands, deforming the collodion wall. This slight deformation is readily detected by an optical system whereby an image of a grid is cau.sed to move across another grid, vary- ing the light received on the face of a photocell, as the membrane is deformed. The detector is intended to be used with chopped radiation. With .such a cell, energies of 5 X 10~" watt may be detected (Golay, 1949). Ultimate Sensitivity of Thermal Detectors. The sensitivity of any ther- mal detector of radiation is ultimately limited by the random fluctuations to be expected on thermodynamic grounds, in the temperature of any body in equilibrium with its environment (Myers, 1946; Jones, 1947). It would be impractical to attempt to measure a temperature change due to incident radiation, which is small compared to these random fluctuations. Alternatively, from a different but equivalent point of view, one may regard the sensitivity as limited by the inevitable statistical fluctuations in the heat radiation emitted and received at all times by any body. Since a thermal detector is sensitive to radiation of all wave lengths, it is sensitive to the thermal radiation emitted by its surroundings. Any attempt to detect a radiation beam of energy less than the fluctuations to be expected in the thermal radiation energy received from (and emitted to) the surroundings (Fellgett, 1949) would be impractical. Such considerations set a lower limit to the sensitivity of thermal detectors expo.sed to a surround at ordinary temperatures, at about 3 X 10~'"^ watt for a detector of area 1 mm- and response time of 1 sec (Jones, 1947). To achieve higher sen.sitivity in any radiation detector, it is necessary to limit the wave-length region to which it is sensitive, in order thereby to reduce the fluctuation in the detector output, due to the fluctuation in incident thermal radiation energy. Thus, for instance, for objects at ordinary laboratory temperatures, the intensity of emitted thermal radiation of wave length less than 6000 A is negligible. There- fore, a photoelectric detector which is .sensitive only to radiations of wave length less than 6000 A is entirely insensitive to the fluctuations in ther- mal radiation. PHOTOCHEMIC.\L DETECTORS If monochromatic radiation is employed or if the spectral energy dis- tribution of the radiation concerned is known, photochemical processes may be conveniently used as a mea.sure of radiation intensity. For absolute determinations, the quantum yield of the photochemical reaction must fir.st have been determined at all wave lengths of interest by calibra- tion again.st a standard thermal detector. The use of a photochemical ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 133 detector, instead of direct use of a thermal detector, is frequently found advantageous for reproducing the geometry of an irradiation experiment, or for reasons of economy and convenience. In ultraviolet, the photochemical decomposition of uranyl oxalate is widely used as an actinometer (Leighton and Forbes, 1930; Forbes and Heidt, 1934; Bowen, 1946; Launer, 1949). The quantum yield for this decomposition has been carefully measured at a series of wave lengths from 208 to 434 m/x (Leighton and Forbes, 1930; Brackett and Forbes, 1933). The initial oxalate content of the stock solution, and the residual oxalate after irradiation, are measured by permanganate titration and the decomposition determined by difference. From the quantity of oxalate decomposed and the quantum yield, the number of photons absorbed can be calculated, and from this figure, the incident intensity determined, knowing the absorption of the solution and making appropriate correc- tions for such factors as reflections at cell windows. It is convenient to use a solution of sufficient concentration to absorb all the incident radia- tion; adequate stirring must be provided to equalize the irradiation of all volumes of the solution. The photochemical reaction has been shown to have nearly unity temperature coefficient. A rather unusual photochemical transformation of certain triphenyl- methane dyes (Lifschitz, 1919; Lifschitz and Joffe, 1921; Harris et al., 1935) from a colorless to colored form has been used as a means of com- paring ultraviolet intensities in the spectral region 2400-3200 A (Calvert and Rechen, 1952; Harris and Kaminsky, 1935; Weyde, 1930; Weyde et al., 1930; Miyake, 1949). The quantum yield for the transformation has been shown to be unity over this spectral region and is independent of temperature (Weyde and Frankenburger, 1931; Calvert and Rechen, 1952). This actinometer can be used at considerably lower light inten- sities than can the uranyl oxalate. Under appropriate conditions, the transformation is quantitatively autoreversible, permitting the same solution to be used repeatedly (Weyde, 1930; Weyde et al., 1932). PHOTOGRAPHIC DETECTORS The photographic plate or film has a number of advantages as a radia- tion detector. It is a sensitive detector and can integrate the radiation received. Furthermore, it is unique ^ among ultraviolet radiation detec- tors in that it is a two-dimensional detector, thus permitting an entire spectrum or microscopic image to be recorded in one exposure. While it cannot be regarded as a precision instrument for the measurement of radiation, with appropriate auxiliary techniques, accuracies of the order of ± 3 per cent can be obtained. Since individual plates or films vary unavoidably in sensitivity and in contrast, for quantitative work it is necessary to standardize each plate ^ With the potential exception of the image orthicon tube. 134 RADIATION mOLOGY (for a general description of the properties of photographic materials, see James and Higgins, 1948). Furthermore, since the sensitivity and contrast vary with wave length, the plate must be calibrated at all wave lengths for which measurement is desired. (For data on the ultraviolet characteristics of spectroscopic plates, see Harrison, 1925a; Jones and Sandvik, 1920; Johnson and Hancock, 1933; Amstein, 1944. Fraser, 1950, has obtained data on the ultraviolet characteristics of several types of motion picture film.) For homochromatic photometry, in which the only desire is to compare radiation intensities at a given wave length, the plate or film may be cali- brated by means of any device which produces a scale of plate blackening versus source intensity on which the desired unknown intensities may be (a) (b) Fig. 4-2. Rotating sectors: (a) stepped, (b) continuously variable. (Reproduction from Practical Spectroscopy, by G. Harrison, R. Lord, and J. R. Loofbourow, Prentice- Hall, Inc., 1948.) read (Harrison, 1934b). Calibrated step wedges or rotating sectors are commonly used for this purpose. Thin films of platinum (Merton, 1924; O'Brien and Russel, 1934; Uber, 1939) or Chromel A evaporated onto quartz (Banning, 1947a) are frequently used for the former, since they are nearly constant in optical density over a wide range of wave lengths, but for accurate work they must be calibrated. The rotating sector may either be stepped or it may vary continuously in exposure time (Fig. 4-2). It is truly a "neutral-density" device. For heterochromatic photometry, in which it is desired to compare radiation intensities at different wave lengths, the relative sensitivity of the plate as a function of wave length must be determined. This is most easily done with a source of previously determined spectral energy distri- bution, preferably one with a spectral continuum, such as the hydrogen discharge tube. Ordinary photographic plates are sensitive to ultraviolet radiations to wave lengths as short as 2300 A. The faster of these plates reciuire a net exposure to 0.1 1.0 ergs/cm- to produce a plate density of 0.5-1.0. Below 2300 A, the absorption of the gelatin matrix for the silver halide grains prevents the radiation from penetrating beyond the upp«n- layer of ULTHAVIOLET SPECTROSCOPIC TECHNIQUE 135 emulsion and thereby greatly reduces the plate sensitivity. For work at shorter wave lengths, very thin emulsions heavily laden with silver halide grains may be used, such as the Ilford "Q" plates or the Eastman Kodak SWR film (Schoen and Hodge, 1950). As an alternative, the surface of the film may be coated with material that will fluoresce under the short- wave-length radiation, so that the exposure is actually produced by the fluorescent radiation (Harrison, 1925b). Such thin-emulsion or fluores- cent-coated plates may be used far into the vacuum ultraviolet (Harrison and Leighton, 1930). By the use of a fluorescent coating with constant quantum yield of fluorescence, independent of exciting wave length, problems of heterochromatic photometry may be greatly simplified. Such coatings also eliminate the variation of contrast with wave length (Harrison and Leighton, 1931). For photomicrography, however, the use of fluorescent coatings generally leads to some loss of plate resolution. PHOTOELECTRIC DETECTORS Photoelectric detectors useful in the ultraviolet are of two general types: the photovoltaic or barrier-layer cell, and the photoemissive detector. The photovoltaic cells, which do not require an external power source, are convenient and useful in instances where relatively large amounts of radiant energy are available. The photoemissive detectors require more elaborate accessory equipment but are far more sensitive and are effective in a wide variety of applications. Photovoltaic Detectors. Upon illumination of a photovoltaic cell, a potential difference appears across a semiconductor (usually iron sele- nide), which potential can be used to drive a current through an external circuit (Lange, 1938; Zworykin and Ramberg, 1949, Chap. 11). Elec- trically, the photovoltaic cell acts as a source of current which is shunted by an internal resistance and capacitance. The shunting internal resist- ance is not constant, but decreases with increasing illumination and with increasing current flow. Although the photocurrent generated within the semiconductor is, at moderate light levels, linearly dependent on light intensity, because of the internal resistance and its variation with light intensity, the external current is a linear function of light intensity only if very low external resistance is employed (Wood, 1934). As a conse- quence, the output of a barrier-layer cell as a function of fight intensity, with various external resistances, is as shown in Fig. 4-3. Electronic circuits have been developed to permit the use of larger external resistance, if desired for purposes of amplification, without introducing appreciable nonlinearity. Such circuits (Rittner, 1947) employ negative feedback to effectively reduce the apparent resistance external to the photocell. The internal capacitance of the photovoltaic cell also acts to shunt the external resistance if an oscillatory photocurrent is produced by a modu- 130 RADIATION BIOLOGY 700 600 500 400 300 200 100 lated light beam. This capacitance siuiiit Hmits the useful range of modulation frcciuencies to below 10, ()()() cycles/sec. Although all commercially available barrier-layer cells have peak sensi- tivity in the \isible spectral region, they are available in quartz envelopes which permit apprecial)le response to wave lengths as short as 270 m/i. These cells are somewhat temperature sensitive, and may display an initial "fatigue" for 15-20 min on exposure to radiation (Lange, 1938; Barbrow, 1940). For use in appro- priate applications, matched pairs of cells are available. Photoemissive Detectors. Because of their high sensitivity, linearity and speed of response, and convenience of operation, photoemissive detectors have become the most widely used means for quantitative measurement of ultraviolet radiation. The elec- tric currents deri\^ed from these de- vices are easily amplified and may then be used to operate meters or au}^ of various kinds of automatic recording devices. With modern techniques (Engstrom, 1947a; Som- mer and Turk, 1950) it is possible to reduce the extraneous sources of elec- trical fluctuation, such as the thermal emission of electrons from the photo- cathode and the thermal motion of electrons in the amplifier input cir- cuit, to levels sufficiently low for the principal limitation on the precision of measurement of w'eak beams of radiation to arise from the (juantized nature of the radiation itself and from the concomitant statistical fluctua- tions in radiation intensity (Johnson and Llewellyn, 1934). The operation of photoemissive cells depends on the release of electrons from a photosensitive surface on incidence of quanta of adequate energy. Since the energy of a quantum is proportional to the frequency of the radiation, there is for any surface a minimum value of frequency — or a maximum value of wave length — below (or above) which the quanta will not have sufficient energy to release electrons. This maximum wave length is known as the threshold wave length for the photosurface in ciuestion. For many metallic svafaces, the threshold wave length lies in the ultra- violet. This circumstance has made possible the design of photocells / / C/l / f ^ < f/ y "' ^/ Lv / _y y 7 o.-^ y y ^ y y ^ oo^ .^ -^■^522- ^ ^ \600_ --' / 3000 — = / ■ — " D.OOO A l( ^~ 0 50 100 150 200 250 300 350 ILLUMINATION, ft-C Fig. 4-;J. Influence of external circuit resistance upon current output of photovoltaic cell. Photocurrent char- acteristics with several external resist- ances; rectangular cell Model lOA; active area 0.70 sq in. Figures on curves, ohms. {Bradley Laboratories, Inc.) ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 137 which are sensitive only within well-defined spectral regions; the upper wave-length limit is defined by the quantum threshold and the lower by the absorption properties of the photocell envelope. Thus a cell with a cadmium-magnesium surface and a Corex-D window has a spectral response curve closely paralleling the action spectrum for erythema production, and is of considerable utility in the measurement of the erythemal effectiveness of various sources (KoUer and Taylor, 1935; Kerr, 1947; Taylor, 1944). Other cells with magnesium (Coblentz and Cashman, 1940), titanium (Coblentz and Stair, 1935; Kuper et al., 1941), or uranium (Rentschler, 1930) surfaces have found use for the measure- ment of the intensity of extreme ultraviolet radiation from the sun. Other metals such as zirconium, thorium, tantalum, platinum, and alloys such as beryllium-copper have threshold wave lengths at various places in the ultraviolet spectrum and might be used to provide photocells with specific spectral response characteristics (Rentschler et al., 1932; Glover, 1941; Andrews, 1945; Morrish et al., 1950; Piore et al., 1951). The precise threshold wave length and spectral response curves of these metallic surfaces depend considerably on the particular method of prepa- ration (Dejardin, 1933). Most metallic surfaces, however, have relatively low quantum effi- ciency, emitting one electron per lO'^-lO^ incident quanta (Sommer, 1947). Hence, most modern photocells are made with composite surfaces, such as the cesium-antimony surface which has a quantum yield of approxi- mately 0.1-0.3 at the wave length of maximum response (400 m/x) (Janes and Glover, 1941; Sommer, 1947; Morton, 1949; Zworykin and Ramberg, 1949, Chaps. 5 and 6) and maintain a high yield well into the ultraviolet (Fig. 4-4). Glass-jacketed photocells begin to decline in sensitivity at wave lengths less than 3500 A. Commercially available ultraviolet-sensitive photo- cells have envelopes of Corex-D, Corning 9741, or Vycor glass. The Vycor glass provides good transmission to approximately 210 m/x, but begins to absorb appreciably at shorter wave lengths (Nordberg, 1947). Some response may be obtained to wave lengths as short as 160 m^ (Dunkelman and Lock, 1951), owing in part to fluorescence of the glass. Special quartz-jacketed photocells have high sensitivity to 175 mu and will respond to wave lengths as short as 155 m^t. In the vacuum-type photoemissive cell, the current developed is directly proportional to radiation intensity over several decades of intensity range. The current developed, obtained in a typical photoemissive cell as a func- tion of anode voltage, is shown for several radiation intensities in Fig. 4-5. Evidently at any anode voltage greater than 25 volts, the full photocur- rent is collected, and the current is thus substantially independent of anode voltage. The total current which may be drawn from a photo- surface without damage is limited to values of 5-10 ^a/cm-. 138 100 2000 RADIATION UIOLOGY 4000 6000 WAVE LENGTH, A (C) 8000 Fig. 4-4. Spectral response characteristics for three types of photosurface: (a) type 1P22, S-8 response; (6) types 931-A and 1P21, S-4 response; (c) type 1P28, S-5 response. (Engsirom, 1947a; Journal Optical Society of America.) 250 150 AI\JO0E, volts Fig. 4-5. Current-voltage characteristic of RCA 935 (ultraviolet sensitive) (Radio Corporation of America.) photocell. ULTRAVIOLET SPECTKOSCOPIC TECHNIQUE 139 At low levels of radiation intensity, the photociirrent generated will be small. This current may be amplified external to the cell by conven- tional vacuum tube circuits (Zworykin and Ramberg, 1949, Chaps. 12-14) or it may be amplified within the cell, either by gas multiplication or by the use of secondary emission, as in the photomuitiplier tubes. The 30 50 70 10,000 100 200 300 500 700 1000 2000 4000 ACCELERATING VOLTAGE OF PRIMARY ELECTRONS Fig. 4-6. Secondary-emission characteristics of typical photosurface materials. {Reproduction from Photoelectricity and Its Application, by V. K. Zioorykin and E. G. Romberg, John Wiley d- Sons, Inc., 1949.) advent of photomuitiplier tubes has largely supplanted the use of gas- filled tubes. The direct or amplified photocurrent may be measured with a galvanometer or ammeter, may be recorded, may be integrated in dis- crete quantities and counted (Douglas, 1947; Launer, 1949), or may be used to operate such devices as relays and motors. Photomuitiplier Tubes. In the photomuitiplier tubes, the primary current from the photosurface is multiplied by a factor which may be as large as 10^ by repeated use of multiplication at secondary emis- sion surfaces. Many surfaces, in- cluding those commonly used as photosurfaces, will, when struck by an electron of appropriate energy, emit several electrons. The num- ber given off per primary electron depends on the surface and the voltage applied to the primary elec- tron (Fig. 4-6) (Zworykin et at., 1936; Morton, 1949). In the focused photomuitiplier tubes (Rajchman and Snyder, 1940), the primary photocurrent is focused by an electrostatic field onto such a surface, called a dynode. This process is repeated nine or ten times until the vastly amplified current from the last secondary emitting surface is collected on an anode (Fig. 4-7). FOCUSING GRILL MICA SHIELD' Fig. 4-7. Construction of nine-dynode focused photomuitiplier tube. 0, photo- cathode; 10, anode; 1-9, dynodes. {Engstrom, 1947a; Journal Optical Society of America.) 10 RADIATION HIOLOGY An alternative design, such as is used in the " Venetian bhnd" photo- multiplier tubes (Sommer and Turk, 1950), does not attempt to focus the electrons from each dynode upon the next, but merely uses an accelerating field to draw the majority of secondary electrons (70-8o per cent) to the succeeding dynode (Fig. -4-8). Thus a conventional photomultiplier tube consists of a photocathode, a series of 9-11 secondary emission dynodes, the first of which is main- tained at a potential 75-150 volts above that of the photocathode while each succeeding dynode is elevated another 75-150 volts in potential in sequence, and a final anode which is maintained 50-100 volts above the potential of the last dynode. With a multiplication of 3-5 per dynode, the over-all amplification of a 3- 2- 0-1 I —v/yy/'/'T- — KSNWssr- —ryyyyy'yr- — kWVCsV^— —vyyyyyyr- — K\\\x\si— —vyyyyy^~ — KX\\\V1— —Y///y/r- — KVX\XV\I — > i?| to 27„ or 0 ^ umM 9 dynode tube can range from 3* to . 20,000 to 2,000,000. Because the current capacity of the last dynode or anode is limited, there is, at normal gain, a maximum current which may be drawn from the photosurface, and hence a maximum illumination to which it should be exposed. This limit, which will be less than 1 /xw for a 1P28 photomultiplier tube operated at a cur- rent amplification of 10^, may be raised if the voltage applied per stage is reduced. The response of photomultiplier tubes is a linear function of light intensity over many decades. Fatigue is inappreciable at low light Jevels. Because of the variations in secondary emission with dynode voltage, the voltage supply for the photomultiplier tube potentials must be held stable to an order of magnitude better than the stability desired in the output current. Batteries may be used, or regulated elec- tronic supplies have been described (Ply male and Hansen, 1950; Higinbotham, 1951; Hill, 1945; Mautner, 1947). The over-all amplification of the photomultiplier is very closely a logarithmic function of the voltage applied per dynode, over several decades of gain (Fig. 4-9). As a consequence of this circumstance, it can be shown that, for varying levels of illumination, the voltage per dynode necessary to maintain a constant output current is proportional to the logarithm of the reciprocal of the intensity of the illumination. This property may be used in the design of circuits intended to measure absorp- tion directly in terms of optical density (Sweet, 1946). The time resolution of a photomultiplier tube is limited only by the Fifi. 4-8. Design of "Vene- tian blind" type photomul- tiplier tube: T, photosensi- tive surface; D, dynodes; E, collecting anode. (Sommer and Turk, 1950; Journal of Scientific Instruments.) ULTRAVIOLET SPECTUOSCOPIC TECHNIQUE 141 variations in time of transit of electrons from photosurface to anode, which are of the order of 6 X 10~^ sec (Morton, 1949). As a consequence, the photomultiplier tube will faithfully respond to very brief pulses of light, as short as 10~^ sec. The amplified photocurrent from the photomultiplier tube easily over- whelms the random fluctuations in electric current arising in the external circuit, as a result of thermal agitation, so that the only limitations on the sensitivity of a photomultiplier detector are those arising from the ran- dom fluctuation of the "dark current" which is actually the thermal emission of electrons from the photosurface, and from those inherent in 1.000,000 125 150 75 100 VOLTS PER STAGE Fig. 4-9. Amplification characteristic of focused type photomultiplier. (Ent/strom, 1947a; Journal Optical Society of America.) the statistical nature of the radiation intensity itself. The thermal- emission dark current and its corresponding fluctuations may be reduced by choice of a photocell with a small photosensitive surface, or it may be minimized by refrigeration of the photocell, without appreciably influencing the sensitivity to radiation (Engstrom, 1947a, b). It should be recognized that photoelectric detectors vary considerably from tube to tube (of the same design) with regard to sensitivity, to variation of sensitivity with wave length, and to the dark current. Because of these variations, if it is desired to use photoelectric detectors for the comparison of two beams of radiation, one of two courses is neces- sary: (1) some artifice whereby one detector may be used must be employed, or (2) if two detectors are used, either matched tubes must be found, or some means of compensating for their differences (which may be expected to be reasonably stable) must be provided. A single detector may be used, if it is alternately exposed to the two beam.s in time, or if the electrical signal arising from each beam can be distijiguished by virtue of a frequency or phase modulation (Wright and Herscher, 1947; Savit- zky and Halford, 1950; Wyckoff, 1952). 142 RADIATION BIOLOGY The use of modulated radiation, giving rise to an oscillatory current, permits the use of alternating-current amplifiers and thus simplifies the associated electronic circuitry. Alternatively, a steady photocurrent may be converted to an oscillatory current, either by magnetic modula- tion (Kalmus and Striker, 1948) of the photocurrent, or by mechanical interruption (Liston et al., 1946; Lash, 1949). Image Orthicon. The application of television techniques (Zworykin and Ramberg, 1949, Chaps. 16, 17) to ultraviolet spectroscopy through the medium of ultraviolet-sensitive image orthicon tubes has significant potentialities. The orthicon is in effect a two-dimensional photoelectric detector which permits the application of photoelectric techniciues to problems that previously could be adecjuately approached only by photo- graphic means. The use of the image orthicon by Pai-part and Flory for the \isualization of ultraviolet microscope images (Purpart, 1950; Flory, 19")1). and the study of source spectral characteristics (Benn el al., 1949; Agiiew et al., 1949) may be cited as examples of the potential applications. METHODS OF SPECTRAL ISOLATION One of the important data in any problem in radiation biology is the variation of the subject under investigation (absorption, fluorescence, photobiological or photochemical effect) with the wave length of the radiation concerned. To obtain these data, spectrally defined beams of radiation must be available. A wide variety of devices have been devel- oped to provide such spectrally defined beams ; these devices differ in basic principles and in range of application and, in general, may have specific advantages or disadvantages for a particular application. For the iso- lation of well-separated spectrum lines from a discontinuous source, much simpler technicjues can be employed than are necessary to isolate narrow spectral band widths from a source of spectral continuum. For some pur- poses, high intensity or large total energy of radiation are more important than purity of wave length. For others, flexibility and the possibility of easy, rapid change of wave length are important. The optimum means of spectral isolation can be chosen only after the research objectives are clearly defined. The various means employed for spectral isolation may be somewhat arbitrarily grouped into two classes: filters, which by one means or another block or prevent transmission of all save the selected band of wave lengths, and dispersing systems, which transmit all wave lengths, but disperse them in space so that })arti('ulai' regions may be selected. FILTERS Absorption Filters. The simplest filters are absorption filters, liy virtue of the al)sorption spectra of their components these filters absorb, ULTRAVIOLET SPECTROSCOPIC TECHNIQUE TaBI.K 4-2. Ul.TRAVlOLKT FILTERS M.I 11(1 pass, transmission Reference iiiyu: 143 Wave-length logion transiiiitti'i 190-290 230-265 230-330 230-420 240-280: >350 245-275 245-290; >340 255-290 270-325 290-340 295-330 300-340 300-400 320-360 320-390 340-390 For specific spectral lines, mn: llg 254 Hg 254 + 265 Hg 280 Hg 313 Hg 334 Hg 366 Cd326 Band pass, absorption Wave-length region absorbed, niju: 280-390 (CI,) 350-540 (Br,) 290-360 (CSo) 340-800 Long-wave pass Approximate cnt-off wave length, niju: 190-200 210-230 220-260 230-250 245-260 260-280 260-310 2t)5-275 270-280 Heidt, 1939 Backstrom, 1940 Backstrom, 1940: Mazza, 11)40 Corning, 1948 Kasha, 1948 Kasha, 1948 Kasha, 1948 Kasha, 1948 Dorcas and Forbes, 1927 Kasha, 1948 Backstrom, 1940 Kasha, 1948 Corning, 1948: Schott-Jena, 1952 Kasha, 1948 Corning, 1948 Kasha, 1948 West, 1946 Bowen, 1946 Backstrom, 1940: Bi'icher and Kaspers, 1946 Backstrom, 1940; Bowen, 1946; Biicher and Kaspers, 1946; Hunt and Davis, 1947; West, 1946 Bowen, 1946; Biicher and Kaspers, 1946 Bowen, 1946; Biicher and Kaspers, 1946; Corning, 1948; West, 1946 Bowen, 1946 Gibson and Bayliss, 1933; von Halban and Siedentopf, 1922 Acton et al., 1936 Bowen, 1946 Backstrom, 1940 Haas, 1935 Maclean et al., 1945 Corning, 1948 Bass, 1948; McLaren and Pearson, 1949 Bass, 1948 Kasha, 1948 Corning, 1948 Bass, 1948; Maclean et al., 1945 Bass, 1948; Bowen, 1940; Maclean et al., 1945 144 RADIATION moLOGY Tabke 4-2. Ui/rRAVioLET Filters. — (Continued) l-oiiH-wavc |>ass Reference 280-300 Bass, 1948; Itowcn, lltKi; Maclean el al., 1045 280-:i20 Corning, 1U18; Scliult-.lena, 1952 290-310 Polaroid, 1951 300-310 Kasha, 1948; Ley ami Wingdien, 1934; Saiiiidcrs, 1928 300-330 Corning, 1948; 8chott-Jena, 1952 310-330 Kasha, 1948; Polaroid, 1951; Schott-Jena, 1952 315-365 Maclean et al, 1945; Schott-.Iena, 1952 340-300 Kaslui, 1948; Schott-Jena, li)52 340-380 Corning, 1948; Schott-Jena, 1952 ;^50-380 Polaroid, 1951 360-400 Bowen, 1946; Corning, 1948; Schott-Jena, 1952 365-430 Corning, 1948; Schott-Jena, 1952 380-410 Eisenlirand and von Hallian, 1930; Pola- roid, 1951 420 Bowen, 1946 more or less strongly, all wave lengths other than those of the selected region. Such filters are simple to use, may be made in large dimensions, and place no limitations on the angular spread of the radiation to be transmitted. On the other hand, it is difficult to obtain absorption filters which can provide both a narrow transmission band and high transmis- sion within the band; further the design of an absorption filter for any particular spectral region is a wholly empirical enterprise. Absorption filters may be made of glass, of liquid cells, or of gas-filled cells (chlorine and bromine) or combinations of these. In general, a filter need not (and will not) transmit only a narrow band of wave lengths out of the entire electromagnetic spectrum. Consideration must be given to the characteristics of the radiation source and the radiation detector, or biological subject, to be employed. Transmission bands in far-removed wave-length regions, such as the infrared, might well be of no consequence in particular investigations. References to band-pass and long-wave-length-pass absorption filters for various regions of the ultra\'iolet are summarized in Table 4-2. Appropriate combinations of these may be employed for isolation of par- ticular spectral lines from various sources. If the absorption of the ultraviolet radiation involves a photochemical decomposition of some component of the filter, the filter may have to be renewed frequently. This is particularly likely with liciuid filters includ- ing organic components. Such decompo.sition may sometimes be mini- mized by placing the sensitive component farthest from the source in the sequence of filter elements. ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 145 Interference Filter. The action of an interference filter is based on the cancellation of coherent light waves when they are added together in phase opposition. In its action, each ray of the light beam is divided into a large number of weaker rays, with regular shifts in phase between adjacent rays, by the use of multiple reflections between lightly silvered surfaces appropriately spaced by dielectric, these surfaces allowing a slight transmission of energy at each contact (Fig. 4-10). When these rays are recombined by a lens, the resultant intensity at the focus of the lens will depend on the phase difference between adjacent rays, being maximal when all rays are in phase, and minimal when adjacent rays are exactly out of phase. If a stop is then placed about the focus of the lens, Fig. 4-10. Figure illustrating the splitting and multiple reflection of light rays origi- nating from Pi by the silvered surfaces Ei and £"2, to provide a monochromatic image of Pi at P-i. {Reproduction from. Fundamentals of Optics, 2d ed., by F. A. Jenkins and H. E. White, McGraw-Hill Book Company, Inc., 1950.) only those wave lengths for which adjacent rays are exactly or nearly in phase will be transmitted in appreciable intensity. By controlling the thickness of dielectric between the reflecting surfaces, the variation of phase difference between adjacent rays with wave length can be controlled and thus the wave length or wave lengths of maximum transmission selected. The simple interference filter can be regarded essentially as a crude Fabry-Perot etalon (Jenkins and White^ 1950, Chap. 14). Thus a simple interference filter consists of two lightly silvered reflect- ing surfaces, spaced by an appropriate thickness of dielectric (frequently magnesium fluoride). The spectral selectivity of such a filter is depend- ent extrinsically on the angular aperture of the radiation with which it is employed (increasing with decreasing aperture), and intrinsically on the reflectivity of the reflecting surfaces and the number of wave length paths in the dielectric spacer (Mooney, 1946; Hadley and Dennison, 1947, 1948). Under favorable optical conditions, such filters can provide a peak transmission of about 35 per cent with a band width of about 100 A (at half-maximum transmission) when peaked for various wave lengths in the visible region. The transmission of interference filters does not, however, drop to zero outside the transmission band (or bands) but to a minimum of about 1 per cent. The wave length of peak transmission 146 RADIATION HIOLOGY is specified on the assumption that the filter will be used with radiation at normal incidence (Buc and Stearns, 1950). More complex "multilayer" interference filters (Banning, 1947b; Bolster, 1949, 1952), which rely on the cancellation of rays multiply reflected between sandwiches of dielectric layers of appropriate thickness aiul refractive index (replacinji; the silvered surfaces), can provide higher transmission (70 80 per cent) and narrower band widths (50-00 A) at half-maximum transmission) . The simple interference filters cannot be satisfactorily made for wave lengths less than 3600 A because of the decline in the reflectivity of silver. Aluminum reflectors have not proved satisfactory. It would seem possi- ble to extend the range of the multiple layer dielectric filters farther into the ultraviolet if dielectrics combining proper refractive indices and ultra- violet transparency can be found. Christiansen Filters. If rough chips of transparent dielectric are sus- pended in a cell containing a transparent liquid, the resultant mass will be highly scattering and hence of low transmission, except at or near the wave length at which the refractive index of the liquid matches that of the dielectric. When employed with an appropriate optical system, such a cell constitutes a Christiansen filter (Christiansen, 1884). Such filters can be made with large cross section. Their spectral selec- tivity depends inversely on the angular divergence of the radiation pass- ing through them, directly on the difference in the slopes of the refractive index versus wave-length curves of the liquid and solid at their point of intersection (Raman, 1949) (the curve for the liquid always has the greater slope), directly on the thickness of the cell, and also on the size of the dielectric chips, for which there appears to be an optimum (Denmark and Cady, 1935). As the refractive indices of liquid and dielectric gen- erally vary at different rates with temperature, the wave length of peak transmission of Christiansen filters is strongly temperature dependent. Appropriate dielectric and liquid mixtures have been described for the visible region by MacAlister (1935), for the 3100 4000 A region by Kohn and von Fragstein (1932), and for the 2300-3100 A region by Sinsheimer and Loofbourow (1947). A filter for the mercury 2537 A line has been described by Minkoff and Gaydon (194G); von Fragstein (1938) mentions filters centered at 2610 and 2450 A. It should be emphasized that the transmission and spectral selectivity of these filters depend strongly on the optical system in which they are employed (Weigert and Staude, 1927; von Fragstein, 1938). The transmission of Christiansen filters does not decline to zero outside the transmission band but to a minimum d(;pendent on the opti(!al system employed. Focal Isolation Fillers. The focal length of a simple uncorrected lens depends on its refractive index and hence on the wave length of the radia- tion. At the focal plane of any gixcii wave length, radiation of othei' ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 147 wave lengths is necessarily not in focus and is spread out more or less dif- fusely. If the image of a small source is sharply masked at the focal plane of the wave length desired, this wave lenj^th will be favored in the radiation transmitted. If this process is repeated several times (the same lens may be used in autocollimating schemes), quite narrow spectral band widths may be obtained. Such a device is known as a focal isolation filter. The selectivity of such a filter will depend on the dispersive power of the lens material, on the number of lenses employed, and on the size of source and angular aperture of transmitted radiation. Fluorite or quartz lenses have been employed in this fashion to isolate spectrum lines in the vacuum ultraviolet (Forbes et al., 1934; Duncan, 1940). Miscellaneous Filters. A simple ultraviolet filter which can be used in well-collimated light to reject all wave lengths greater than an arbitrarily chosen boundary, has been described by Regener (1936). A thin film of paraffin oil is sandwiched between the long sides of two 90° quartz prisms. Since the refractive index of the paraffin oil is less than that of the quartz, total internal reflection can occur at the first oil-quartz interface; owing to the greater refractive dispersion of the paraffin oil, there will be, for any angle between the entrant beam and the oil-quartz interface, some critical wave length above w^hich all wave lengths will undergo internal reflection. This critical wave length can be varied by rotation of the interface. Transmission is not complete at wave lengths imme- diately less than the critical w^ave length, but increases rapidly with decreasing wave lengths. Various types of light filters have found employment for special pur- poses in the visible portion of the spectrum, and could undoubtedly be adapted for use in the ultraviolet, but for one reason or another have not been so used. Among these might be mentioned the polarization inter- ference filter and the rotary dispersion filter. The former is based on the interference between two orthogonal com- ponents of a beam of polarized light after passage through a birefringent crystal; the retardation (in wave lengths) of the one component relative to the other will depend on the thickness and birefringence of the crystal and on the actual wave length, and hence varies wdth wave length, pro- ducing maxima and minima of transmission throughout the spectrum, as the interfering Avaves combine constructively or destructively (Billings, 1947; Evans, 1949a, b). Filters of this type, using cascaded birefringent elements of appropriate sequence of thickness, have been made with a band width of 1 A at half-maximum transmission (Billings et at., 1951). Such filters are designed for use at a particular w^ave length. Rotary dispersion filters rely for their action on the variation in rotary power of an opticall}^ active material, such as quartz, with wave length. A piece of such material, placed between similarly oriented polarizing 148 RADIATION niOLOGY elements, will transniil coinplotely only those wave lengths for which the total rotation ;lit , 1939). Minor optics may also be employed to solve the problem of achromatization. Multiple prism cascades may be used etTectively io obtain greater prism l)nse and hence greater resohing power. Double monochromators, employing essentially two single monociiromators in tandem, may be used tor greater purity of radiation and freedom from scattered radiation at the COLLIMATING AND TELESCOPE LENS REFLECTING PRISM FOCAL PLANE REFLECTING SURFACE Fig. 4-11. Littrow moiiiiiiiig for prism iiistnunciit. {Reproduction from Practical Spectroscopy, by G. Harrison, li. Lord, and J. li. Loofbourow, Prentice-Hall, Inc., 1948.) (a) Fig. 4-12. Constant dovialion prism instrum('nt.s usiii^: (a) Pcllin-Broca prism; (h) \\ adsworth mounting for prism. {Reproduction from Practical^ Spectroscopy, by G. Harrison, R. Lord, and J . R. Loofbourow, Prentice- Hall, Inc., 1948.) expense of energy transmission (Sawyer, 1951; Harrison ef al., 1948). Cascaded Pellin-Broca prisms may also be employed in such a way as to minimize stray radiation (Benford, 1936). By reflecting the radiation back through the prism, as in the Littrow moimting (Fig. 4-11), twice the dispersion and resolving power may be obtained. The collimating lens then may serve also as telescope lens. With this arrangement the exit slit is spatially near to the entrance slit, and scattered radiation may be a problem. Constant deviation monochromators may be made with the Pellin- Broca prism (Fig. 4-12a), with the Wadsworth mounting for the ordinary ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 151 prism (Fig. 4-126), with the Young and ThoUon spht-prism arrangemont (Kurtz, 1926), or with an adroit mirror arrangement recently described by Makishima et al. (1951). In these instruments, the entrance and exit slits (and hence the source and monochromatic image) are maintained constant in position while the dispersing element(s) is rotated to vary the emergent wave length. Grating Instruments. If a wave front of radiation is broken into a number of narrow, parallel zones evenly spaced by appropriate distanc-es, the waves propagating from each zone will interfere with those from all other zones so as to produce a diffraction pattern. For any given wave length, there will be some direction or directions in which the waves from each zone will all be in phase to pro- duce a maximum of intensity. In another direction, waves of another wave length will be in phase to yield a maximum of intensity, whereas the waves of the first wave length will largely cancel each other. A device to thus disrupt a wave front is known as a diffraction grating. By thus deviating radiation of different wave lengths into different angles, a grat- ing can serve as a dispersing element. A grating may consist of a large number of thin, parallel slits, in which case it is a transmission grat- ing, or of a similar number of thin, parallel reflecting strips, in which case it is a reflection grating. If, in a direction of maximum intensity for a given wave length, the waves from one slit (or strip) are exactly one wave length retarded or advanced with respect to those from the two adjacent slits, this direction is referred to as that of the first-order maximum. If the phase difference between waves from two adjacent slits is just two wave lengths, the direc- tion is that of the second-order maximum. In general, directions of maximum intensity will occur whenever /)X = f/(sin a + sin i3) where n — order number (an integer) X = wave length d = spacing between slits. (See Fig. 4-13). For a given wave length, the distribution of intensity in angle al)Out a direction of maximum intensity, will d(^p(Mid ultimately (Hi iho ratio of th(^ Fic. 4-13. Diffraction by a plane trans- mission grating. 152 HADIATION BIOLOGY ovtM-all width of tlio grating to the wave length, or more specifically on the number of wave lengths of phase dilTerence between the waves, pro- ceeding in a given direction from the extreme slits of the grating. As this phase dilTerence increases, as with increasing order number, the width of the angular intensity distribution decreases, and hence the spectroscopic resolving power increases. The angular dispersion of a grating — the variation of wave length of maximum intensity with angle — likewise increases with increasing order number, and also w-ith decreasing spacing between the slits. Analytically, where jS = angle of diifracted beam with grating normal n — order number d = spacing between slits. While both spectral resolution and angular dispersion are favored by the use of higher ditTraction orders, the problem of overlapping orders becomes acute for high-order numbers. The third diffraction order of Xi will overlie the second order of 1.5Xi and the first order of 3Xi, etc. Fre(iuently filters or elementary prism devices may be added to sur- mount this difficulty and permit the use of second- or third-order spectra. The grating, transmission or reflection, may be on a plane surface, in which case it is illuminated with parallel light from a collimating lens or mirror, and the emergent beams are focused to a spectrum with a tele- scope lens or mirror. Or the grating, if reflecting, may be ruled on a concave surface, in which case it will serve as its own focusing element, permitting the elimination of the collimating and telescope elements (Beutler, 1945). Such concave gratings are then effective throughout any wave-length region for which a reflecting surface may be made, since the need for any transparent dielectric is eliminated. The images formed by such gratings are, however, generally astigmatic, unless the grating is illuminated with a parallel beam, as in the Wadsworth mounting (Sawyer, 1951, Chap. 6; Harrison et al., 1948, Chap. 4). Gratings may be produced with higher resolving power than any prism instrument. By the use of replica technicjues, many copies can be made from one master at moderate cost. However, grating instruments neces- sarily waste light in unused orders, although this drawback can be mini- mized by proper ruling of the reflecting strips which can serve to direct most of the energy into one order (Wood, 1944; Babcock, 1944; Stamm and Whalen, 1946). The use of grating dispersing elements in monochromators is a rela- tively recent development (French et al., 1947), although commercial designs employing plane reflection gratings are now available (Bausch and Lomb, 19.")1) (see Fig. 4-14). The u.se of such monochromators may ULTRAVIOLET SPECTROSCOPIC TECHNIQUE 153 LIGHT SOURCE GRATING OBJECTIVE MIRROR, 33-86-40 GRATING MONOCHROMATOR Fig. 4-14. Optical path of Bausrh and Lomb grating monochromator. {Bausch ami Lomh Optical Compani).) become more widespread as high-quality gratings become more generally available. REFERENCES Acton, A. P., R. G. Aickin, and x\. S. Bayliss (1936) The continuous absorption spectrum of bromine: a new interpretation. J. Chem. Fhys 4- 474-479 Agnew J. T., R. G. Franklin, R. E. Benn, and A. Bazarian (1949) Combustion studies with the orthicon spectrograph. J. Opt. Soc. Amer., 39- 409-410 Aldington, J. N. (1949) The gas arc: a new light source. Trans. Ilium Eng Soc London, 14: 1951. Allen, A. J. 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Morton, and L. Malter (1936) The secondary emission multiplier — a new electronic device. Proc. Inst. Radio Engrs., 24: 351-375. Zworykin, V. K., and E. G. Ramberg (1949) Photoelectricity and its applications. John Wiley & Sons, Inc., New York. Manuscript received by the editor Sept. 22, 1952 CHAPTER 5 Ultraviolet Absorption Spectra Robert L. Sinsheimer Department of Physics, Iowa State College Ames, Iowa Paravieters of absorption spectra: Position in the electromagnetic spectrum — Width of absorption band — Intensity. Absorption and chemical constitution: Empirical correla- tions— Theoretical developments. Specification of absorption: Intensity — Spectral posi- tion— Band undth. Environmental factors influencing absorption: Solvent — pH value — Concentration — -Temperature — Orientation — Scattering — Local concentrations. Ultra- violet absorption spectra of important biological substances: Proteins and amino acids — Nucleic acids and nucleotides — Steroids — Carotenoids — Porphyrins — Flavins — Pterins — Vitamins — Plant pigments. References. The absorption of ultraviolet radiation by a molecule results in a change in the electronic configuration of that molecule and therefore in a change, usually transient and reductive, in the stability of the molecule. The ability of any molecule to absorb ultraviolet radiation of a particular fre- quency is dependent on the electronic configuration of the molecule and the electronic configurations of the possible higher energy states of the molecule. This absorptive ability is thus intimately related to the detailed molecular structure (Lewis and Calvin, 1939; Ferguson, 1948; Bowen, 1946, 1950; Maccoll, 1947). An absorption spectrum of any substance is a quantitative description of the absorptive ability of the molecules of that substance over some range of electromagnetic frequencies. A knowledge of the absorption spectra of the major components of a living organism makes it possible to limit the number of possible primary receptors of radiation that are found to produce some biological effect. Only those substances that absorb the radiation found to be responsible for the effect need be considered. The correlation of the measured rela- tive efficacy of radiations of various wave lengths in producing the effect, with the variation of absorptive power with wave length of the substances under consideration as primary receptors, can, under favorable circum- stances, further limit the possibilities as to the nature of the receptor. A knowledge of the influence of absorption on the stability of the 165 166 RADIATION BIOLOGY \arious substances considered may aid still further in identification of the primary receptor or receptors. PARAMETERS OF ABSORPTION SPECTRA Any absori)tion spectrum, however complex, may he regarded as a summation of a set of individual absorption bands, each corresponding to a transition between two particular electronic configurations (Fig. 5-1) (Sheppard et al., 1941; Wulf and Dcming, 1938). It is usually possible to group these individual bands, each group consisting of transitions invoking nearly the same energy difference. The dilTcrcnt bands within a group then represent transitions involving a common change in basic electronic configuration, together with varied associated secondary changes in the distribution of energy among the molecular vibrations. 475 500 525 550 575 600 625 650 675 700 725 750 FREQUENCY X IO-'2 Fig. 5-1. Resolution of the absorption spectrum of merocyanine in hexane into six bands, each representing a particular vibrational transition associated with the funda- mental electronic transition. (Sheppard et al., 1941.) The parameters of an absorption spectrum are properly the sum of the parameters of the individual bands. Any individual band (representing a transition between two distinct electronic states plus vibrational states) may be described by three parameters: (1) the position in the electro- magnetic spectrum, (2) the breadth of the electromagnetic spectrum occupied by the band, and (3) the intensity of absorption. POSITION IN THE ELECTROMAGNETIC SPECTRUM The position of an absorption band in the electromagnetic spectrum Js dependent on the energy difference between the initial and the e.xcited electronic configurations since this energy dilTerence must be supplied by the absorbed photon, the energy of which is related to its frequency (v) by Planck's relation E = hv = ^^ (5-1) ULTRAVIOLET ABSORPTION SPECTRA l(i7 where h = 6.61 X 10~" erg-sec h = 4.13 X 10-'-^ ev-sec c = 3 X 10'" cm/sec. The energy differences corresponding to absorption bands in the ultra- violet region (wave length <4000 A) are of the magnitude of 3.1 ev or greater. Since these energies are greater than those that corre- spond to the energy of formation of many chemical bonds (C — C bond energy = 2.54 ev; C— N bond energy = 2.11 ev) (Pauling, 1945), the rupture of such bonds in molecules raised to an excited level by absorp- tion of an ultraviolet photon is energetically possible. Such rupture may lead to the formation of free radicals or of oppositely charged groups or. in molecules containing atoms with unbonded electron pairs, to photo- oxidation and semiquinone formation (Waters, 1948; Lewis and Lipkin, 1942; Lewis and Bigeleisen, 1943b). The farther into the ultraviolet the absorption band is located, the greater is the excess of excitation energy over the minimum necessary for bond rupture. With absorption bands in the far ultraviolet (wave length <2000 A) the absorbed energies gen- erally become adequate to produce molecular ionization (8-12 ev) (Price, 1947). WIDTH OF ABSORPTION BAND The width of an individual absorption band is dependent intramolecu- larly on the duration of the excited electronic state (Heitler, 1944, pp. 110#) and extramolecularly on the statistical distribution of the fre- quencies of the particular absorption band among the assemblage of absorbing molecules, each exposed to a certain randomness of molecular environment. Considering any one molecule in a given molecular environment, the width of its absorption band is inversely dependent on the duration (mean lifetime) of the excited electronic state. This may be formulated by the "uncertainty principle" AE At ^ ^ ZTT where, in this instance, AE is the uncertainty, i.e., variation, in the energy difference accompanying the transition and A^ is the duration of the transition. For the usual absorptive process in an isolated mole- cule, At is of the order of magnitude of IQ-^ sec, A^ is about 10"' ev, AE/E = 10-^ per cent as is AX/X. However, any of several processes may shorten the duration of the excited state (A^, thus increasing the uncertainty in energy of the transi- tion {AE), and hence may broaden the absorption band. Disruption of the molecule may take place within the duration of a single moleculai' vibration and thus reduce the excitation lifetime to as short as 10"'^ sec. 108 RADIATION BIOLOGY II' disrupt ion is less likel}', tlir inolccule may remuiu iiituct for several luiiulrod or thousand vibrational periods, or lO^'^-lO"'^ sec. The spec- tral hroadeniii}; in this case will not he so great and will give rise to so-called "predissociation " l)ands (Rice and Teller, 1949). The duration of the excited state may be reduced because of a high probability of a transition to a third electronic state (Fig. 5-2). The dura- tion of this state will have no influ- ence on the width of the original absorption band. A number of such instances of transitions to a "triplet" state have been reported (Kasha, 1947). Transitions from this triplet state to the original ground state are of very low probability so that the molecule may retain energy as an excited triplet state for appreciable lengths (seconds) of time (McClure, 1949). Under conditions of appreciable intermolecular contact (solutions and solids) the energy of excitation may be rapidly dissipated by conversion to vibrational energy which, in turn, is simply transferred by collisions or electromagnetic damping to neigh- boring molecules and ultimately ap- pears as thermal energy (Massey, 1949). Such dissipative effects, which reduce the duration of the ex- cited state, are in part responsible for the broadening of absorption bands of substances in solution as compared to their vapor absorption spectra. If the molecule retains its excita- tion energy for a time comparable with the probability^ of transition from the excited to the ground state, the energy will be reradiated as "fluorescence." This fluorescence radia- tion may then escape, or it may, under appropriate conditions, be reab- sorbed by other chromophores within the solution or biological system (Arnold and Oppenheimer, 1950; Forster, 1948; Franck and Livingston, 1949). In addition to these primarily intramolecular factors, the electronic INTERATOMIC DISTANCE ALONG CRITICAL COORDINATE Fig. 5-2. Illustration of the possibility of a radiationless transition from the initial excited electronic state (T*) to a second excited state (T), in this instance a triplet state which would have a long duration and from whicih return to the ground state could occur by delayed emission of radiation or phosphorescence (P). The curves rep- resent the variation of potential energy of the molecule as a function of the inter- atomic separation for a diatomic mole- cule. (Kasha, 1947; copyright, 1947, by The Williams and Wilkins Company.) ultraviol?:t absorption spectra 169 configuration of the individual molecules and thus the energy associated with a particular electronic transition will be influenced in a condensed system by the electric and magnetic fields associated with nearby mole- cules. Since the spatial orientations involved will be random (except in crystals) and will be varying, owing to thermal motion, there will result a statistical distribution of electronic configurations and of transition energies of the absorbing molecules, thus broadening the observed absorp- 60.000 60,000 40,000 20,000 - 12 3 4 5 INTERATOMIC DISTANCE r, A Fig. 5-3. Illustration of the Franck-Condon principle. Horizontal line.s within the well of each potential-energy curve represent various vibrational-energy levels. A transition from the ground state (V") to the excited state (V) would most probably leave the molecule in the second excited vibrational level (point A) since the inter- atomic distance cannot change appreciably within the duration of the transition. (Reproduced by permission of the publishers front Practical Spectroscopy, by George R. Harrison, Richard C. Lord, and John R. Loofbourow, copyright, 1948, by Prentice-Hall. Inc.) tion band. These effects will be reduced if the fields involved are reduced (as in nonpolar solvents) or if the extent of the variations due to thermal motion is reduced, as in spectra of substances at low temperatures (Sin- sheimer et al., 1950a). For many substances in solution, the effects described widen the indi- vidual absorption bands associated with a given electronic transition so as to produce a fusion of these bands into an apparently single band of considerable breadth. The individual bands, representing transitions from vibrational energy states accompanying the normal (lowest energy) electronic state to various vibrational states accompanying the excited 170 RADIATION HIOLOGY electronic conligurutioii, are thus concealed, reducing the amount of infor- mation available in the spectrum. The possible vibrational transitions are limited by the Franck-Condon principle (Rice and Teller, 1949), which simply recognizes that the duration of the electronic transition is brief compared to the duration of a molecular vibration, so that the posi- tion of the atoms camiot change appreciably during the act of absorption. Hence only transitions to excited-state vibrational levels, involving atomic configurations similar to those in the vibrational levels associated with the ground state, are probable (Fig. 5-3). Broadening of this type may also be reduced by a reduction of the temperature of the absorbing substance; the reduction in thermal molecu- lar energy decreases the molecular population in the higher vibra- tional energy levels and thus reduces the number of possible transitions. Indeed, at li(iuid-air temperature or below, all molecules must commence a transition from the lowest vibrational energy level. INTENSITY The total intensity of an absorption band, i.e., the integrated absorp- tion over the band, is dependent on the difference in scale and symmetry of the electronic configurations for the initial and the excited states (Heitler, 1944). A net time-average displacement of charge along some molecular axis must accompany the absorption of radiation. If the elec- tronic configurations of the two energy levels are of such a symmetry that a transition from one to the other does not provide such a time- average displacement, then a transition between these levels cannot be induced by radiation, i.e., absorption cannot occur. Such a transition is said to be "forbidden." In benzene, the electronic configurations of the ground and the first excited singlet energy levels are of such a symmetry that a transition between them is forbidden (Sklar, 1942). This transition, which is associated with the benzene absorption maximum at 2550 A, can occur only if accompanied by a particular molecular vibration which so dis- torts the molecule as to alter the symmetry of either the ground or the first excited energy levels and thus gives rise to a small time-average dis- placement of charge. The intensity of such forbidden absorption bands, which re(iuire the participation of a molecular vibration, is generally low. Thus for the benzene absorption maximum at 2550 A, € = 120; this may be contrasted with the intensity of the "allowed" benzene absorption band at 1835 A which is about 380 times as great (c = 4G,000) (Piatt and Klevens, 1947). In general, the greater the time-average displacement of charge, the greater the integrated absorption. For a given integrated absorption, the intensity of the al)sorption maxi- mum will ob\iously depend in\ersely on the width of the l)and and thus directlv on the duration of the excited state. ULTRAVIOLET ABSORPTION SPECTRA 171 WAVE LENGTH, A 3250 3000 2750 2500 2250 3.0 - 2.5 ■ 2 0 - o o ABSORPTION AND CHEMICAL CONSTITUTION EMPIRICAL CORRELATIONS The empirical correlation of the spectral position of absorption bands in the ultraviolet with certain chemical structures was begun about 1885 with the work of Hartley (1885) and has been steadily continued and expanded as improvements in technique have simplified the task of measuring absorption spectra (Braude, 1945; Lewis and Calvin, 1939; Ferguson, 1948; Erode, 1943; Jones, 1943). The broader long-recog- nized empirical correlations have now been given a theoretical basis by the development of approximate wave-mechanical methods of calcu- lation of electronic-energy levels in complex molecules. These correlations early indi- cated that the absorption bands of compounds, composed exclu- sively of saturated linkages, oc- curred generally below 2000 A, usually in the vacuum ultraviolet below 1850 A. The long-wave arms of such peaks extended above 2000 A, increasing in amplitude with increase in size of the molecule. Because many spectrographs and spectrophotometers do not record below 2000-2200 A, these long- wave limbs of bands of saturated compounds, rising in absorption with decreasing wave length, are often referred to as "end" absorption. Compounds with single unsatu- rated bonds such as C'=C, C=0, or C=N were found to have ab- sorption bands, usually weak, in the region 1900-3000 A, the actual wave length being dependent on the adjacent parts of the molecule. Strong absorption bands in the region 2000-4000 A are always corre- lated with molecular structures containing chains or rings of conjugated double bonds; in general, the larger the conjugated structure, the stronger is the absorption and the longer the wave length of the maximum absorp- tion. Ring structures with conjugated double bonds, as in aromatic compounds, often possess particularly high absorption. 0.5 - 30,000 35,000 40,000 45,000 Fig. 5-4. Ultraviolet absorption spectra of some .simple derivatives of benzene. I, aniline; II, phenol; III, chlorobenzene ; IV, benzene; all in heptane. (Adapted from Wolf and Herold, 1931.) 72 RADIATION BIOLOGY The absorption o{ coiijusatcd-boiul groups separated within a {^ivcn molecule by two or more saturated bonds is usually independent and simply additive. The absorption of such groups can, of course, be affected by the addition of side chains or auxiliary groups, especially if the latter may be charged (Nils and OH). Such groups may distinctly affect the spectral position and intensity of absorption of a given conju- gated system (Fig. 5-4). In large polar macromolecules, such as proteins and nucleic acids, the near-ultraviolet absorption spectrum of the polymer is often not strictly WAVE LENGTH, A 6000 4000 3000 2500 I I I 2000 6000 WAVE LENGTH, A 4000 3000 2500 2000 20 >- (/) I 5 u I 0 Q. O 05 1 1 1 - - - - - - - - r/,n - _ // - - /'N // - - u\ / - - f\ / - - ' - : - - - _ - 1 J 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J 1 1 1 1 1 1 15 20 25 30 35 40 45 50 -3 Q I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 15 20 25 30 35 40 45 50 WAVE NUMBER, Cm"'xi0"' WAVE NUMBER, Cm''xiO" (a) (b) Fig. 5-5. (a) The ultraviolet dichroism of hexamethylbenzene crystals. I, Electric vector perpendicular to the plane of the ring; II, electric vector parallel to the plane of the ring, (b) The ultraviolet dichroism of tobacco mosaic virus particles, oriented by .streaming. I, Electric vector perpendicular to the direction of streaming; II, electric vector parallel to the direction of streaming. (Scheibe et al., 1943; see also Wilkins et al, 1950.) the linear sum of the absorption of its component conjugated groups, even though these component groups are separated by appreciable lengths of saturated bonds. This nonadditivity appears to be due to the formation of labile intergroup bonds to the conjugated groups, either of ionic or hydrogen-bond type. Thus the ultraviolet absorption spectra of proteins are often not simply the sum of the absorption of the compo- nent amino acids. The phenolic group of tyrosine, for instance, fre- quently appears to be involved in some type of loose bond (Crammer and Neuberger, 1943; Sizer and Peacock, 1947; Finkelstein and McLaren, 1949; Schauenstein and Treiber, 1950). The ultraviolet absorption of highly polymerized deoxyril^onucleic acid is about 25-30 per cent less than that of the depolymerized form, which form is very nearly the linear sum of the absorption of the component nucleotides (Kunitz, 1950; ULTRAVIOLET ABSORPTION SPECTRA 173 Tsuboi, 1950; Loofbourow, 1940; Siiisheimer, 1954). The absorption spectrum of ribonucleic acid also increases in intensity and shifts slightly toward shorter wave lengths during depolymerization (Kunitz, 1946; Tsuboi, 1950). If the radiant energy is plane polarized, absorption will be greatest when the plane of the electric vector is parallel to the direction (s) of greatest electron mobility and maximal induced dipole moment, i.e., parallel to the direction of a chain of conjugated bonds, as in carotene, or parallel to the plane of the ring in a planar aromatic or heterocyclic mole- cule, as in benzene (Fig. 5-5). Spectrally distinct absorption bands may appear, corresponding to transitions involving mutually perpendicular changes in dipole moment (Lewis and Bigeleisen, 1943b; Scheibe and Kandler, 1938; Scheibe et al., 1943; Coulson, 1948; Nakamoto, 1952). In solution the random orientation of molecules will prevent detection of any such preferred directions; however, in cellular structures or in crystals, uniform molecular orientation may permit a preferential absorp- tion for light polarized in these directions (Butenandt et al., 1942; X^ ^\ \/ \/ /\ /\ /\ \ KEKULE DEWAR Fig. 5-6. The canonical structures of benzene. {Sklar, 1937.) Schauenstein et al., 1949). This phenomenon of differential absorption dependent on the plane of polarization is known as dichroism and can be useful as an indication of molecular orientation. THEORETICAL DEVELOPMENTS As indicated, these empirical correlations have received support from modern theories of the electronic structure of organic molecules and of the change in electronic configuration attendant on the absorption of radiation. These treatments have developed along two lines, the valence- bond concept (MaccoU, 1947; Heitler, 1945; Pauling, 1945; Van Vleck and Sherman, 1935; Sklar, 1937) and the molecular-orbital concept (Coulson, 1947; Herzfeld, 1947; Mulliken and Rieke, 1941). In the valence-bond concept, the electrons involved in chemical bonds are assumed to remain in atomic orbitals, which overlap with orbitals of the neighboring atoms, and the energy of each bond may be calculated from wave-mechanical principles. The energy level of the molecule is dependent on the summation of the energy levels of each bond. It is recognized that, with conjugated structures, the formulas usually written represent but one of several possible canonical forms (Fig. 5-6), all of which may be considered to contribute in varying degree to the actual 174 RADIATION BIOLOGY structure. By combining these possible forms in various proportions, a combination may be found which produces the lowest energy state. The energy level of this state is generally less than that of any of the indi- vidual canonical forms, a result known as "resonance stabilization" (Whohind, 1911) and due to "exchange energy" (Hcitler, 1945). Other combinations, with varying proportions of the possible canonical forms, give rise to higher energy levels to whi('h the molecule may be excited on the absorption of radiation. Resonance among possible structures in the excited state can lower the energy of the excited state and thus reduce the energy difference between it and the ground state, thereby increasing the wave length of the absorp- tion associated with the electronic transition. The intensity of absorp- tion will be greatest for transitions between states involving resonating structures which have appreciable dipole moments. The molecular-orbital method, which has been the more succes.sful in regard to the correlation of calculated with ob.served spectra, has been based on the assumption of molecular orbitals for the valence electrons of the atoms involved in the chemical bonds. In this development the molecular orbitals are usually made up formally of linear combinations of appropriately chosen atomic orbitals — LCAO method' (Mulliken and Reike, 1941; Chirgwin and Coulson, 1950; Lennard-Jones, 1949; Matsen, 1950; Dewar, 1950; Piatt, 1950; Longuet-Higgins d al, 1950). The con- struction of the molecular orbitals may involve only the atomic orbitals of two atoms, as is usually the case with single bonds and with isolated double bonds, in which case they are referred to as "localized orbitals." or, as in the case of conjugated chains of double bonds, the molecular orbitals may involve contributions from the tt orbitals of all the atoms involved in the chain. In this latter case, the orbital is said to be "unlo- calized," and electrons in such orbitals are considered to migrate freely along the chain (Fig. 5-7). Varying the combinations of atomic orbitals will produce molecular orbitals of various energy levels. The electrons available for bonding (all in the outer atomic shell) are then disposed in successively higher energy levels, two to a molecular orbital with spins opposed, until all electrons are accounted for. Absorption of radiation may then cause an excitation of an electron from the highest filled molecular orbital to the lowest unfilled orbital. The energy difference between orbital levels ' Refinements of this procedure involve the ii.se of "antisyiiuiietrized inoiccuhir orbitals" to reduee the apparent contribution of configurations including multiply ionized atom.s ((loepixTt-Mayer and Skiar, 1938; Roothaan, 1951) and recognition of configurational interaction (Jacobs, 1919; Craig, 1950). .\notiier theoretical ajiproach which has had considerable success is the "free-electron model" in which all the ir electrons are considered to be able to migrate freely throughout the molecule, along the atomic bonds, in a jiotential field that is constant, or, in some instances, sinusoidally varying (Bayliss, 1918; Piatt, 1949; Kulm, 1949; Simpson, 1949). ULTRAVIOLET ABSOUI'TION ttl'lOCTUA t .) decreases with increasing); orbital energy. As a result, the transitions induced by absorption in large conjugated systems, which are transitions between high-level orbitals, re((uire less energy, and thus a longer wave photon, than the transitions induced by absorption in smaller conjugated systems. This deduction accounts generally for the increase in wave length of the absorption peaks with increase in size of the conjugated system. Absorption of radiation may also induce transitions from the highest filled molecular orbital to some of the higher unfilled orbitals; these tran- hiiiljiMiMiiiiiiiiiiiiiiriirpiii *''y iiiiiiiiiiiiiiiiiriiiii'^ (C) Fi- c - - I + + ! + 1 - >-c A >C' V + :a + •c-^ + )! - >-c li I \ I / w II X - )1 + 1! - c -< c -< - ; + Fig. 5-8. A schematic representation of the wave functions describing the four molecxi- hir orbitals that may be synthesized by Hnear combinations of the four tt atomic orbitals of butadiene CHo=CH— CH=CH.,. On and outside the dotted Hues the wave functions are practically zero. Within the dotted lines the wave functions have finite values, with sign as indicated, and thus these are the regions with an appreciable probability for the presence of an electron (the pluses and minuses refer only to the sign of the electronic wave function and not to the charge). (Reproduced from Chemical Aspects of Light, by E. J . Bowen, copyright, 1940, by Oxford University Press.) ./^v ^n/ V > \ /^s V ) (, .-s 1 ( -* I- V 1 < \y V \ \ V ,-, ; / / Fig. 5-9. The molecidar orbitals of anthracene. (Bmren, 1950.) nodal planes for the molecular orbitals of a simple conjugated chain (butadiene) are shown in Fig. 5-8 (Bowen, 1940). Absorption induces a transition of an electron from orbital 2 to orbital 3. In aromatic molecules the new nodal plane may intersect the molecular ULTRAVIOLET ABSORPTION SPECTRA 177 plane along either of two mutually perpendicular axes (.r, y), producing different molecular orbitals. If the molecule is asymmetric (as in naph- thalene), these orbitals will represent different energy levels, and transi- tions from the ground state to these orbitals will be associated with spec- troscopically distinct absorption bands, which will be strongly dichroic (Coulson, 1948). The intersections of the nodal planes with the molec- ular plane for the molecular orbitals of anthracene are indicated in Fig. 5-9. The transitions that correspond to the two prominent ultraviolet absorption bands of anthracene are labeled with the band wave length. It can be shown that these two approaches, valence-bond and molec- ular-orbital, in their simple forms, probably bracket the correct solution; the valence-bond method does not allow enough weight to possible ionic structures, i.e., those in which two or more of the bonding electrons may be concentrated on one atom; the molecular-orbital method allows too much. More advanced developments of both theories have tended to narrow the gap between them. SPECIFICATION OF ABSORPTION INTENSITY The ultraviolet absorption spectra of substances of biochemical interest are usually obtained with solutions of these substances in transparent solvents. The measured absorption at any wave length will then be dependent on the concentration of the substance in the solution and the length of the light path in the solution. The specification of absorption spectra may be standardized by referring all measured spectra to the spectrum that would be obtained from a solution of a standard concentra- tion and a standard light path. This conversion of measured to standard spectrum is rendered easy by the simple nature of the formula relating absorption to concentration and light path. Since the absorption of a photon by a molecule is an all-or-none act and since all molecules may be assumed to have, statistically, the same proba- bility of absorption of an incident photon of a given wave length, any layer of a solution of thickness dl, transverse to the light beam, may be expected to absorb the same fraction of radiant energy of one wave length as any other such layer, and if dl is small, this fraction will be proportional to dl. Thus jT ^ dt. This statement is known variously as Lambert's or Bouguer's law. If the absorbing molecules may be assumed to act independently, the fraction of incident energy absorbed in a given layer will be expected to be proportional to the concentration of absorl)ing molecules in the solution. 178 RADIATION BIOLOCY Combining this with the previous expectation, — -,- a cl is ohtained. This icljilion is known variously as the LainlxTt-Hccr or Bouf2;uer-]ieer law. Integratiiifz;, it is t'ouiid that (J I ell, or also In -J- *^ c/, logiu Y °^ ^^• The proportionality constant in this etiuation, which applies at eacii wave length, is, of course, characteristic of the absorbing substance. Numerically, it will depend on the logarithmic base employed and on the T.vble 5-1. Symbols for the Proportionality Co.nsta.nt Log base Optical path length (/), cm c, g /liter c, moles/liter r, moles /ml e 10 k; a, (specific extinction) E t\ a„, (molar extinction) 13 units for concentration (c) and optical path length [1). X'arious com- binations of base and units have been employed, and the most commonly used symbols for the proportionality constant in the various systems are indicated in Table 5-1. Thus ^ logiu jh/I) ' cl where c is in moles per liter and / is in centimeters. The (juantit}' logio (lo/I) = eel is often referred to as the optical den- sity^ (/)) of the solution. Thus e is the optical density that would be measured for a 1-cm path of a solution containing 1 mole/liter.''-^ ' Also referred to as the extinction or the absorhance (Gibson, 1949; Brode, 1949). ' Another symbol occasionally used to specify absorption is Ef which signifies the optical density of an /-cm path of a solution containing p per cent of tlie absorbing substance. ' In some circumstances, lr logu, J) may be plotted either to compress a ui(h- pinge of vahies into rea.soiial)U' dimensions or to obtain a curve the shape of whicii is independent of the concentration or path length, wliich may be unknown. Since logio D = logii) t f- logio (• + logiu X, the latter will enter only as additive constants. ULTRA VKJLKT ABSOKPTION SPKCTRA 170 The Lambert or Boiiguer law is valid under all foiiditions of normal use. The modification due to Beer is accurate as long as the condition for its validity is met — that the absorbing molecules act independently. In concentrated solutions there is often a tendency toward dimerization or other forms of molecular association. In these instances the nature of the absorbing entity really changes with changing concentration, so that Beer's law will not apply. Another method of indication of the absorbing power of a substance is the specification of the "absorption cross section" (a) of molecules of the substance as a function of wave length. The absorption cross section is a measure of the probability of absorption of a photon, known to be crossing a unit area transverse to the beam, by a single molecule known to be con- fined within that area. This quantity is useful in calculations concerning the possibility of radiative-energy transfer from the primary receptor to other receptors within a cell (Arnold and Oppenheimer, 1950). The absorption cross section is related to e by the following formula: a = 3.83 X 10-21^ where a is in square centimeters. The cross section a does not necessarily bear simple relation to the physical cross-sectional area of the molecule, although in certain instances a good correlation has been demonstrated between an "effective" geometrical cross section and absorptive power (Braude, 1950). SPECTRAL POSITION The position of an absorption band in the electromagnetic spectrum is usually defined by the position of the absorption maximum. This posi- tion may be expressed in terms of (1) wave length, in Angstroms (1 A = 10-« cm) or millimicrons (1 m^ = lO"" cm); (2) wave number,^ in cm-^ or mm-i; or (3) frequency, in vibrations per second or in fresnels (1 f = 10'2 vps). Thus the longer wave maximum in the absorption spectrum of methyl- cholanthrene at 77°K occurs at 2995 A. 299.5 m/x, 33,390 cm-', 10.02 X 101^ vps, or 10,020 f. BAND WIDTH The width of an absorption band is usually considered to be the spectral separation between the points of half-maximal absorption. This separa- tion may be expressed in any of the units used to express the position of the absorption maximum. '• The wave number is defined as the number of wave lengths per centimeter (or millimeter) of path in vacuo. 180 RADIATION inOLOGY ENVIRONMENTAL FACTORS INFLUENCING ABSORPTION In general, a variation of any factor that influen(;e^s the electronic (^on- fi}z;in'ation of the absorbing molecules, either uniformly or with a statistical (listrii)ution, will affect the absorption spectrum (Sheppard, 1942). In addition, certain factors may alter the technical conditions of the absorp- tion measurement and thereby affect the spectrum. SOLVENT The choice of solvent can influence the position, width, and intensity of absorption bands. Changes in position are to some degree correlated 4 8 I 6 V = 900 1000 HOC 1200 1300 1400 I/X= 30,000 33,330 36,670 40,000 43,330 46,660 A = 3333 3000 2727 2500 2308 2143 1500 1600 f 50,000 cm'' 2000 A Fig. 5-10. The absorption spectrum of phthalic anhydride. I, in hexane; IT, in alcohol. {Mcnrzcl 1927.) with the dielectric constant of the solvent according to Kundt's rule which states that, with increasing dispersion of the solvent, the absorption maximum is shifted toward longer wave lengths. Although Kundt's rule is generally valid for nonpolar solvents, there are serious deviations with polar solvents (Sheppard, 1942). This shift may be interpreted as indicative of the increased role played by ionized structures in resonance stabilization of the excited state in media of high dielectric constant (Wheland, 1944) or in terms of the influence of the reaction field of the oscillating dipole on the electric field ULTRAVIOLET ABiSOllPTION SPECTRA 181 of the light wave in a dielectric medium (Bayliss, 1950; Hartmann and Schlafcr, 1950). As indicated, absorption bands are widened in polar solvents because of increased molecular interaction with conseciuent perturbation of the elec- tronic configurations (Fig. 5-10). The total absorption intensity, the je dv, can also vary with the solvent and would be expected, in general, to increase moderately with increasing solvent refractive index (Chako, 1934) owing to augmentation of the 220 230 240 250 260 270 280 290 300 320 340 WAVE LENGTH, m/( Fig. 5-11. The absorption spectrum of cytosine at three different vahies of pH (« = 8.0G X lO'D). I,pH 1.2;II, pH6.0;III, pHl2.7. (Scott, unpublished data, 1951.) exciting electric field by the field of the induced dipoles in the medium. However, this expectation is frequently not fulfilled for unknown reasons (Jacobs and Piatt, 1948). pH VALUE In aqueous solutions of substances containing dissociable groups, the pH of the solution will usually have a marked effect on the absorption spectrum. Ionization of any such group, resulting in gain or loss of charge, will certainly alter the basic electronic configuration of the mole- cule and thereby the spectral distribution of absorption. An example is the absorption of a solution of cytosine at various pH values (Fig. 5-11), lJ^-2 UADIATION mOLOGY iiulicatiiifi; the olTccts of ionization of the amino sroup (i)K' = 4.()0) and of the enolic group (pK = 12.16) (Levenc and Bass. I'.Kil). CONCENTRATION As was mentioned in the discussion of Beer's law, in concentrated solu- tions the association of solute molecules may cause modification of their absorption spectrum. This elTect may give rise to a nonlinear relation between the optical density of such solutions at certain wave lengths and Fig. 5-12. Variation of the absorption spectrum of pinacyanol chlorides in water at 20.0°C with concentration. I, 4.44 X 10"^ M; II, \.••^'^ X 10"^ M ; III, 4.44 X 10"^ M; IV, l.:^3 X 10-'' .1/; V, 4.44 X 10-« M . (Scheibe, 1938; reproduced from Kolloid- Zeitschrift. ) the solute concentration, as is observed with the Nessler test for ammonia (Hawk etal, 1947). In more extreme cases, extensive molecular association, possibly involv- ing electron transfer through intermolecular hydration, may cause the development of entirely new absorption bands. An example of this is the "mesophase" J band (Fig. 5-12) of the cyanine dyes (Sheppard, 1942). TEMPERATURE The temperature of an absorbing substance significantly affects its absorption spectrum by controlling the statistical distribution of mole- cules among various vibrational energy states associated with the lower energy electronic state and by influencing the velocity of Brownian ULTRAVIOLET ABSORPTION SPECTRA 183 motion, which in turn determines the frequency of molecular collision. The latter influences the duration of the excited state and the extent of the distortion of the molecular electronic configurations by the electro- magnetic fields of neighboring molecules. In addition, variation of tem- perature may vary the relative statistical contribution of various possible tautomers (Freed and Sancier, 1951) or resonating states. In general, reduction of the temperature, by reduction of the variety of initial energy levels in an electronic transition induced by absorption and by reduction of the perturbing effect of extramolecular fields, will reduce the width of the individual and fused absorption bands (Fig. 5-13). This effect may be particularly marked if the absorbing substance is in a crystalline WAVE LENGTH, A 2600 2800 3000 3200 2400 2600 2800 3000 3200 1— 1 — I — I — I — r THYMINE (FILM) — I r H I I rHN^^CHj T — 1 — I — i — I — I — r THYMINE (EPA) r/oK (b) 42 4.0 3.8 3.6 32 3.0 3.4 3.2 3.0 4.2 4.0 3.8 3.6 3.4 WAVE NUMBER, cm"' X 10"^ Fig. 5-13. Effect of reduced temperature on the absorption spectrum of thymine. {Sinsheimer et al., 1950b.) form so that the molecules have a uniform environment (Scott et al., 1952). ORIENTATION If the molecules of a dichroic substance are uniformly orientated, as might occur in a cellular structure, the absorption spectrum would depend on the plane of polarization of the incident radiation. If unpolarized light is used, as is ordinarily the case, the influence of the molecular orientation on the observed spectrum will depend on the degree of dichroism. In an extreme case, such as might occur if there were no absorption at all of light for which the electric vector lay in a particular plane, the maximum possible light absorption would be 50 per cent, cor- responding to an optical density of 0.3 at that wave length (Commoner and Lipkin, 1949). Obviously, intermediate cases would permit various maximal values of optical density. SCATTERING If the absorl)ing solution (or living cell) contains objects of dimensions comparable with those of the wave length of light employed, appreciable quantities of light may be lost from the l)eam by scattering out, as well as 184 RADIATION HIOLOGY by absorption. For simple spherical particles of diameter i)litl(' liiikjit?(>s, alT(>ctiiiK the absorhing .structuros (Fi^j;. .'»-l(i). The spectra of the iiuli\ idiial miclcot ides are easily disliuKuished (Kig. 5-17); they are generally similar to the spectra of the component purine and pyrimidiiic hases, which hases are primarily responsible for the absorption of these compounds in the spectral region above 2300 A. The 200 240 320 280 WAVE LENGTH, rr\ p. Fig. 5-15. Absorption spectrum of trypsin in acid and in alkaline sohition, and of the trypsin-trypsin inhibitor complex (e = :3fi,700 K). • •, trypsin in .V/lOO H2SO4; O O, trypsin in A/10 HCl; ® ®, trypsin after 24 hours in A710 HCl; 3 9, heat-inactivated trypsin; O O, trypsin in A/10 NaOH; O O, trypsin inhibitor complex. (Schonnuller, 1949.) spectra are sensitive to changes in pH, especnally in the regions of the pK vahies of the functional groups attached to the purines and pyrimidines (Stimson, 1949) . Absorption in these bases is considered to be dependent on the presence of — C^C— C=N— or — C=C— C=0 groupings (Cavalieri and Bendich, 1950). STEROIDS Although all steroids will exhibit end absorption in the region below 2100 A, only those steroids that contain sequences of conjugated double ULTRAVIOLET ABSORPTION SPECTRA 187 bonds will show appreciable absorption in the region 2200 3000 A (Dannenberg, 1939; Fieser and Fieser, 1949). Thus steroids that contain only isolated ketone groups (androsterone) have only a very weak absorp- 1.2 1.0 0 8- o 0.6 0 -^ 02 \ -1 - \\ /"^•^. : :V<^ 1 V 220 230 240 290 300 310 250 260 270 280 WAVE LENGTH, mjl Fig. 5-16. Change in ultraviolet absorption spectrum of thymu.s deoxyribonucleic acid upon digestion with deoxyribonuclease. I, digested; II, undigested. {Kunitz, 1950, reproduced from the Journal of General Physiology.) 16.000 14.000 12.000 10.000 o o 2 8000 - < 6000 -J o s 4000 - 2000 210 220 230 240 290 300 310 320 250 260 270 280 WAVE LENGTH, mji Fig. 5-17. Absorption spectra of five deoxyribonucleotides at pH 4.30. I, deoxy- guanj'lic acid; II, deoxyadenylic acid; III, deoxycytidylic acid; IV, deoxy-5-methyl cytidylic acid; V, thymidylic acid. {Sinsheimer, 1954.) tion at about 2900 A (e = 43). Steroids with a and /3 unsaturated ketones will have a strong absorption in the region 2300-2000 A (for pro- gesterone, Xmax. = 2370 A, and e = 17,000) and a much weaker band near 3200 A (e ~ 10-20) (Morton, 1942). 188 RADIATION BIOLOGY N I o /I r\ 100 : s n V Av/ \\ 20 '" \ ^ If \y ! , \-^ A 320 360 Jr^ m/i ■ / bO - 1 1 1 1 1 1 1 1 V 220 240 280 300 260 WAVE LENGTH, mjd Yu\. 5-18. Absorption speetrum of orgostorol. , ergosterol in CsHsOH; , crgostcrol in isooctane; X, T-dchydrtx-liolostcrol. {Ajler Hognrss et al., reproduced from The AppUeation of Absorption Speelra to the Stinlii of Vitamins, Honnones and Coenzymes, by R. A. Morton, 1942.) 2200 3000 3800 WAVE LENGTH. A Fig. 5-19. Absorption spectrum ofequilenin. (Jones, 1948.) 300 500 400 WAVE LENGTH, rr\ju Fig. 5-20. Absorption spectrum of lycopcuc with varying degree of cis-lrans isomerization. I, mix- tvu-e of stereoi.somers after iodine catalysis at room temperature in light; II, fresh solution of the all- trans compound ; III, mixture of stereoisomers after refluxiiig in darkness for 45 min. {Zeehmeister, 1944; eopyriijht, 1944, by the Williams and Wilkins Company.) ULTRAVIOLET ABSORPTION SPECTRA 189 5.70 5.50 5,00 - Steroids with diene and triene chains, such as ergosterol (Fig. 5-18) and calciferol, will have strong absorption maxima in the region 2200 2900 A (for calciferol, \,„^^. = 2650 A, and e = 18,200). Steroids that contain aromatic rings, such as the estrogens, will, in general, have strong absorp- tion bands in the region 2400-2800 A and may have absorption intensity- well above 3000 A if the aromatic grouping includes more than one ring, as in equilenin (Fig. 5-19) (Morton, 1942; Jones, 1948). CAROTENOIDS The long conjugated double-bond chains of the carotenoid compounds give rise to from one to three ultraviolet absorption bands at varying posi- tions (Karrer and Jucker, 1948). One band is usually found in the region 260-320 m^u. Absorption in the region 320-380 m/x has been demonstrated to be depend- ent on the number and position of cts-configurations in the chain (Fig. 5-20) (Zechmeister, 1944). These ultraviolet bands (e = 10,000 50,000) are considerably weaker than the intense set of three bands com- monly found in the visible absorp- tion spectra of these compounds (e = 50,000-200,000). PORPHYRINS The intense Soret absorption band of the porphyrins is found in the near ultraviolet in the simpler members of the group, such as porphyrin itself (Fig. 5-21). With increasing substitu- tion of the tetrapyrrole ring, this band moves into the visible-spec- trum region. A number of shoul- ders or weak maxima are usually to be found on the descending short wave limb of this peak. Porphyrins usually show a minimum of absorption in the region 2500-3000 A and then increasing absorption again at wave lengths <2500 A (Pruckner and Stern, 1937; Theorell, 1947; Holden, 1941). The Soret band of the dihydroporphyrines, such as the chlorophylls, is o o 4 50 - 4 00 - - r, ■ 1 ! 1 1 1 1 1 I 1 : 1 1 - /i / V 1 1 t\\\ 1 '\\ - // ^l i ! '11 ' i 'r— . ™ n/ ; i "^ •■-^1 i \ - \ • 1 in \l. / 1 ■\ \ ^^ \^ ; \\ 1 ; N 3,50 450 250 300 350 400 WAVE LENGTH, m/i Fig. 5-21. Ultraviolet absorption spectra of several porphyrins in dioxane. I, porphyrin; II, aetioporphyrin; III, copro- porphyrin-II-tetramethyl ester; IV, rhodo- porphyrin-XV-dimethyl ester. {Pruckner and Stern, 1937.) 100 RADIATION HIOLOGY moved woll out into the visible reRion ((Iranick aiul (Jilder. 1047). 'i'iie ehlorophylls show several minor absorption peaks in the speetral region •28tK) :i8(H) A (Fis- .V22). Chlort)phylls a and b differ notably in their absorption at about 3S(H) A (Harris and Zscheile. 1043). 76 68 - 60 - 52 - gAA X UJ ^ 36H u Ul a. V) 28 - 20 12 C-~V?0\£\T i 0 o c FS£FARAriO\ 8 ll (1950) The formation of 6-hydroxy- and 7-hydroxypteridines from 4,5-diaminopyrimidines and a-ketoacids and esters. J. Am. Chem. Soc., 72: 78-81. Ellinger, F. v. (1937) Absorptions-Spektroskopie im lUtraviolett. I: Ab.sorptions- spektra der Eiweiss-Korper, Kohlehydrate und Fette einschliesslich ihre Aufbau- und Abbau-Substanzen. Tabulae Biologicae, 12: 291-343. 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Phys., 6: 702-711. Zechmeister, L. (1944) Cis-trans isomerization and stereochemistry of carotenoids and diphenylpolyenes. Chem. Revs., 34: 267-344. Manuscript received by the editor Feb. 20, 1952 CHAPTER 0 A Critique of Cytochemical Methods Arthur W. Pollister Department of Zoology, Columbia University, New York, New York Introduction. Lairs of absorption. Localization of substances in cells: Preservation of intracellular substances in situ — Nucleic acid staining and tests — Protein staining and tests — Ultraviolet absorption of nucleic acids and proteins. Quantitative micro- scopical methods: Visual comparison — Photometric technique — Some errors of quantita- tive microspectrophotometry — Quantitative applications, absolute and relative. References. 1. INTRODUCTION The bulk of the extensive researches in cytology has been aimed pri- marily at demonstrating the morphological features of the cell. By 1875 modern microscopes had become available which reached close to theo- retical limits imposed by the properties of visible light, and within less than two decades the application of this tool, in conjunction with an increasing number of special microtechniciues for preparing cells for examination, had demonstrated a wide diversity in intracellular mor- phology and many striking correlations of cell structure with physiology. From these studies the concept of the histological unit, the cell, emerged with increasing clarity (Figs. 6-1, 2A). A similar morphological picture of most cell types could be drawn from the researches of three-quarters of a century of cytology. Such a descrip- tion, of course, is compfetely unsatisfactory from the standpoint of intracellular biochemistry; indeed, it is merely an invitation to further research which might lead to more nearly complete understanding of the physiological processes by which such a cell can synthesize a secretory granule or can elaborate the material to duplicate itself. One obvious way in which to complete the picture is to attempt to determine the chemical composition of the cell. To a cytologist this problem emerges as one of demonstrating how the various substances, which biochemists obtain by such procedures as extraction from minced organs, are dis- tributed among the various visible cellular constitutents and in the apparently structureless material which fills spaces between the iniclei, mitochondria, granules, and other formed elements of the cell. Broadlj' regarded, this appears to be the field of what has come to be called 203 204 RADIATION BIOLOGY "cytochemistry," and in this chapter an attempt will be made to describe and to evalnate critically some of the approaches to this problem of chem- ical organization of the cell. 2. LAWS OF ABSORPTION Since cytological stndies are necessarily carried out at high magnifica- tion with a compound microscope the preparation, a section or smear of an organ, is always examined by transmitted light. For this reason the Fig. 6-1. Diagram of the structural features of a serous glandular cell at an early stage in restitution of the secretory granules, c, centriole; mg, mature zymogenic granule; ig, immature zymogenic granule; n, nvideus with masses of chromatin and a large spherical nucleolus; m, mitochondrion. technique of cytological microscopy is readilj^ adapted to photometric chemical analysis, in which the nature and amount of material may be determined from the spectral characteristics and intensity of the light which emerges from a semitransparent mass. This fraction of the light is said to be "transmitted"; that portion which entered the object but did not emerge is said to have been "absorbed." Application oi laws of absorption (see Chap. 5, this volume) not only make it possi- ble to use on slides qualitative and (juantitative methods of chemi- cal analysis, but these laws also apply directly to the visual examination of cells since the visibility of a natural or artificial color in a cell depends on whether the colored object absorbs enough light to make it distin- guishable from the surrounding nonal^sorhing regions. A CRITIQUE OF CYTOCHEMICAL METHODS 205 Fig. 6-2. A series of photomicrographs of pancreatic glandular cells, showing structure and the results of various techniques of localization of nucleic acids and proteins. (A) phase contrast, no stain or reaction; (B) Millon reaction for total protein; (C) nucleic acids stained specifically by basic dye, azure A; (D) ultraviolet photograph (254 m/u) showing absorption in regions of nucleic acid concentration; (E) digested with ribonuclease before staining with azure A [compare with (C)]; (F) Feulgen's nucieal reaction for deoxypentose. {Pollister et al., 1951.) Inferences from the more general laws of relation of mass to absorption of radiant energy are conveniently summarized by some simple equations, in which the following symbols are used: /o = light intensity (galvanometer reading) when no absorbing object is in the optical path (in cytological measurements, the reading through an empty part of the slide) ; Ix = intensity when the absorbing object or sample is in the optical pathway (in cytological measurements, the reading through the cell component) ; T = transmission, h/h (7c T = \00T); •JOG UADIATIU.N lllULOGV E = extinction, also called optical density, logio 1/7' or logio I d h; X = wave length; E\ = extinction at a given wave length, e.g.. A'b-.o niyu; {E\)2 = extinction for unit amount per unit area; c = concentration; / = length of absorbing pathway; .1 — area of absorbing mass in plane perpendicular to the absorbing pathway; V = volume of absorbing mass; A- = specific e.xtinction, e.g., (^^x)^^ cm- Beer's law deals with the relation of light lo.ss to concentration (c) ; Lambert's law deals with the relation of light loss to absorption path (/). A useful simple e(iuation which expresses the fact that extinction is directly proportional to the number of specifically absorbing chromo- phores (k) in the absorbing path, as determined by concentration and thickness is E = kcl. In most routine (juantitative photometric chemical analj'sis the sample is in a carefully measured cuvette or absorption cell (generally 1 cm thick). The unknown concentration (Cx) may be computed in terms of the extinction of a standard of unit thickness and concentration, e.g., (■^x)i.ncra» where E^ is the extinction of the unknown from the following formula. ^x % ('E'x)l.'oc In cytological preparations the thickness is always but a few microns. When this thickness has been measured, computation from a standard extinction must take into account the relative thickness. An equation by which, assuming the validity of Beer's law over the concentration range of which the two measurements are the extremes, the percentage concentration in a cytological preparation may be computed from the standard (£/x)L^°e„ is 10,000 (£:x), (^x /c ~ 7/Tn\l% h(E,)\, cm where l^ is the thickness of the cytological structure, in microns. Instead of using the .standards ol)tained in the cuvettes of a colorimeter or spectrophotometer, it is more convenient to compute special cyto- logical standards. Most useful is Ea computed as the extinction for 10"'" rfig/M", {E\)li ""~', in which Caspersson has expressed the results of his (juantitative cell analyses. In a thickness of 1 ^ this is equivalent to 10 per cent, to 100 mg^cc, or to 10~'" mg/;u^. This standard has been com- A CRITIQUE OF CYTOCHEMICAL METHODS 207 putcd from in vitro (l;it;i tor a number of naturally colorc^d .substances commonly found within cells [\\\i\ foi- some speeitic stains and tests for proteins and nucleic acids (columns 1, 2, and 3, Tables G-l, 2). The sources of the \alues from which computations were carried out are indi- cated in column 5.' These values in Tables 6-1 and 2 are not claimed to be necessarily close approximations of any physical constants of the intracellular substance. They are almost certainly subject to considerable revision as more is learned of the effects on absorption of high concentrations and of special intermolecular associations within the cell (see p. 215 and Chaps. 1 and 5, this volume). In the meantime, they are useful relative values from which the possibilities of seeing, or measuring photometrically, an intra- cellular substance under the microscope can readily be estimated; they are likewise the only method of translating results of intracellular absorp- tion measurements into the familiar values of chemical analysis, and in radiation experiments they can serve as the basis for an approximate esti- mate of the amount of energy absorbed per cell or cell part. Since the errors introduced by high concentrations and other special conditions within the cell tend, as a rule, to reduce the specific extinctions, it seems fairly safe to assume that the values in column 3 are maximal and those in column 4 are minimal. The great usefulness of the cytological stand- ard, {E\)a, arises from the fact that of itself extinction is a direct measure of the number of molecules in the absorbing path and can be used as such when neither concentration nor thickness of the absorbing area is known. From the standard {E\)lt '""'* the amount per total area of the part of the cell measured (^4) can be computed simply as {E\)a-A {A being meas- ured in square microns). Likewise, because extinction times area is the equivalent of amount, it can be used in simple arithmetical compu- tations to compare compositions of cytological objects in purely arbitrary amounts (see Swift, 1950). So far W'C have been considering photometric methods with measure- ment in a more or less restricted spectral region, a procedure which, some- what paradoxically, is often called colorimetry. .When measurements are made at many wave lengths, a picture is obtained of the etTect of the absorbing substance on the light, which expresses in objective data the phenomena which cause the visual sensation of color. Such data are often plotted as absorption curves, with some measure of relative light ^ The cytological standard, {E\)ll ^"'"^ is computed as follows, from a cuvette standard, e.g., for deoxyribonucleic acid (DXA) where {E2i4)l',o^ "" is 20. It is given that 20 is the E^hi of 1 yu^ of a standard solution in a thickness of 1.0 cm, lO"* m (for extinction depends on thickness and concentration and is independent of area). Each cubic centimeter of cuvette standard contains 1.0 mg of DX.\; 1 cc is 10'^ fx^, and the volume of a mass which is 1 m^ in area and 1 cm thick is 10^ n^. This volume of the standard solution then contains lOVlO'^, or lO"**, mg of DNA. Therefore, (Ei,,)lV'"'"^ is 20/102 or 0.200 (Table 6-1). 20S RADIATION HIOLOGY Tabi.k ()-i. N.\n K.M, Absokption of Substances Occurring in Cells Wave Concentration 8iil>.-;t;iii('i! length, m^ (E^y.r""' (%) in 1 M to give E\ of 0.030 Kcfcicncc Deoxyribonucleic acid . . 254 0.200 1.5 Ris (1947) DeoxvrilxJiiucleic acid. . 260 0.220 1.4 Caspersson (1940a) Deoxyril)()imcl('ic acitl , . 275 0.180 1.7 Caspersson (1940a) Ribonucleic acid 260 0 . 230 1.3 Thorell (1947) Tryptophane 275 0.270 1.1 Fig. ()-2 1 ; Caspersson (1940a) Tyrosine (acid) 275 0.065 4.6 Fig. 6-21; Caspersson (1940a) Tyrosine (alkali) 290 0 090 3.3 Fig. 6-21 ; Caspersson (1940a) Serum albumen 275 0.006 50.0 Fig. 6-5 Serum albumen 260 0.0016 Not detectable Fig. 6-5 Ascorbic acid 265 0.355 0.85 Stearns (1950) Thiamine 232 265 0.312 1.78 0 . 9() 0.17 Stearns (1950) Ril)()tlavin Stearns (1950) Vitamin A 324 1.82 0. 16 Stearns (1950) Oxyhcinofrlobin 413 0 . 069 4.3 Thorell (1947) Chlorophyll a 665 2.90 0.10 Zscheile (1934) Chlorophyll b 640 1.22 0.25 Zscheile (1934) Cytochrome c 415 0.665 0 . 46 Dixon et al. (1931) Table 6-2. Absorption of Cytochemical St.\ins and Tests Concen- Wave tration Substance length, m/x / 7,1 NlO^lOinK (%)inla to give E\ of 0.030 Reference Deoxyribonucleic acid, Feulgen reaction. . . . 546 0 200 1.5 Alfert, 1950 Tryosine, Millon reac- tion 365 0.220 1.4 Pollister (1950) Protein, Millon reac- tion (6.25% tyrosine) 365 0.014 21 0 Pollister (1950) Protein, Millon reac- tion (6.25% tyrosine) 490 0.007 43.8 Pollister (1950) Fast green, pH 2.0 ... . 625 1.6 0. 19 Bryan (1951) Egg albumen, fast 625 0.568 0.53 Computed from Fraenkel- green Conrat and Cooper (1944); Bryan, 1951 A CRITIQUE OF CYTOCHEMICAL METHODS 209 loss on the vertical axis, and wave length or frefjuency on the horizontal axis. The shape of the absorption curve is of qualitative value in identi- fying the specific absorbing atomic configuration, the chromophore. When a sample contains two or more nonreacting absorbing substances, the compound absorption curve results from addition of the individual components. If the curves of the chromophores are considerably differ- ent in absorption coefficient at some wave lengths, the compound curve can be analyzed into the individual curves of its components by solving simultaneous equations (Stearns, 1950). As Tables 6-1 and 2 indicate, in order to be detectable in the cell by absorption (i.e., by contrast), substances must reach a concentration many thousand times that which is sufficient for analysis in an absorption cuvette. This imposes a fundamental limitation on the whole method of interpreting the chemical composition of cellular components from visual microscopical appearance or, indeed, from the most careful microscopical absorption measurements with an objective photometer. Very small quantities can be detected, as little as 10~''* g in a single small granule, which may be perhaps no more than a millionth of the entire cell volume. Therefore, in one sense, these are very "sensitive" techniques in the vocabulary of the microchemist (see Benedetti-Pichler and Rachele, 1940). But, as a means of demonstrating a complete picture of distribu- tion of a substance within the cell, these microscopic methods are deplor- ably inadequate. Obviously, if the concentration within a small granule just reaches the threshold for detection (which experience shows to be roughly equivalent to an extinction of 0.03 or about 7 per cent absorption; see Tables 6-1 and 2, column 4), then outside this spot of high local con- centration there can be a relatively enormous amount of substance which is below the detectable absorption or contrast. It is a simple matter to compute in any cell, from data like those of Tables 6-1 and 2 and the volume relations within the cell, the maximum possible amount of sub- stance that could escape visualization or measurement. Conclusions about localization and distribution of intracellular substances from micro- scopical data must always take into account this interrelation between absorption and intracellular geometry. It must be emphasized that microscopic methods alone can prove neither the exclusive localization of a substance within a small intracellular structure nor the complete absence of a substance from any part of the cell.- Such conclusions can come only from a combination of methods of chemical analysis of cell isolates and cytology, as pointed out by Pollister, Himes, and Ornstein (1951). ^ One escape from tliis limitation on microscopic detection and estimation lies in developing stains which are fluorescent and tests based on fluorescence. Since fluorescence is seen or measure4 as total intensity against a dark field, i.e., zero inten- sity (instead of by subtraction from a field of high intensity), the dye or color reaction can be detected readily in concentrations as low as one one-thousandth of the minimum 210 UADlATlU.N JJlOLUCiV 3. LOCALIZATION OF SUBSTANCES IN CELLS It imisl not l)c' supposed that an awareness of the difficulties just suin- mari/ed lias operated to inhibit the development of a chemical cytology. (Juite the contrary has been the case; from the earliest days of cytology there have been attempts to supplement the morphological descriptions of cells by some idea of the chemical composition. A few substances such as hemoglobin or chlorophyll are visible in the living cell because of the natural color, but in most cases a substance can become visible only because of a color reaction carried out on a micro- scopic slide. To be useful cytochemically such a test or stain must ful- fill certain criteria: 1. It must be possible to carry out the test under conditions which will not seriously distort the cell morphology, a requirement which excludes a great many of the color reactions of analytical chemistry. 2. The specificity of the reaction must be known from data obtained in vitro. 3. The reaction must be one which will proceed without interference in the presence of large amounts of proteins, and often in the presence of nucleic acids or lipids. 3-1. PRESERVATION OF INTRACELLULAR SUBSTANCES IN SITU Lison (193G) has considered critically many of these so-called "histo- chemical" techniques; additional ones are briefly mentioned by Glick (1949). The microchemical tests for inorganic ions, such as ferric, chloride, and phosphate, and for smaller organic molecules, such as uric acid and oxalic acid, are for the most part closely analogous to those of microchemistry and cause the appearance on the slide of a colored pre- cipitate or crystals only when the reacting group is in solution. For cytological studies this means that the group will not react Avhen it is a part of a large molecule, such as that of protein, but only after it has been split off. These tests can undoubtedly be interpreted as an indication that the reacting substance is present in the section of tissue, but this is but a poor imitation of the precise conclusions which are possible from the methods of analysis of tissue masses (Hogeboom, 1951) or from refine- ment of microchemical methods to reach down to the level of a single cell (Xorberg, 1942). For cytological studies the intracellular localiza- tion is most important, and there are good reasons to (juestion the validity of methods of microscopical demonstration of small diffusible chemical detoetable by absorption. Tlic |)ossiI)iliti('s have not been widely explored, but a fluoiosceiit Scliiff reatfciit lias hccii dcNcloiM'd and found to dcnioustrate marked aldfliyde (jjlasnial?) reaction in cells where the test api)eared couipletely negative by ab.sorption contrast technique (L. Ornstein, unpublished). Fluorescent cytological techniques also offer one possibility of avoiding the distributional error (p. 235). A CRITIQUE OF CYTOCHEMICAL METHODS 211 entities by color reactions. Within the dimensions of the cell, diffusion — 11 slow process at the macroscopic lev^el — is practically instantaneous, and there is every reason to suppose that extensive redistribution takes place, either as tissue is being fixed or as the test is carried out on the tissue. This is particularly misleading because the precipitates tend to be adsorbed on the extensive internal surfaces of the denser parts of the cell such as nuclei, myofibrillae, or the thick distal borders of some epithelial cells. Thus many early observers erroneously reported that the nucleus contained considerable iron, an element in which it is actually notably deficient. The nucleus was repeatedly described as the site of the enzyme alkaline phosphatase, since phosphate split off from a substrate, glycero- phosphate, appeared as an intranuclear precipitate, but Jacoby and Martin (1949) have demonstrated that this is largely a secondary accumu- lation of the phosphate (see also Novikoff, 1951, 1952). Redistribution during the fixation process is effectively prevented by the freeze-dry method of preparing sections of tissue, which was suggested by Altmann (1890) and elaborated by Gersh (1932), Hoerr (1936), Simp- son (1941), and others. In this technique fresh tissue is quickly frozen at such low temperatures ( — 190°C) that ice crystals do not form (the water appears rather to be practically ^^itrified). Tissue is then dehy- drated at low pressure and temperature and is finally embedded in paraf- fin and sectioned. Up to this stage, it is generally agreed that little redistribution of intracellular chemical constituents can have taken place, and two excellent methods of elementary chemical analysis apparently can be carried out without producing any essential change in this dis- tribution. The paraffin sections may be burned in an electric furnace (microincinerated; Policard, 1923; Scott, 1943), and the appearance and amount of the ash indicate the distribution of the mineral elements in the cells (e.g., iron is a yellow to red ash, silicates are crystalline, and calcium and magnesium are amorphous and dense white ashes.) In another method the paraffin is removed from the section, and the tissue is dried and subjected in vacuo to X-ray absorption analysis (Engstrom, 1946, 1950). Although it involves immense technical difficulties, the latter appears to be an extremely promising approach since it offers the possi- bility both of determination of cell mass from polychromatic X-ray absorption data and of analysis for a large number of individual elements from absorption of monochromatic X rays in the wave length range 2-50 A. The distribution in frozen-dried sections would also be expected to remain essentially unchanged, except for lipoidal constituents, if the paraffin is removed — for instance, by chloroform — and the section kept in nonaqueous solvents such as alcohol and glycerin. This procedure has been recommended in preparing material for ultraviolet absorption studies of intracellular proteins (Caspersson, 1947) principally because 212 UADIATIO.N HlOLOGV the optical coiKlitioiis within the tissue arc tlicii more i'ax'orablc loi' tlu-.so. measurcnuMits. This aclvaiitufj;c would appear to ho somewhat olTset by the confusion which is introduced into the absorption picture hy the probable presence, in unextracted material, of many absorbinji, com- pounds of low molecular wci<>;lit, a situation like that which makes the results of ultraviolet absorption stud}'' of li\iiiji; cells so inconclusive. When precipitation or a color reaction on the microscopic section necessarily invoh'es use of a(|ueous reagents, then the frozen-dried sec- tions must be rehydrated. This in effect appears to eliminate about every advantage of the freeze-drying technicjue because extensive redistribution can take place at once and can continue dining any subsecjuent steps of the technifjue (Iloerr, 1943). Unless these technical difficulties can be overcome, the sound view seems to be to admit that, except for micro- incineration and X-ray absorption, precise intracellular localization is practically limited to substances of high molecular weight, which diffuse slowly and are easily converted into relatively insoluble masses, and to the use of tests which demonstrate the smaller chemical groupings when they are parts of these large molecules. Two substances of this charac- ter, proteins and nucleic acids, bulk large in the composition of all cells. Indeed, it is easy to see that, if these two constituents are removed, as can be done with enzymes, the remainder is but an unrecognizable ghost of a cell. As a matter of fact, the whole concept of the fixed cell is mainly that of a nucleoprotein mass. Except for some of the special mito- chondrial methods which preserve lipids, the vast majority of cellular studies have been made on cells fixed in strongly acid fluids which, while precipitating admirably proteins and nucleic acids, at the same time must wash out smaller unattached cellular constituents, organic or inorganic, to such an extent that the residue can hardly be great enough to be cyto- logically detectable (see Pollister, 1952a). This predominance of nucleo- protein in cellular composition has always been so obvious that it is possi- ble to overlook its significance to cytochemistry. For example, in Lison's Histochemie animale (1936) this point is not stressed, and there is a distinct impression that the primary concern of histochemists should l)e the localization of simple chemicals such as iron and amino acids. Whatever the special objectives of a study of intracellular localization, a very obvious fact about cellular composition is that, in all cells, proteins and nucleic acids occur in high enough concentration so that tests for them fall within the visible, or measurable, range, and consequently tech- nifiues for nucleoprotein demonstration are applicable to a great variety of problems. 3-2. NUCLEIC ACID STAINING AND TESTS Although the major features of morphology are distinct in living cells and in fixed uncolored cells, the bulk of cytological researches have been A CRITIQUE OF CYTOCHEMICAL METHODS 213 made with the advantage of artificial contrast which is introduced by .staining or developing a metallic precipitate in c(>ll c()mpf)ncnts.'' One of the oldest of these techniques is l)asic staining, which involves the use of dye salts which, upon dissociation, carry the ctjlor, the chromophore, in the cation and which therefore form colored salts with the anions of strongly acid substances within the cell (Fig. 6-2C). It was early recog- nized (e.g., Mathews, 1898) that, if basic dyes were applied in acid solu- tion (after appropriate fixation), this property of cell substances, which is included in the general term "basophilia" of tissues, constituted in effect a test for strongly acidic substances. Specifically, in animal cells these include the relatively uncommon sulfuric acid esters of polysac- charides and the phosphoproteins plus the universal cell constituents nucleic acids, w^hich are orthophosphoric esters of nucleosides. There were many early cytochemical researches based on the supposition that basophilia indicated the intracellular distribution of nucleic acid. One outstanding example is the so-called "chromidial hypothesis," which as applied to metazoan cells held that the basophilia of cytoplasmic struc- tures was evidence of their origin from the nuclear chromatin (see Wilson, 1925, pp. 700^.). However, most cytologists used basic staining so as to achieve maximum contrast for morphological studies (e.g., iron hema- toxylin, applied to material which had been fixed in reagents containing chromic acid). Such technique departed widely from the strict criteria laid down by Alathews for specific staining by salt formation between basic dyes and nucleic acids, and undoubtedly the increased contrast was to a large extent due to adsorption of dye rather than chemical staining (Pollister, 1952a). This distinction was rarely appreciated, however, either by cytologists themselves or by others interested in the chemistry of the cell, and as a result there developed a widespread distrust of attaching any chemical significance whatsoever to the basophilic reac- tion. This was not dissipated even when van Herwerden (1913, 1914) developed the nuclease technique for identification of intracellular nucleic acid basophilia. The modern use of basophilia for localization of nucleic acids (Mazia and Jaeger, 1939; Brachet, 1942; PoUister, 1950; Kaufmann et al., 1951) stems directly from van Herwerden's work but rests on a much firmer biochemical basis since it is now known that: 1. There are actually two nucleic acids, the pentose type — ribose (RNA), plasmonucleic acid — found in the cytoplasm, nucleolus, and to some extent in chromatin, and the deoxypentose type — deoxyribose (DNA), chromonucleic acid — which is normally restricted to chromatin of the nucleus (Davidson, 1950). ■^ The development of phase contrast microscopy fsee Fip;. 6-2.4 and Bennett et al., 1951) has nearly freed cytologists from the necessity of introducing artificial contrast by these methods. 211 K \1)1 AI'lON HI()I,<)(;V ■J. Thcic arc specific enzymes, rihoiiuclease and deoxyrihonuclease, which act to detiiadc each type of nncieic acid. 3. The total nucleic- acid content may he renio\c(l specifically hy chemi- cal ajj;ents such as trichloroacetic acid. Thus, sites of nucleic acid are readily identifiable a.s parts of the cell with basophilia which is removable by acid extraction, and the type of nuclease susceptibility shows which of the two nucleic acids is present (cf. Figs. 6-2C, F and 5a, /;). Such specific nucleic acid basophilia adds to cell morphology an impor- tant chemical datum, showiiif^ that, in a cell such as that of Fig. G-1, for example, there is undoubtedly considerable nucleic acid in the basal zone and nucleolus as well as in the chromatin. More precisely, from such a cytological preparation as Fig. (3-20 it can be concluded that, in these parts of the cell, the nucleic acid concentration is so high that the dye bound as dye nucleate is in high enough concentration to appear as strong visible color in structures no more than o ij. thick (p. 209). To what extent does this approach a complete picture of the distribution of major polynu- cleotide concentrations within the cell? It must be emphasized that basic staining does not lead to localization of nucleic acid by any of its natural physical properties in the same manner as the natural green color indicates the sites of chlorophyll. Instead, visualization by basophilia depends on the capacity of nucleic acid to bind the cations of basic dyes, which may be mainly through displacement of protein from its natural combina- tion with the residual phosphoric acid valencies of the polynucleotide. This staining reaction may therefore be very complex, and the relation between color and amount of substrate may by no means necessarily be a simple one. From basophilia alone it is impossible to answer such questions as: How strict is the proportionality between basophilia and nucleic acid concentration? Is this proportionality constant or highly variable? What interpretations may be assigned to negative basophilia? Can there be considerable accumulations of polynucleotide which are unaccompanied by any basophilia? Is an increase or decrease of baso- philia due to change in amount of nucleic acid or to change in the number of phosphoric acid valences which are available for dye binding? The transition from a cytological to a cytochemical viewpoint poses all such questions and immediately reveals the danger inherent in uncontrolled cytochemical use of staining reactions. Only by an independent method which measures nucleic acid directly can these questions be answered, and in natural ultraviolet absorption of nucleic acid such a method is available (see Sect. 3-4). In general, regions of strong nucleic acid ultraviolet absorption have been found to coincide with those of pronounced baso- philia (see Figs. 6-2C, D), but there are indications that the amount of dye bound in cells for a given amount of pentose polynucleotide is variable (M. H. Flax, unpublished data), and one extreme case has been reported A CRITIQUE OF CYTOCHEMICAL METHODS 215 ill which there was nearly negative cytoplasmic basophilia in cells which, by ultraviolet absorption, were shown to contain considerable nucleic acid (Pollister . . . Breakstone, 1951). In view of these difficulties it is per- haps best to regard basophilia as a useful indicator of sites of major nucleic acid concentration, which then becomes a guide to application of more satisfactory (lualitative and c^uantitative methods. In fact, this has been the role of l)asophilia in development of modern concepts of the intracel- lular distribution of nucleic acid. Although the methods of identification by the ultraviolet absorption spectrum measure nucleic acid directly and are therefore potentially applicable to situations where polynucleotide basophilia might be misleading, for the most part ultraviolet studies have proceeded along lines which were clearly foreshadowed by old findings of the basophilic reactions of cells. This is strikingly emphasized also by the fact that practically all the fundamental conclusions about intracellu- lar distribution of nucleic acids which the Caspersson school reached by use of ultraviolet absorption techniques and used in elaborating compre- hensive theories of cell function (see Caspersson, 1950) were arrived at independently by Brachet and his collaborators (Braehet, 1944) with only nuclease-digestible basophilia as a guide. Indeed, since Brachet and coworkers used nucleases in combination with basophilia, they were able to detect ribonucleic acid in chromatin, while it was necessarily overlooked in the less specific ultraviolet absorption studies. There are certain applications of basophilia to (lualitative cytochem- istry which are of special interest because not only do they demonstrate the presence of strongly acidic substances, but also by specific color changes they appear to indicate something of the intramolecular structure of the acidic substrate with which they combine. The best known of these is the so-called "metachromatic" basophilia (metachromasia) by which certain cellular structures stain red with dyes which appear blue in solution (e.g., toluidine blue and azure). This method was empirically recognized long ago (Ehrlich, 1877; Hoyer, 1890). It has been the sub- ject of a number of chemical and spectrophotometric studies (e.g., Kelley and Miller, 1935a, b; Lison, 1935; Bank and Bungenberg de Jong, 1939; Wislocki et al., 1947; Michaelis and Granick, 1945). Spectrophotometric analysis shows that, whenever these dyes are in water solution, three states are in ecjuilibrium. An a absorption peak in the red part of the spectrum represents unaggregated dye in the "monomeric" state; a 0 absorption peak (green) is believed to represent dye in the two-molecule aggregate, or "dimer," state. A /x absorption peak (in the blue-green) supposedly represents highei- states of aggregation than the dimer which may for conv^enience be called a "polymer" state. The aggregation, as would be expected, is dependent on concentration, with the result that these basic dyes notably fail to follow Beer's law. In stained cells, basic dyes are, of course, removed from solution and combined with the solid 210 RADIATION BIOLOGY coagulu or i)iecipitates of the substrates. This suggests that the sub- strates \\ liich are colored red have bound the dye in such steric relation that the dimor and polymer association occur, while those colored blue have the dyr molecules more widely separated so that substantially all the color is due to the blue monomer. According to this interpretation, the color of the dye is in effect a reflection of the intramolecular structure of the substrate. The polysaccharide sulfuric esters (e.g., chondroitin sul- fate of connective tissue) are decidedly metachromatic. A careful spec- trophotometric analysis has shown that a metachromasia distinguishes RXA from DNA, a difference which presumably is related to the highly branched structure of the former (Flax and Himes, 1950, 1952). Methyl green basophilia is another staining reaction which appears to reflect the intramolecular configuration of the substrate. This dye stains normal DNA; with i-are exceptions it does not stain UNA. In vitro the formation of the salt methyl green-deoxyribonucleate is depend- ent on the nucleic acid being in a state which forms highly \'iscous solu- tions (Kurnick, 1947, 1949; Kurnick and Mirsky, 1949), and therefore reduction or loss of methyl green basophilia of nuclei has been interpreted as evidence of a physical change in the DNA molecule which is similar to that which is accompanied by loss of viscosity of solutions of the acid, a change which is usually called " depolymerization " (Pollister and Leuchtenberger, 1949; Leuchtenberger, 1950; Leuchtenberger e/ a/., 1949; Harrington and Koza, 1951). Such changes in methyl green basophilia of nuclei have been noted to result from experimental treatment (heat, deoxyribonuclease digestion, ionizing radiation) and also to accompany pathological nuclear degeneration. 3-2a. Nucleal Reaction. Goldschmidt (1904) and the other adherents of the views embodied in what was called the "chromidial hypothesis" believed that they had in basophilia a sort of qualitative test for chro- matin by which they could detect this substance even after its extrusion from the nuclei into the cytoplasm. This was an over-optimistic point of view and led to widespread distrust of cytochemical conclusions from staining I'esults. Feulgen and Uossenbeck (1924) developed a specific cytochemical test foi' chromatin (Fig. i\-'2F) which not onl,y finally dis- posed of the chromidial hypothesis, sensu strictu, but also led to great strides in clarification of the whole problem of intracellular distribution of luicleic acids. For nearly thirty years two different nucleic acids had been recognized by chemists. One, obtainable in (juantity from yeast and often called yeast nucleic acid, had been shown to contain a pentose; the other, identical with the acidic component of Miescher's iniclein but usually later obtained from the thymus gland, contained a sugar that was clearly not a pentose and was generally considered to be sort of hexose (see Le\'ene and Bass, 1931; Dax'idson, 1950). Feulgen and Rossenbcck discovered that mild acid hydrolysis, which was known to split off the A CUlTUjUE OF CYTOCHEMICAL METHODS 217 purine bases from nucleic acid, changed the thymus nucleic acid so that it gave a positive Schiff reaction for aldehydes — a restoration of color to a reduced leukofuchsin, and the}^ demonstrated that this reaction could he carried out not onl}^ in vitro but also on tissue sections in which it colored brilliantly the chromatin of the cell nuclei. For this reason they called it the " nucleal reaction. " C'ytologists were quick to try this new technique, and in a few years the reaction had been demonstrated to be positive on the tissues of a wide variety of animals and plants (Milovidov, 1936). This is an ideal (lualitative chemical reaction, highly specific (Fig. 6-2F) for the unique substance of chromatin which Levene et al. (1930) even- tually showed to be deoxypentonucleic acid (DNA), not a hexose polynu- cleotide. The intense color (Table 6-2) is one of its important character- istics. The Feulgen reaction at once demonstrated that the major basophilic component of chromatin and chromosomes of both plants and animals is always DNA and that the cytoplasm never contains this in detectable amount. This was eventually fully confirmed by the analysis of isolated nuclei and cytoplasm (Feulgen et al., 1937; Hogeboom et al., 1948). It thus became quite clear that, whatever the nature of the basophilic substance in the cytoplasm, it was certainly not the same as the nucleinic acid of Miescher and Altmann. When, therefore, as a result of the earlier discoveries of pentoses and purine bases in the cytoplasm of developing eggs, Brachet (1942) was led to reintroduce on a wide scale van Herwerden's (1914) long-forgotten nuclease technique and conclude therefrom that the Feulgen-negative basophilic substance of the cyto- plasm was a pentose nucleic acid, it was not seriously questioned; like- wise, no objections were raised when Caspersson and Schultz (1939) stressed the fact that the ultraviolet absorption spectrum of these same basophilic areas must be due to pentose nucleic acid because these parts of the cell were Feulgen negative. Like basophilia, the Feulgen reaction is not directly dependent on a physical property of nucleic acid but on a chemical reaction which the acid can give after removal of the purines from a part of the nucleotides. (From the results of Stacey et al, 1946, it appears that the deoxypentose undergoes considerable intramolecular change to become a substance which is capable of recolorizing the Schiff reagent.) It must be understood that the very useful specificity, which rarely has been seriously cjuestioned, resides in the release of reactive groups as a result of the hydrolytic process, and it is customary to stain simultaneously a control slide which has not been hydrolyzed. 3-3. PROTEIN STAINING AND TESTS Although the bulk of the solid matter of the cell is protein, the prospect for fruitful cytochemical protein analysis is by no means as bright as for nucleic acids. It is certain that this protein mass must be a very hetero- geneous mixture, including countless intracellular enzymes and other pro- 218 RADIATION HIOLOGY tein.s of special fuiiction. Most of these may never be hifi,hly coiiceiitrated ill aii>' one part of (he cell. Even if such an accinnuhitioii did occui, the correlation of specific function with icadily accessihh' asjiects of the chemistry of the protein molecuk! is rarely so definite! as to offer hope of localizing many specific proteins by techni(|ues similar to those which have just l)een tlescribed for nucleic acids. Natural color, as in hemoglobin, of course, ofTers one opportunity for a microscopic approach (Thorell, 1947). For the most part, however, methods of microscopic analysis of proteins cannot be expected to give more than information concerning the approxi- mate total amount of the protein mass, the fractionation of which on a microscopic slide is possible to but a very limited extent. For example, a considerable proportion of the histone of chromatin is split off readily (PoUister and Ris, 1947). Nevertheless, the approximate analysis of total protein is information of considerable importance to the broad fiucs- tion of protein synthesis as the prime chemical achievement in growth, cell division, and secretory activity (Caspersson, 1950; Pollister, 1954). Most methods for protein are not nearly so sensitive as are basophilia and the Feulgen reaction for nucleic acids since the special reactions are almost entirely those of groups at the omega ends of the amino acid resi- dues, and in most proteins (protamine being one exception) no specific reacting group makes up more than a small fraction of the total rmmber of amino acid residvies. The reactive groups which have been used cyto- logically are (a) the dibasic amino acids arginine (Serra, 1944; Thomas, 1946) and histidine (acidophilia, p. 219); (6) the dicarboxylic amino acid glutamic acid (by alkaline basophilia, Dempsey and Singer, 1946) ; (c) the sulfur-containing amino acids cystine and cysteine (see Lison, 1936; Bennett, 1948) ; and (d) the aromatic amino acids tyrosine, tryptophane, and phenylalanine (see p. 222). Specific reactions for proteins in cytological material are all adapta- tions of well-known spot tests. One of the oldest of these is the Millon reaction for tyrosine and tryptophane (Fig. 6-2B), which was usedbyLeit- geb to identify the nature of crystals in plant cells as early as 1888. No Millon test is impressive under the microscope, partly because the protein cannot possibly be concentrated enough to give a strong visible reaction on a slide and partly because, since everything is colored, the observer does not have the benefit of the contrast to which he is accustomed in a stained preparation. The test material, if present, is, of course, readily detectable microscopically by objective photometric measurements. The sensitivity of the Millon reaction in visible light is low (Table 6-2). At the visible absorption peak (490 m/x), for a protein assumed to give a Millon reaction ecjuivalent to 6.25 per cent tyrosine, the £"490 is 0.007, and the protein would have to reach a concentration of over 40 per cent to give a detectable extinction, 0.030, in a thickness of 1 m- (At the natural ultraviolet absorption peak, 275 m^i, the absorption is no more intense.) A CRITIQUE OF CYTOCHEMICAL METHODS 219 The sensitivity of the Milloii reaction is nearly doubled if it is measured at 365 mn, near the peak in the near ultraviolet (Table 6-2 and Fig. 6-13). By contrast with the specific spot tests, the widely used method of con- trast by acid dyes (in which the color is carried in the anion) gives readily detectable color in parts of the cell where protein is concentrated (Table 6-2). For this reason, a dye of color roughly complementary to the basic dye for polynucleotide is often used in preparing microscopic slides for histological or pathological examination (e.g., basic methyl green and acid fuchsin or basic hematoxylin and acid eosin). It has long been known that this acid dye staining probably has a sound chemical basis (Mathews, 1898), that it is essentially the binding of the dye anions by the cationic groups (NH^) of the diamino acids of the protein to form a salt (for example, what we may call, for convenience, protein fuchsinate with acid fuchsin). There is evidence for the chemical basis of this stain- ing reaction in demonstrations that in vitro protein binds acid dyes stoichiometrically (Chapman et al., 1927; Fraenkel-Conrat and Cooper, 1944). The specificity of acidophilia for the amino groups of protein has been demonstrated by Monne and Slautterback (1951), who showed that deamination removes the acidophilia. As with basophilia the staining must be carried out in acid solution (pH 1.5-2.0) so as to preclude binding of the dye in other than salt formation (Mathews, 1898; Leuchtenberger and Schrader, 1950). The basic amino acids constitute so large a propor- tion of the composition of nearly every protein that acidophilia should be a more sensitive test for proteins than the Feulgen reaction is for DNA (e.g., fast green in Table 6-2). Since protein acidophilia is a measure of diamino acids, it can be employed, in conjunction with either the Millon reaction or ultraviolet absorption, to localize proteins of basic character (Leuchtenberger and Schrader, 1950). All the protein methods just discussed determine the presence of pro- tein only indirectly, through using one of its side groups to develop color in a reagent, or to bind a dye. Hence, any semiquantitative conclusions drawn therefrom are subject to all the possible errors which have been discussed earlier (p. 214). Protein can be measured directly by the natural ultraviolet absorption spectrum of its aromatic amino acids (Chap. 5, this volume, and Fig. 6-3). The microscopic techniciue for this has been developed by Caspersson (1940a, 1950), and, as usually carried out, the data obtained are absorption curves of masses of nucleoprotein. The qualitative and semiquantitative ultraviolet methods for these two major cell components, nucleic acid and protein, are therefore discussed together in the next section. 3-4. ULTRAVIOLET ABSORPTION OF NUCLEIC ACIDS AND PROTEINS While chromatin is quite colorless, in the ultraviolet spectrum — at a wave length a little shorter than the region transmitted b}'- glass — it can 220 RADIATION BIOLOGY be seen that it absorbs heavily, as first shown by Kohler (1904). Thus, if the cell is studied \isually on the fluorescent screen or phot(jf^raphed by ultraviolet lifiht, the nucleus is dark and is often fully as sharply con- trasted with the lighter background as it would be if the chromatin had been stained with a basic dye (Fig. (i-ID). Of the many observers who wvvv im})iessed with the strikinj^ contrast shown by chromatin under ultra\iolet examination, none seems to have realized that this was due to a physical property of luicleic acid until Caspersson published his thesis on the chemical composition of structures of the cell nucleus (1930). In 2200 2400 2600 2800 WAVE LENGTH, A 3000 220 300 Fig. 6-3. Ultraviolet absorption curve of a 0.02 per cent deoxyribonucleic acid solu- tion (I) compared with that of a 0.2 per cent solution of serum albumen (II). Curves were measured with a Beckman spectrophotometer in a cuvette 1 mm thick. (After Thorell, 1947.) 240 260 280 WAVE LENGTH, m/^ Fig. 6-4. Ultraviolet absorption curves of the salivary gland of Dro.sophila. Curve I is through a chromosome band ; II is through an adjacent part of the cell outside the chromosome; III is computed for a 10 per cent solution of nucleic acid. (After Caspersson, 1936.) this landmark in cytochemistry, Caspersson pointed out that nucleic acid has so strong a natural specific ultraviolet absorption that it can account for the great contrast of ultraviolet pictures (Chap. 5, this vol- ume; Table 6-3 and Fig. 6-3); he showed that the absorption curve through a single chromosome (Fig. 6-4) closelj^ resembles that of nucleic acid, not protein; and he confirmed the opinion that protein could account for but little of the absorption, by digesting the protein with little effect upon the ultraviolet contrast. Xumerous subseriuent publications of Caspersson and his coworkers have made it amply clear that regions of strong ultraviolet contrast in cells are, as a rule, sites of high nucleic acid con('entration (as seen in Figs. 6-2C, D). However, it is a mistake to sup- pose that such ultraviolet contrast is necessarily an accurate reflection of the intracellular nucleic acid distribution, for in each region the density A CRITIQUE OF CYTOCHEMICAL METHODS 221 .shows only the total light loss. In ultraviolet light of wave length near that of nucleic acid absorption a large part of the light lo.ss, particularly in fixed preparations, may he of nonspecific character mainly as a result Table (i-3. Beer's Law for Nucleic Acid (After Thorell, 1947.) Deoxyribonucleic acid Ribonucleic acid Concen- tration, % Layer thickness, mm E k Concen- tration, % Layer thickness, mm E k 2 0.01 0.01 3.17 0 . 37.3 0.600 1.86 1.89 2.5 0.005 0.01 3.17 0.550 0.340 2.20 2.15 The extinction {E) and absorption constant (A-) of RNA and DNA at concentra- tions of 0.005-2.5 per cent. Preparation according to Hammarsten (1924). Table 6-4. Ultraviolet Absorption of Maize Nucleoli (After Polli.ster and Leuchtenberger, 1949b.) Experiment No. Number measured Nucleotides removed by Em Percentage reduction of extinction 1428E-3 30 30 23 10 31 31 31 17 0.690 ± 0.011 0.347 + 0.011 0.847 ± 0.010 0.407 ± 0.009 1.000 ± 0.014 0.860 + 0,011 0.472 ± 0.008 0.843 ± 0.016 0.395 + 0.010 1428E-3 1428E-7 Hot TCA 50.0 1428E-7 1428E-5 Hot TCA 53.4 1428E-5 1428E-5 1428E-6 Cold TCA Hot TCA 52.8 1428E-6 Ribonuclease 52.8 The reproducibility of the effect of the enzymatic and chemical (trichloroacetic acid, TCA) removal of nucleic acid upon the extinction {E-za) of whole nucleoli of pollen mother cells of Zea mays. (In experiment 1428E-3 the extinction is lower because a very large central cylinder was measured; in 1428E-5 the higher extinction results from using a smaller cylinder.) In each experiment the percentage reduction of extinction indicates the proportion of the light loss (specific and nonspecific) due to ribose polynucleotide. The residual light loss (46-50 per cent) is largely non- specific, owing to high protein concentration. of scattering by the dense protein mass (see Table 6-4 and Fig. 6-56). Specific nucleic acid absorption may be recognized from the shape of the absorption curve (Caspersson, 1950, and Fig. 6-3). Also, the specific polynucleotide absorption can readily be dissociated from the absorption OO') UADIA rioX UK )!.()(; Y aiul scattering of protein by making two photographs or absorption measurements of the same cell, one before and the other after enzymatic or chemical rem()\al of the nucleic acid (see p. 231, Table 0-4, and Fig. 6-5a, 6). Since the difference between the two is (hrectly dependent upon the natural absorption of the purine and pyrimidine components of the nucleic acid, the latter technicjue provides an easy and sure method of obtaining evidence of intracellular nucleic acid distribution. The sensi- tivity is comparable with that of the Feulgen reaction (Tables 6-1, 2). In the specificity of the nucleases lies the possibility of overcoming the (a) (6) Fig. 6-5. Test, left, and blank for nucleic acid determination by ultraviolet absorption. Photographs, at 254 mn, of a maize pollen mother cell (No. 14281'>3-54) taken before (a) and after (b) the section had been subjected to hot 5 per cent trichloroacetic acid to extract all polj'nucleotide. The change in density of the spherical nucleolus is marked. By direct measurement of this nucleolus (Pollister and Leuchtenberger, 1949b) it was found that the extinction of a central cylinder through a was 0.750, that through b was 0.405, the difference being 0.345. These three values are assumed to represent, respectively: (1) total specific and nonspecific light loss in the part of the nucleolus measured; (2) light loss due to protein, mainly nonspecific; and (3) the light lo.ss due to polynucleotide, mainly specific absorption. major disadvantage that ultraviolet absorption alone does not discrimi- nate between RNA and DXA (Davidson, 1947; Pollister, 1950). None of the ultraviolet absorption curves of cell structures is exactly like the curve of pure nucleic acid; there is always distortion, certainly due in part to the associated protein, which characteristically exhibits specific absorption in the region of 2750 A owing to its content of aromatic amino acids (Chap. 5, this volume). Since these constitute but a small percentage of the total amino acid content, the specific absorption of proteins is very low in comparison with that of nucleic acid (Table 6-1 and Fig. 6-3), and, within the region of nucleoprotein absorption which has been most studied (2500-2800 A), protein must be present in 20-50 A CRITIQUE OF CYTOCHEMICAL METHODS 223 times the coiicentratioii of the nucleic acid to cause an equivalent ultra- violet light loss. The distortion of the nucleic acid curve means therefore that in the cell the nucleic acid is always accompanied by at least several times as much protein. The intracellular nucleoprotein curves published by the Caspersson group are of two distinctly different types (I and II, Fig. 6-6). In type I the nucleic acid peak is broadened, and the whole right shoulder is shifted toward the longer wave lengths. This shape is not unex- pected for nucleoprotein; it seems to be simply the summation of a nucleic acid curve and that of a common protein type, hke serum albumen (Fig. 6-3). On this basis, cell regions showing this type ab- sorption have been interpreted as sites where nucleic acid and a typical acid pro- tein ("globulin type") occur together. The type II curves are very different and puzzling; there is less broadening of the nucleic acid peak, and within the long- wave-length slope a second peak is indi- cated by a distinct shoulder. For a variety of reasons (see Caspersson and Thorell, 1941), curve II has been held to localize nucleic acid accompanied by markedly basic protein, called "histone type" or "diamino-acid-rich" protein (Caspersson, 1940a, 1950). The strongest evidence for this interpretation of the type II curves was that certain nucleohistone prepara- tions showed a protein peak apparently shifted toward 2900 A. However, when histones cjuite free of nucleic acid were finally obtained, it was found that they did not show such a shift of absorption, the peak being near 2750 A as in typical pro- teins (Mirsky and Pollister, 1943, 1946). There remains therefore no certain expla- nation of the peculiar shape of the type II curves. The nucleoprotein curves have been the basis for detailed speculations concerning the roles of basic and acid proteins in cellular physiology (Caspersson, 1950). For the general cytologist, perhaps the greatest significance of these curves is that they emphasize unmistakably what 2400 2600 2800 3000 WAVE LENGTH, A Fig. 6-6. Absorption curves of the cytoplasm of cells of dif- ferentiated (I) and undifferen- tiated (II) renal tubules of the chick embryo. Curves of the I type are considered to repre- sent ribonucleic acid with pro- tein of the "globulin type"; the type II curves are believed to indicate the presence of ribonu- cleic acid accompanied by con- siderable basic protein, "histone type." {After Caspersson and Thorell, 1941.) '22 \ RADIATION HIOLOGY inifj;lit otherwise be overlooked, namely, thiit, however striking the baso- philia and ultraviolet absorption of nuclcotide-rich parts of the cell may be. this by no means signifies that they are strongly acidic pools of nearly pun^ nucleic acid. Instead, the al)sorption curves clearly show that pro- tein is usually present in much greater (|uantity than the nucleic acid. This situation must always be recalled in any attempt to interpret chem- ically the results of staining reactions, and it must be the starting point for all speculations concerning the role of nucleoproteins in intracellular physiology (see Pollister, 1952b). 4. QUANTITATIVE MICROSCOPICAL METHODS l-l. VISUAL COMPARISOX The (jualitativc c,ytological methods which have just been discussed lead to localization of a substance within a cell by its absorption, "con- trast" in the language of a microscopist, which is detectable visually. Cytologists often speak of the intensity of a stain or color reaction as weak, strong, very dark, etc. Of course these terms imply semicjuantita- tive evaluation of the concentration of the component which is responsible for the color. When two similar objects are side by side in an evenly illuminated microscopic field, or in the two half fields of a comparison eye- piece, visual matching appears to be as accurate as objective photometric measurements. For example, with the comparison eyepiece, Bauer (1932) arranged a series of slides in order of intensity and was thus able to work out the relation of intensity of the Feulgen reaction to time of hydrolysis, which was essentially like that later worked out by Di Stefano (1949) from photoelectric measurements. For objects of the same size proper visual comparison is, then, a rough indication of relative amounts — if two objects are equally dark the}^ may be assumed to have approx- imately the same amount, and if they are different, the darker one may be said to contain more reacting substance. The same conclusions regarding relative concentrations may be drawn of two bodies of eqwdl vertical thickness (equal absorbing path). More often, the (juantitative question which faces a eytologist cannot be answered, even roughly, by visual comparison. For example, one often wishes to know relative amounts in two objects of very different size. It is uncertain to what extent by visual study a microscopist can determine whether two such une(iual objects have the same intensity (a rare condition probably); for the relati\'e sizes of the contrasting surroundings introduce considerable difficulty. If this match could be accurately made, a fairly good estimate of how much more the larger object contained could be computed from the dimensions of the objects. The examples cited illustrate the range of visual microscopic com- parison. If, to mention a very common experience, one cytological A CRITigi'E OF CYTOCIIEMICAL METHODS 225 object is both larger and more lightly colored than another, the cytolosist is almost completely helpless to answer the obvious (juestion of whether the decrease of color is entirely due to dilution in the larger mass. The relation of volume to light absorption is easily computed from an actual figure, a measurement of extinction, but such a (juantitative datum is absolutely necessary. No amount of experience can train a cyto- logist's eye to operate as a microscopic photometric device. These measurements must be made with objective photometers, with which a transmission is measured from which concentration may be estimated and amount computed (where the form and homogeneity of the cytological object are favorable). 4-2. PHOTOMETRIC TECHNIQUE The simplest photometer is a photographic plate, which can be used to determine relative intensities, from comparison with a density-intensity calibration curve. This curve may be independently measured and used for a whole series of plates (Caspersson, 1936; Pierce and Nachtrieb, 1941) or may be measured from a series of intensities through a rotating sector (Cole and Brackett, 1940) or a calibrated wedge (Uber, 1939) which is photographed on the same plate with the cells. The photographic method seems at first glance easy and obvious, a simple modification of the technique of photomicrography. For accurate results, however, it is far more complicated. Plate exposure and development must be rigidly standardized, and the negative density must be measured with a fairly elaborate photoelectric apparatus. The latter must, in fact, be nearly as sensitive as a photometer for direct measurement of microscopic slides — hence, in most cytological w^ork, the latter is an easier technique. Photographic photometry is indispensable for some problems, for example, where ultraviolet absorption measurements are to be made upon living cells (Thorell, 1947; Malmgren and Heden, 1947; Mellors et al, 1950). For direct absorption measurements of fixed preparations at a single wave length, the relatively simple device indicated in Fig. 6-7 is adequate (Pollister and Moses, 1949). Photomultiplier tubes are sensitive enough to allow measurement of areas less than 1 m', tor all methods for nucleic acid and protein. Other devices employing photomultiplier tubes have been described by Lison and Pasteels (1951) and by Pollister (1952c). The data obtained are pairs of measurements, a first (h) through part of the cell, a second (h) through an empty part of the slide, outside the sec- tion. From these, transmission (7') can be computed (as Ix/h) and extinction either computed (as logio /o /x) or obtained from a conversion table (Erode, 1943). If absorption curves are to be measured, the apparatus described must be supplemented by means of dispersing the spectrum either before or 22G RADIATION HIOLOGY after the microscopic preparation. There are many possible types of apparatus for this sort of procedure (see Caspersson, 1950; Loofl)()urow, 1950; Mellors et al., 1950; Blout et al., 1950). Since in the visible spec- trum there are objectives and condensers, corrected both for chromatic and spherical aberrations, the measurement of visible absorption spectra is relatively simple, involving merely movement of the wave-length drum of the monochromator. For ultraviolet absorption measurements, Caspersson (193G) originally used the Zeiss-Kohler apparatus and meas- ured absorption at each wave length by photography. This whole instrument was designed for photography at one wave length, quite MAGNIFIER PHOTOTUBE POWER SUPPLY Fig. 6-7. Diagram of a simplified apparatus for microphotometric study of cytological preparations. {After Swift, 1950.) uncorrected for chromatic aberration, and its use for absorption curve measurement by photography is extremely tedious. For each wave length both condenser and objective must be refocused, and it is necessary to carry out two measurements or make two photographs at each wave length. This uncorrected optical system has been used for all the exten- sive work of the Caspersson school, with many additions (Fig. 6-8), such as an achromatic grating monochromator, extremely sensitive photore- ceivers (measuring currents of the order of lO"'^ amp) and a polarizing prism and special mechanical stages (Caspersson, 1950). The develop- ment of achromatic reflecting objectives and condensers (Fig. 6-9) (Brumberg, 1943; Burch, 1947; Grey, 1950; Norris et al, 1951; Barer, 1951) makes the problem of optical apparatus for ultraviolet absorption measurements essentially as straightforward as in the visible spectrum. With the instrument which has been developed by Sinsheimer a com- A CRITIQUE OF CYTOCHEMICAL METHODS 227 plete absorption curve can be run mechanically, without refocusing; a density-wave length curve is recorded on a drum; and by a beam- splitting mechanism and chopping the beams at two frec^uencies it is possible to compensate for the transmission of the empty part of the slide so that no second curve is necessary (Loofbourow, 1950). Many details of techni(iue will be found in such references as Caspersson (1936, 1950); Fig. 6-8. Diagram of main instrument used in measuring ultraviolet absorption with high accuracy and stability. A, mercury lamp; B, tungsten band lamp; C, mono- chromator; D, exit slit of monochromator; E, lens; F, movable 90° quartz prism; G, quartz plate, used with photocell to compensate for changes in the lamp; H, condenser; /, object on slide; K, objective; L, ocular with adjustable diaphragm; il/, accurately movable prism of fused quartz; N, rotating sector; 0, telescope for centering; P, Kohler's rotating spark gap arrangement; R, photocell; *S, electrometer; T, leakage resistance; U, four-step potentiometer; X, camera; F, Kohler focuser for the ultra- violet, interchangeable with prism M. (After Caspersson, 1950.) Gersh and Baker (1943); Thorell (1947); Pollister and Ris (1947); Pol- lister and Moses (1949); Swift (1950); and Pollister (1952c). An extreme simplification of the problem of instrumentation for microspectrophotometry is to regard the whole apparatus as merely a somewhat more complicated optical pathway than that in the conven- tional devices which use absorption cuvettes, and to consider the micro- scope as no more than an aid to locating an extremely small analytical sample symmetrically in the optical pathwa}^ and delimiting the area to be measured. If computations of concentrations and amounts are to be 228 RADIATION UIOLOGY made, it is obviously necessary to take into account the problems raised by such factors as the angle of the illuminating cone from the condenser, the probable variations of path Icnj^th, and scatter into or outside the area of measurements as this cone is changed. These questions are con- IMAGE OF SOURCE 4-nim DIA. HERE 31-10-02 OBJECTIVE \ CONDENSER SHOULDER 31-15-02 EYEPIECE J. DIAPHRAGM Fig. 6-9. (a) Diagram of the optical components of a microscope with reflecting- refracting condenser and objective, for use in the visible and ultraviolet spectrum. (The Bausch and Lomb Optical Company.) (b) Diagram of a totally reflecting objec- tive, a design with chromatic correction over a wide extent of the visible, ultraviolet, and infrared regions of the optical spectrum. (Courtesy of A. J. Kavanagh and The American Optical Company.) sidered at length, from both the theoretical and experimental viewpoints, in such references as C^aspersson (193G, 1950); Uber (1939); Thoroll (1947); Swift (1950); and Davies and Walker (1953). Caspersson, especially, has discussed at some length the problems raised by image formation, a treatment which seems to make the whole ques- A CRITIQUE OF CYTOCHEMICAL METHODS 229 tion of microspectrophotometry considerably more difficult than in the simplification suggested. He believes that if the intensity distribution in the microscopic image is to correspond in every detail with that in the cell, a requirement for absorption measurements, then the demands on the microscope are essentiall}^ identi('al with those set forth by Abbe for highest resolution. Others have suggested that an absorption micro- scope can perhaps be a compromise between the simplified optical system of such instruments as colorimeters and that for the sharpest images at high magnification (Norris et al., 1951; Grey, 1952; Kavanagh, 1952). 4-3. SOME ERRORS OF QUANTITATIVE MICROSPECTROPHOTOMETRY In practice, an apparatus for microscopic absorption is indeed much like a colorimeter or a spectrophotometer, except for the introduction of a microscope into the optical pathway, and the actual absorption measure- ment is essentially the same — Ix is intensity measured through the cell while /o is a second reading through an empty part of the slide, outside the section. Such quantitative absorp- tion data are easily obtained, but the successful evaluation of the results must take into consideration many possible sources of error which arise from the nature of cytological mate- rial and the fact that the microscope is used. Most substances within the cell are in a physical state very differ- ent from the dilute solutions meas- ured in a colorimeter or spectropho- tometer. The proteins and nucleic acids are very concentrated and if fixed, possibly even when unfixed, are more like solid precipitates or gels than solutions. Very little is actu- ally known about the extent to which this physical state can affect the operation of Beer's law because no extracellular model for such a study is available. Solutions of nucleic acid which approach that which occurs in cells (1-5 per cent) appear to give the same k value as dilute solutions (Table 6-3), and the absorption curve of such concentrated solutions meas- ured in the microspectrophotometer matches very closely that obtained on dilute solutions (Fig. 6-10). Nevertheless, it is difficult to escape a sus- 2400 3000 2600 2800 WAVE LENGTH, A Fig. 6-10. Absorption spectrum of a 2.5 per cent ribonucleic acid solution, obtained (1) in a cellophane bag with a microspectrographieal arrangement (soHd circles) and (2) in a 10-/x Scheibe cuvette with a photoelectrical absorp- tion spectrograph according to Warburg- Negelein (open circles). (Redrawn after Thorell, 1947.) 230 RADIATION BIOLOGY picion that, in the cuinplex iiucleoproteiii association within the cell, the resonance conditions may be significantly different from those of the iso- lated nucleic acid and protein in solutions (see Chap. 5, this volume), and hence any computations of absolute concentration or amounts from stand- ards obtained on solutions must be considered provisional. For relative concentrations this is less important, for it is a much less unlikely assump- tion that A- is constant throughout the limited range under study. Deviations from Lambert's law that extinction is proportional to thick- ness are uncommon sources of error if photometric analysis is properly carried out, since there are no conditions within a thick sample which differ from those within a thin one. It has been suggested that, within a cytological preparation, error may arise because the color reaction occurs only at the surface of the section, or because a colored product piles up to form a sort of opacjue screen on the surface. This has not yet been found. Conformity to Lambert's law is easily tested in cyto- logical preparations, and it has been repeatedly demonstrated that light loss is proportional to thickness of the absorbing layer (Pollister and Ris, 19-1:7; Pollister and Swift, 1950). A possible source of failure is dichroism as a result of orientation of chromophores (Commoner, 1949; Commoner and Lipkin, 1949). No case of such error has yet been detected in visible and ultraviolet studies (Pollister and Swift, 1950), and there is evidence that moderate nucleic acid orientation would have little effect on the ultraviolet absorption measurements of cells carried out with unpolarized light (Thorell and Ruch, 1951). It is perhaps a safe rule that dichroism is unlikely to be a complicating factor except in objects which are con- spicuously birefringent (e.g., skeletal muscle). Marked dichroism is a potential tool for study of molecular orientation within the cell. For example, Caspersson (1940b) has demonstrated that the ultraviolet dichroism of grasshopper sperm heads is due to orientation of the pyrimi- dine chromophores, and infrared dichroism has been employed to detect orientation of protein polypeptide chains (Goldstein, 1950). When carrying out in vitro photometric analysis, the usual method of isolating specific absorption from nonspecific is by subtracting the light loss of a blank, which is either the solvent alo?ie, or a solution of the sample substance in which a color test, which is the basis of the photometric analysis, has not been developed. The wide usefulness of the photo- metric approach in ([uantitative chemical analysis depends largely upon these simple methods of extracting the essential datum from what is actually in most cases an extremely complicated optical phenomenon (see Chaps. 1 and 5, this volume). The cytological use of photometric analysis likewise depends on, in one way or another, the relation of light loss within the cell to specific absorption of a given chemical substance. The elimination of the nonspecific component has been l)est achiexed in methods of photometry of color reactions for nucleic acids and proteins, A CRITIQUE OF CYTOCHEMICAL METHODS 231 which may be measured by visible light (Pollister, 1950, 1952a). It happens that when tissues are so fixed that the cell consists of little more than nucleoprotein, all cellular structures have very nearly the same optical dispersion, and it thus becomes possible to mount the specimen in a medium (an oil) which matches the refractive index at any wave length. Under these conditions unstained structures are invisible even by dark- field or phase contrast, showing that nonspecific light loss is negligible. If such material, colored by a reaction, is measured while mounted in oil at or near the appropriate refractive index, practically all the light loss may be assumed to be due to specific absorption by the chromophore of the test. [One possible source of error is that of anomalous dispersion near the absorption peak of the chromophore as pointed out by Scott (1952) and Ornstein (1952).] Another approach is to measure a cell twice, first before (a blank) then after (a test) development of color, a procedure which has been followed with the Millon reaction for proteins (Pollister and Mirsky, 1946; Pollister, 1950). Another method is that used in photometry of the natural absorption of nucleic acids, in which the blank is the second meas- urement made after removal of the nucleic acid by nuclease digestion or chemical extraction (Fig. 6-5 and Table 6-4). This is somewhat less satisfactory than the protein blank because the component of the non- specific light loss due to nucleic acid is also removed and thus becomes added to the apparent specific chromophore absorption. When the refractive index of the mounting medium is markedly differ- ent from that of the section (e.g., when an unstained section is in water), the nonspecific light losses become appreciable. The methods of ultra- violet microspectrophotometry have been applied either to living cells or to sections which are mounted in glycerin, after either freeze-drying or fixation (e.g., in acetic alcohol). In the two former materials nonspecific light loss is believed by Caspersson (1950) to be minimized in some cases by the absence of sharp phase boundaries. In nearly all fixed material the nonspecific light loss is always considerable (Fig. 6-2D and 6-5). Apparently no mounting medium for ultraviolet studies closely matches the refractive index of such fixed sections. Hence ultraviolet absorption studies must always grapple with the problem of estimating the scatter and internal reflections. As Caspersson (1950) has said, "the most important of all conditioning factors for quantitative microspectrograph}^ is the elimination of the sources of errors caused by these factors." Cas- persson elected to estimate the nonspecific light losses, where appreciable, in the preparations, not by a measured blank as described, but by the unusual method of computing them from analogy with the light losses which he had previously studied in solutions of colorless salts — in which, of course, all light loss was nonspecific. In spite of the urgency and priority of this problem for any quantitative interpretation of the nucleo- protein ultraviolet absorption curves, there has never been a complete 232 RADIATION UIOLOGY explanation of how llic nonspccilic li^ilit losses can be estimated and .sul>- lract(Kl from the compound measured curve of intracellular nucleopro- lein. The nonspccilic lifl 1 \\ ID / < 40 - 1 « 1- j 1 I 1 I o 1 i \ ^20 \ j ^ i\ 1 i Ll_ 20 30 MICRONS 40 50 Fig. 6-11. intraviolot absorption measureiucnts at a series of points across the diameter of a grass- hopper spermatocyte (soHd line), compared with the computed ab- sorption (uirve of an absorbing sphere (broken line). (Redranm from Caspersson, 1939.) 4-4. QUANTITATIVE APPLIC.\TIONS, ABSOLUTE AND REL.\TIVE Because of the potential errors in photometric analysis of cells it is evident that a straightforward computation of absolute amount of sub- stance by referring light loss in a cell to any standard value obtained on A CRITIQUE OF CYTOCHEMICAL METHODS 237 solutions in an absorption cuvette (as described in Sect. 2) is by no means a reliable procedure, although this has frequently been done. The first such attempt at absolute quantitation was made by Caspers- son (1939) who computed the amount of DNA in 24 nuclei of living grass- hopper spermatocytes, assuming that distribution was homogeneous (p. 235 and Fig. 6-10) and that all light loss was due to specific DNA absorp- tion. No correction was made for possible protein or RNA absorption, or for nonspecific light loss, but it now seems evident that these first two must have contributed to the total weaken- ing of the light (Ris, 1947; Caspersson, 1950, Fig. 36). It will be recalled that two kinds of ultraviolet absorption curves (curves I and II, Fig. 6-6) have been obtained for intracellular nucleoprotein. No absolute computations have been made from type I curves except the semi- quantitative nucleic acid-protein ratio, from extinctions near the nucleic acid and protein peaks (best described by Thorell, 1947). Caspersson, however, has made an elaborate effort to com- pute quantities of nucleic acid, tyrosine, and tryptophane from the type II curves, which were formerly supposed to characterize regions of nucleic acid accompanied by histone type or diam- ino acid-rich protein (Fig. 6-12). The method of computation has not been described completely enough so that readers may discover just how the val- ues were reached from the measured curve. It is clear, however, that the curve analysis depends on the validity of the assumption that the protein moiety is of basic type in which the presence of a large proportion of dibasic amino acids, as in histones, brings about a shift of the absorption peak of tyrosine (as suggested very tentatively by Stenstrom and Reinhard, 1925). Therefore, this particular method of curve analysis is in effect invalidated by the demonstration that in histones the peak is not shifted toward longer wave lengths (p. 223). Pollister and Ris (1947) reported computation of the amount of DNA 2500 3100 2700 2900 WAVE LENGTH. A Fig. 6-12. An example of analysis of compound ultraviolet absorp- tion curve of a cell structure con- taining nucleoprotein. Curve I, measured absorption; curve III, nucleic acid component; curve IV, tyrosine coinponent; curve V, tryptophane component; curve II shows the sum of the compo- nents. Results: tyrosine, about 0.1 X 10^"' mg/ix^; tryptophane, 0.04 X 10 '" mg/M^; nucleic acid, 0.6 X 10->" mg/ix\ Light refrac- tion and dilTraction are, in this special case, negligible, as special experiments have shown. (Redrawn after Caspersson, 1940, 1950.) 238 RADIATION BIOLOGY ill isolated thymus nuclei, very much as Caspersson had done earlier for spermatocytes. They reported MX 10~^ mg per nucleus, and, for the first time in cytochemistry, compared a figure obtained by cytological photometry with one obtained by chemical analysis of a mass of known luimbor of isolated nuclei. The two values agreed within 10 per cent, which appeared to be excellent validation of the microspectrcjphoto- metric procedure. It later appeared that this was merely a fortuitous coml)ination of errors, since all other chemical studies indicated the amount per nucleus to be nearer 6.0 X 10~^ mg (see table in Davidson and Leslie, 1950b) ; hence the value computed by Pollister and Ris from ultra- violet absorption is probably considerably less than one-fifth the real amount. This type of study was later carried out under much more favorable conditions by LeiK'htenberger and coworkers (1951). The citrate-iso- lated nuclei were swollen in glycerin to minimize nonspecific light loss and to increase the homogeneity. The ultraviolet absorption was demon- strated by cross-scannings of the spherical nuclei, according to Caspers- son's earlier procedure for spermatocytes (p. 237). The biochemists then determined the DNA, RNA, and protein content, and showed that the last two could have only slight effect upon the absorption. The amount of DNA per nucleus computed from these cytological data (5.4 X 10~^ mg) is much closer to that found independently by the chemical analysis of the remainder of the same sample of isolated nuclei (6.1 X 10~^ mg). It must be pointed out that in such nuclei the DNA is determined under very special conditions of minimal RNA and protein, brought about by the citrate isolation. Such a condition is rarely, if ever, encountered in intact fixed tissue (see Pollister and Leuchtenberger, 1949a, Pollister, 1952b). Di Stefano (1949) also computed DNA in frog cartilage nuclei from ultraviolet absorption data. By comparison with chemical analysis (Davidson and Leslie, 1950b), it appears that these values were deficient by about as much as those given for thymus nuclei by Pollister and Ris. Di Stefano likewise compared the value from ultraviolet absorption with that computed from the Feulgen reaction, on the assumptions that at maximum reaction all purines had been removed and that the fuchsin regeneration from Schiff's reagent proceeded according to the scheme pro- posed by Wieland and Scheuing (1921). The DNA amounts per nucleus computed by these two cytological methods were in satisfactory agree- ment, but, as just indicated, these are far below the value obtained by chemical analysis. Protein-DNA ratios for tissue nuclei were computed from Feulgen and Millon reactions, before the invalidation of the Pollister and Ris and the Di Stefano attemj)ts at absolute (juantitation became known (Pollister and Leuchtenberger, 1949). The Feulgen data of the A CRITIQUE OF CYTOCHEMICAL METHODS 239 last-named authors lead, by the Di Stefaiio type of computation, to DNA vahies about half those obtained by chemical analysis of isolated luiclei (see Pollister, Swift, and Alfert, 1951). As this brief summary shows, in practice the computation of absolute amounts from microscopic absorption measurements has been far from uniformly successful, a result not unexpected in view of the many poten- tial errors. It is apparent, however, that for many problems in cell chem- istry this dubious extrapolation is not necessary; an adeciuate answer is / . ■A ^r\ 1.000 - \ \ A i ny 1 \ 2 1 I o ll \ It 1- 1 \ / / u I \ /I 1 z 1 \ 1 K / \ \ / X / 1 v / \ > UJ 0 500 7 1 J, \ 300 600 700 400 500 WAVE LENGTH, mu Fig. 6-13. Absorption curves, measured in a Beckman spectrophotometer, of various substances which are analyzed photometrically in cytological preparations. Unless otherwise indicated, the length of the absorption path was 1 cm. The light vertical line cutting each curve indicates the spectral region which is measured in the micro- scopical preparations. Curve I, natural absorption of deoxyribonucleic acid, concen- tration about 0.05 mg/cc (redrawn from Pollister and Mirsky, 1946); curve II, tyrosine- mercurial formed by the Millon reaction, tyrosine concentration 0.045 mg/cc; curve III, fuchsin regenerated from standard Schiff's reagent by addition of formalin; curve IV, azure A in distilled water, concentration 0.5 mg/cc, thin cuvette {data of M. Flax); curve V, methyl green (Natl. Anil., cert. No. NG35) in carbol-glycerin-alcohol mixture, concentration 0.01 mg/mm' {data of C. Leuchtenberger). obtainable if the question of relative amount or of relative change can be answered. Numerous quantitative studies with this aim have been published, some of which are reviewed in Pollister (1952a). The specific methods are those which have already been discussed in Sect. 3. The in vitro absorption curves and the wave lengths measured in microscopic material are shown in Fig. 6-13. Since these methods are colorimetric (that is, measurement at a single wave length) and the apparatus is simple (Fig. 6-7), large numbers of absorption measurements on individual nuclei, nucleoli, or other cell structures are readily obtained. On anj^ one type of structure the values are found to vary considerably, the highest extinctions being often as much as twice the lowest. When ?to UADI A'l'ION Hl(H.(KiY 0.200 0 4 00 0 600 0 800 0.600 0 800 100 EXTINCTION Fig. 6-14. Typical distrilnition curves of microspectrophotometric data. Curve I, sections of nucleoli, corn pollen mother cells, Millon reaction ; curve II, nuclei of mouse sarcoma, methyl green; curve III, nucleoli, corn pollen mother cells, ultraviolet absorption; curve IV, mouse spermatocyte nuclcM, Feulgen reaction; curve V, pre- leptotene nuclei from mouse testis, Feulgen reaction. {After Pollister and Swift, 1950.) Table 6-5. Liver Nuclei, Ultraviolet Absorption Slide N umber Mean Eon no. measured A-6 20 0.489 + 0.015 A-6 10 0.474 + 0 024 A-4 20 0.466 ± 0.023 A-9 22 0.507 + 0.016 A-8 25 0.475 ± 0.012 A-5 23 0.499 ± 0 027 Mean = 0.485 The reproducibility of ultraviolet absorption of 5.0-m sections of nuclei of the liver of the salamander, Amhh/stoma, is shown by data from different slides of the same organ, measured at different times. Fixation in acetic acid-alcohol, eml^edded in paraffin, mounted in glycerin, measured as absorption of 254 ni/u radiation isolated from a Hanovia SC2537 mercury-vapor lamp by a Zeiss two-prism quartz mono- chromatic illuminator, with glycerin immersion monochromatic objective, N.A. 0.85, condenser aperture about N.A. 0.40. A CRITIQUE OF CYTOCHEMICAL METHODS 241 enough data are accumulated for statistical analysis it is found that these values group themselves into normal unimodal distribution curves (Fig. 6-14). The cause of this variability is not fully known in any instance. The instrumental and cytological variability can account for but a small part; in many structures (e.g., interphase nuclei) a major portion appears to be due to the distributional error. Until these errors of tec^hnique and unavoidable errors due to cytological structure can be precisely estimated (Sect. 4-3) the magnitude of the biological variation in any one popu- lation of similar cells cannot be estimated; and therefore man}^ of the possible more subtle correlations of nucleoprotein composition with physiological, developmental, pathological, or experimental phenomena cannot be investigated by microspectrophotometry. In the meantime, it has been found possible to make many fruitful studies by utilizing the fact that with proper cytological and photometric techniques the mean values of any population are reproducible to about 10 per cent. This holds when comparing different slides of one material (Table 6-5), when examining the same animal at different times (Table 6-6) , or when study- ing different animals of the same species (Table 6-6). This variability of the means presumably is a measure of the over-all error of the technique of preparing tissues for microscopic examination and of the photometric procedure. In this type of relative quantitative analysis, a major change of composition which may accompany cyclical, experimental, or pathological events is detected as a change of mean value, outside the normal range of variability. This reproducibility of the means has been shown repeatedly for each of the techniques indicated in Fig. 6-13; indeed, it is customary to reexamine it for each new material studied.^ An important aspect of these relative quantitative studies is that in manj'^ cases it has been possible to compare them with results by inde- pendent methods, such as biochemical analysis of samples of known num- bers of isolated cell components. For example, the correlation of amount of DNA with the chromosome complement of nuclei, first indi- cated by biochemical analysis, has been confirmed and greatly extended by cytological studies made with the Feulgen reaction, whereas the pro- tein composition of the nucleolus, suggested by results with the Millon reaction, has been confirmed by both X-ray absorption data and analysis of masses of isolated nucleoli. Many of these essential validations of the cytochemical analyses are summarized in PoUister (1952a). * The approach of the Caspersson school to microspectrophotometry has ])een almost entirely qualitative and semiquantitative. This is emphasized particularly by the fact that in the total of nearly a hundred publications (see list in Caspersson, 1950) there is not one demonstration of the statistical reprodui'il)iIity of the ultra- violet absorption terhiii(|U(', nor is the urgency and priority of such a demonstration ever recognized. 242 RADIATION mOLOGY o < y. a a C a a •< — I 2 "^ s o o n 03 »C ^: IN -f ^T ■»j 8S§8S brt d o o c c ^ SJ ■M 6 +1 +1 +1 +1 +1 -r ««- .«j < JO fC — O 00 ^ fC -f -r '-t -r +J •A O C O C C 6q o ^ o c o o c o c" d u — CJ (N (M IM cr. /^ a 1 CO to >0 CO CO 'I* ■y^ o o o o o o t^. *i o o o o c c; c -«-' o « u 0) S d o c o o c 2 o + +1 +1 +1 +1 +1 — Oj 00 CO d -O — 00 --o o CO ?o -r -f -r c o o c c VM '_S o o O — 'O IN >o CO d 3 (N jv, _ _ — 00 :2: C3 t^ 00 lO lO lO O CD t-- U5 o o o o o o o -»^ o o o o o o o o c t^ d o c o o o o o c — S -1^ +1 +1 +1 +1 +1 +1 +1 +1 +1 (M < 83 t--. Tf (N 00 O 1^ IM lO 00 c 'S* Tf -t r.^ ^ /-y^ -^ «/^ -^ +i O o d C^ (N C4 (N IM (N d d o o o o o tw ■PH o _aj o iC o t^ lO o o o o t^ ^ d o 3 cc IM (N JO -t (N IM ro (M ;^ C C" ) 03 r^ r^ »0 •* lO >0 <£> CD c O O O O O O 2^ a c c -+ > o 1 +1 " o o +1 o o o c o c o -t M O fe d +1 o o o o o o +1 +1 +1 +1 +1 +1 C: O — 00 — -^ ly J "O TtH rr^ -t< -t< JO -f JC 'C -u c 1 (N 5 C o CI (M (M (N IM iM iM O d o o o c o "S .^ o G^ c 3 O c o iC o c o no o o o ^ ^ : c^ "C JO JO JO 'C CO •* T-H >^ S CO '-3 >> o a c 03 o3 C C e3 S3 cc a; .22 "n O c o c § c3 r- c3 r r c3 s3 1 1 1 1 1 1 « 3 O - fc t " =^ tc cc te c o, tc K 5 ii ^ .c _2 c o S 'S "c — O 03 ^ == :5 '5 -| ^ cd o (M CO -t* lO CD l^ 00 C5 O a. •o >> u u rl *J 7J bf ^C C/ fe u t- <1) o *i «*^ c c3 03 o C p 03 O JZ o 03 c 03 a: a 05 c3 ;£ o be U o3 S C 2 S oj S C 03 lU •S 5 .3 03 A CRITiyUK OF CYTUCHKMICAL AIKTJlOD.S 243 REFERENCES Alfert, M. (1950) A ('j'toclu'inical study of ooKoiu-sis Mrid cleavage in the inouso. J. Cellular Comp. 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J. .\nat., 81: 1-31. Zscheile, F. P. J. (1934) .\ (luantitative spectrophotoelectric analytical method • applied to solutions of chlorophylls a and b. J. Phys. Chem., 38: 95-102. Manuscript received by the editor Mar. 12, 1952 CHAPTER 7 The Effect of Ultraviolet Radiation on the Genes and Chromosomes of Higher Organisms C. P. SWANSON The Johns Hopkins University Baltimore, Maryland L. J. Stadler The University of Missouri and U.S. Department of Agriculture Columbia, Missouri^ Introduction. Experimental procedures. Genetic effects of ultraviolet radiation. Direct effects of ultraviolet radiation on chromosomes. Spectral relations. References. INTRODUCTION This review will be concerned with the effects of ultraviolet radiation on the genes and chromosomes of organisms above the microbiological level. The group of organisms thus included is relatively small in num- bers, and the literature is scanty in comparison to that devoted to radia- tion studies on the fungi, bacteria, and viruses. But cytogenetic studies can be made only on organisms with suitable chromosomes. The ultra- violet results obtained with Drosophila 7nelanogaster and Zea mays may be evaluated against an extensive background of X-ray data bearing on problems of cytogenetic interest. Supplementing the data from these organisms are those from Antirrhinum and Sphaerocarpus, which relate to the genetic effectiveness of various wave lengths within the ultra- violet spectrum, and those from Tradescantia and Gasteria, which deal only with induced chromosomal aberrations. Each of these species possesses certain disadvantages, none being wholly satisfactory as a test organism because of difficulties of radiation penetration, accurate dosage measurements at the site of genetic altera- tion, or critical analysis of induced effects. Despite these shortcomings, however, the accumulated evidence from ultraviolet studies has been 1 Cooperative investigations of the Division of Cereal Crops and Diseases, Bureau of Plant Industry, U.S. Department of Agriculture, and Department of Field Crops, University of Missouri. Missouri Agricultural Experiment Station Journal Series No. 1470. 249 250 UADIATIO.N lilol.oCY sufficient to advance malctinllx' tlic uiKicrstandiiiji; of I lie nature of induced hereditary chanjies. 'I'li<> genetic action ot ult ia\iolel radiation hears on threi- major proh- li-nis. riie lirst ot" these is the analysis of the coniph'x of }:;<*"<'tie varia- tions induced l)y the ioni/ins radiations. It is invariably found, where adeciuate cytogenetic tests can he made, that hoth mutations and chro- mosomal aberrations are induced by X radiation. A clear-cut separation of the two phenomena has not yet been accomplished, and the question of their similar or dissimilar nature and origin remains unanswered. If the induction of mutations and chiomosomal rearrangements by the ionizing radiations results from some common, fundamental effect, the two types of genetic alteration should have a common spectral limit. The analysis of induced hereditary variations would then involve determining the alternate pathways of reaction which culminate in a variety of genetical and cytological expressions. If, on the contrary, the diverse effects of X radiation are of independent origin, it is possible that their spectral rela- tions may be sufficiently different to permit the separation and perhaps the selective induction of one type of variation to the exclusion of others. The individual phenomena should then pro\e to lie more amenable to analysis. The second problem is concerned with the determination of genetic effectiveness of specific wave lengths of the ultraviolet spectrum, as a ckie to the chemical nature of the substance within which the energy absorption leading to genetic change takes place. With ionizing radia- tions such a study is not possible since their absorption is independent of molecular organization. The third problem is concerned with the nature of mutations in general, whether spontaneous or induced. Mutations as experimentally identified are a residual class, identified by negative criteria. The analysis of spe- cific X-ray-induced mutations has shown that character changes inherited as if due to gene mutations may be in some instances the result of chromo- somal rather than genie alteration. The category "mutations," as experimentally defined, must therefore be a complex one, including vari- ous extragenic as well as intragenic alterations. Since the mode of action of ultraviolet radiations is so different from that of ionizing radiations, the comparativ^e study of mutations induced by these agents is promising. The relative infrequency of chromosomal derangements induced by ultraviolet suggests that certain extragenic alterations simulating gene mutation may be infre(|uent or absent among the mutations from ultra- violet treatment. The possibility of qualitative differences in the chro- mosomal alterations must also be considered. The spectral analysis of the complex of genetic effects induced by X rays, as outlined above, implies the as.sumption that the mutations and chromosomal alterations induced by X rays are qualitatively analogous GENETIC AND CYTOLOGICAL EFFECTS 251 to those induced by ultraviolet radiation. If they are not, it is possible that the spectral limit of both classes of X-ray alterations is far below the shortest ultraviolet wave length that can be used in biological experi- ments, and that the mutations and chromosomal alterations characteristic of the ultraviolet are of a type not occurring in appreciable frequency in the X-ray progenies. EXPERIMENTAL PROCEDURES Genetic and cytological studies of the effects of ultraviolet radiation on higher organisms are limited by the techni(iues which permit the investi- gator to irradiate the germ cells effectively without killing the organism or the cells being treated. Ultraviolet produces considerable physio- logical damage, setting limits to the dosage; it is also low in penetrating power, a disadvantage when deep-seated sex organs are being treated. Both of these phenomena result from the high absorption of ultraviolet by extranuclear constituents of the cells as well as by the nucleoproteins of the chromosomes. A number of techniques are available, however, and it is fortunate that they permit the irradiation of the germ cells of Droso- pfiila and maize, thus making possible genetic and cytological comparison with other mutagenic agents. Ultraviolet radiation and X rays have also been used extensively with Neurospora, in which significant cytogenetic comparisons could be made, but no comparative studies of the induced alterations have been reported. Perfection of the technique of artificial insemination of Drosophila females with treated spermatozoa would circumvent the difficulties of reaching the sex cells of adult males with ultraviolet radiation, but apparently all attempts to duplicate Gottschewski's (1937) artificial insemination results have so far been unsuccessful. Two other methods, however, have been developed: (1) irradiation of the pole cells of the early embryo in the egg, and (2) irradiation of mature spermatozoa through the ventral side of compressed abdomens of adult males. The polar cap technique was developed by Geigy (1931) after the early work of Guyenot (1914) and Altenburg (1928) had demonstrated that ultraviolet treatment of adult males gave only inconclusive results. The pole cells are destined to enter the germ tract. At 75 min after fertiliza- tion of the egg (at 24°C) the pole cells appear close to the surface at the amicropolar end, w^here they remain for approximately 1 hr (Altenburg, 1934). In this position their nuclei can be readily reached by ultraviolet radiation. The early experiments of Altenburg (1933, 1934) were made with the angle of incidence of the ultraviolet at right angles to the vertical axis of the egg, the lower portion cjf which could be shielded to reduce injury to the developing embryo. More recently, it has been found that a shift in the angle of incidence from a plane of 90° to one more nearly 252 RADIATION mOLOGY parallel to the axis of the egK fj;reatly increases the j;;enetic elTectivencss of a fiiveii ultraviolet dose while lessening the physiological damage to the embryo (Altenhurg et al., I'JoO; Meyer et al., 1950). Dechorionation of the eggs by immersion in a 5 per cent solution of sodium hypochlorite also facilitates penetration of the radiation (Clark, 1948). At the time of irradiation, the pole cells number about 20. Their later incorporation into the germ tract is accompanied by an increase in the number of cells as the sex organs and the reproductive cells are formed. Therefore, if a mutation is induced in one gene at the polar cap stage, it theoretically should appear replicated in about 5 per cent or more of the sex cells from that particular individual. Should the mutation occur after the process of multiplication has begun, a smaller proportion of the germ cells would receive it. Two assumptions are involved if the 5 per cent level of mutation repli- cation among the Fi offspring provides the distinction between induced and spontaneous changes. In the first place, the pole cells at the time of exposure to radiation must be 20 or less in number, and second, an ecjual rate of multiplication of mutated and normal pole cells must be assumed to take place up to the time of formation of the reproductive cells. Unequal exposure of the pole cells through unavoidable shielding as well as through differences in the degree of penetration of radiation would sug- gest that variations in cellular injury are to be expected. These varia- tions would be expressed in unequal rates of cell multiplication, with the most heavily injured cells having the slowest rate of division. The latter cells would also be most likely to possess mutated genes. As a means for demonstrating that ultraviolet is effective in inducing mutations, the technique is entirely satisfactory, but it is unsuitable for accurate deter- minations of the frequency of mutation as a function of dosage. Recessive lethals induced in the X chromosome by the pole cap method of exposure may be determined by testing, through the CIB technique, the Fi daughters of males arising from irradiated eggs. Replicated lethals from any single male must be further tested for identity because of the possibility of two or more coincident lethals. Visible mutations in the X chromosome may be detected in Fi males by breeding the treated Pi males to XX females. Reuss (1935) developed an effective method for the exposure of mature spermatozoa to ultraviolet radiation. The abdomens of adult male Drosophila are compressed gently between (luartz plates, the radiation being applied ventrally. Since the testes are superficially located, the amount of overlying tissue is at a minimum and consists of the chitinous exoskeleton, the wall of the testis, and the intervening connective tissues. Clear areas of chitin, which has chemical and absorptive properties char- acteristic of polysaccharides, are in general highly penetrable by wave lengths from 250-400 m^, with the degree of absorption increasing rapidly GENETIC AND CYTOLOGICAL EFFECTS 253 at shorter wave lengths (Durand el al., 1941). The method, while useful in ascertaining the kinds of gene and chromosome changes which may be induced by ultraviolet in the mature spermatozoa, is not suitable for the accurate (juantitative determination of dosage-mutation frequency rela- tions and wave-length dependence yields. The chitin is of uneven trans- parency and the intervening tissues vary in thickness and position, with the result that considerable differences in the amount of radiation reach- ing the spermatozoa must be expected from one individual to another. The variations in penetration of radiation can be lessened somewhat by the use of light-colored mutant stocks, but there remains the difficulty of accurately determining the amount of energy absorbed by the se.x cells at the site of effective action. Also, the practical limits of dosage are determined largely by the tolerance of the adults to physiological damage leading to sterility. Demerec et al. (1942) have reported induced sex- linked recessive lethals in frequencies as high as 50 per cent, but such high proportions of mutations are a rare exception. The usual frequencies of recessive lethals obtained from optimal abdominal exposures range from 0-5 per cent, with a wide variation among similarly treated flies. The higher plants offer somewhat better technical possibilities for studies of the genetic effects of ultraviolet radiation. The male cells are of minute size, readily accessible, and free from extraneous tissues. Three types of cells have been used: (1) swimming spermatozoids, (2) pollen grains, and (3) generative cells in developing pollen tubes. Swimming spermatozoids of the liverworts, for example those of Sphaerocarpus donnelUi, are particularly favorable objects for ultraviolet studies, since they consist essentially of a naked nucleus approximately 0.5 n in diameter (Knapp, 1938). Absorption of the radiant energy by extranuclear materials is at a minimum, and treatment of the cells can be carried out in water. Their use in the determination of dosage rela- tions has not been exploited to the extent warranted by the excellence of the material. Exposure of the male cells of angiosperms can be readily made by treat- ing a monolayer of loose, dry pollen. Pollens of maize, Antirrhinum, and Gasteria have been successfully employed in ultraviolet studies, the irradiated grains being used to fertilize untreated plants. The maize pollen grain has both sperm cells fully formed at the time of anthesis. One sperm fertilizes the egg to produce the zygote while the other unites with the fusion nucleus to give rise to the endosperm. Maize also pos- sesses the added advantage of a goodly number of clear-cut endosperm marker genes whose presence or absence (or mutated state) can be directly determined by examination of the kernels produced on ears pol- linated by the treated pollen. These markers facilitate the collection of massed data and provide a convenient measure of genetic effectiveness when comparative studies are being made of wave-length and dosage 254 RADIATION BIOLOGY Illations. In both maize and Antirrhinum, mutation data may bo ()btaiiu>d throuj^h tho detection of sefi;re}j;atin}i; characters in F-j popula- tions, (instrria has been used only tor the study of induced chromosomal changes appearing in the cells of I*\ embryos. Compared with the spermatozoids of Hverworts, the pollen grains of angiosperms are relatively large, those of maize, for example, being almost 100 n in diameter. AVhen comparisons are being made of the relative effects of dilTerent wave lengths or of different doses of the same wave length, it becomes necessary to take into account the factor of internal filtration, since the energy incident at the surface of the pollen grain is very greatly reduced by absorption in the extranuclear material. Inter- nal filtration varies greatly with the wave length of radiation employed (ll)er, 1939; Stadler and Uber, 1942), and failure to correct for these ditYerences of penetration may lead to gross error in wave-length com- parisons. The filtration factor can be roughly calculated, as Stadler and Uber have shown, but the difficulties involved stress the need for better genetic materials in this area of investigation. Cytological studies of ultraviolet-irradiated chromosomes have been carried out in the Fi progeny of maize, Gasteria, and Drosophila. The chromosomes of surviving Fi individuals represent a selected group from which all inviable aberrations have been screened. Through genetic technicjues, the types of chromosomal rearrangements induced by ultra- violet may be inferred without cytological examination, but there remains the possibility that certain aberrations may be eliminated after the pas- sage of several cell generations. The pollen-tube technique overcomes this difficulty in that it permits a direct examination of irradiated chromo- somes before the elimination of inviable changes can take place. The technique involves the culturing of pollen tubes on an agar-coated slide, with sucrose or lactose added to the agar as a carbon source. The generative cell, after passing from the pollen grain into the tube, is covered only by a thin cytoplasmic layer and a thin tube wall. Since the pollen tube is narrow (approximately 5 ^ in Tradescantia) , the amount of radiation absorbed before it reaches the nucleus is not great. The chromosomes, undergoing mitotic division in the tube, may therefore be readily exposed to ultraviolet, and an analysis of structural changes may be made at metaphase by blocking the division with colchicine (Swanson, 1940, 1942). The method, as first employed with Tra(l(t 222 control eggs, 13,063 Fi females were tested; from the 239 treated eggs, 14,059 Fi females were tested. The development of the abdominal exposure techiii(iue by Reu.ss (1935) demonstrated the feasibility of inducing mutations in the matiu-e sperma- tozoa of Drosophila. Mackenzie and :\Iuller, using this method, have confirmed and extended the earlier findings of Altenburg, particularly as concerns the comparison of the effects of ultraviolet and X rays (MuUer and Mackenzie, 1939; Mackenzie and Muller, 1940; Mackenzie, 1941). The filtered radiation employed consisted of wave lengths above 280 m^u, a (luality of radiation less damaging physiologically than the shorter wave lengths. A dose of 2 X 10'^ ergs/mm^ w^as found to be optimum for the study, since an appreciable frequency of sex-linked lethals (about 3 per cent) was induced without an accompanying high degree of sterility or mortality. Higher doses raised the frequency of lethals to 9 per cent, but the sterility was disproportionately increased, making extensive observations difficult and (juantitative comparisons unreliable. The principal results of these studies were the following: 1. No translocations affecting the Y, II, and III chromosomes were found in a population which had a frequency of sex-linked lethals of 4.3 per cent. Such a fre(iuency would be induced by an X-ray treatment of about 1300 r. The authors estimate that, in the number of culttn-es GENETIC AND CYTOLOGICAL EFFECTS 257 tested, 40 or more translocations of type II-III alone would have resulted from this X-ray dose. I'hese data were in agreement with an earlier trial in which no translocations were found from treatments which induced sex-linked lethals in such numbers as to indicate an expectancy of at least 25 detectable translocations, on the basis of the relation found with X rays. The discriminatory action of the ultraviolet was thus shown by the absence of detectable translocations in cultures in which at least 65 were to be expected if the relation of mutation and gross chromosomal rearrangement were the same with ultraviolet as with X rays. 2. Similar evidence relating to the occurrence of minute rearrangements was obtained by the use of a special technique, with w^hich minute rear- rangements are induced by X rays in relatively large numbers and are recognizable by mutants at specific loci. In ultraviolet-treated cultures yielding sex-linked lethals at a rate corresponding to an X-ray dose of 1000 r, no mutants at these loci were found. In an X-ray experiment by MuUer and Makki (Mackenzie and MuUer, 1940), these had occurred at significant freciuencies following dosage of 1000 r. MuUer and Mackenzie concluded provisionally that the ultraviolet does not produce minute rearrangements, or at least that it is far less efficacious in this respect than X rays. 3. Wave lengths above 320 m^ were found to be ineffective in inducing mutations. 4. The frequency of mutations was higher when mating of the irradi- ated males followed immediately after treatment. No mutations were transmitted 5 days after irradiation although the supply of mature sperm would not have been exhausted for some days thereafter. Those irradi- ated flies which bred but* died early had higher frequencies of mutations than those which continued to breed over longer periods of time. A correlation was established therefore between the frequency of induced mutations and the amount of physiological damage as determined by the duration of the fecund period. Both phenomena are undoubtedly affected by the degree of penetration of ultraviolet, a factor which varies widely among similarly exposed individuals. 5. Dose fractionation was without effect on the frequency of mutations or the degree of sterility. The occurrence of minute deletions among the mutants induced by ultraviolet was cytologically demonstrated by Slizynski (1942). Among 21 of the sex-linked lethals produced in the experiments of Mackenzie and Muller, 5 Avere found to be cytologically detectable deficiencies, 1 involv- ing the loss of 1 band, 3 the loss of 2 bands, and 1 the loss of 14 bands. All were interstitial deficiencies. Results of studies by McQuate (1950) support the hypothesis of Mackenzie and Muller that terminal deficiencies, if produced, are not recovered in Drosophila populations derived from mature spermatozoa 258 RADIATION BIOLOGY which have boon exposed to ultraviolet, r.sing a stock containing a spcci.-i! ^■ chromosome (y''Y') marked with the normal allele of achaeto, he matccl irradiated males to achacte females, 'rerminal l«»ss of th(> nor- mal allele of acliaet(>, with retention of the remainder of the special Y chromosome, would yield fertile achaete males in the Fi population, l-'rom a total of H),;i01) h\ males, 23 sterile achaete exceptions were found. Such males, however, result from a loss of the paternal X chromosome, or of all or part of both arms of the y-'-Y^ chromosome, losses which could arise through lagging of the chromosomes in division, or by breakage followed by fusion of broken ends to give acentric and dicentric portions having a low survival probability. Breakage of the y^-Y^ chromosome, with loss of the y^ region (which also includes the normal allele of achaete) and foUow-ed by healing, did not occur. The fertile achaete exceptions, two in number, were no more frequent than in the control population. Experiments with Plants. In many species of plants the pollen grain may be effectively treated with ultraviolet radiation. It is therefore feasible to make somewhat simpler tests of the genetic effects of the treat- ment than can be made in the experiments with Drosophila. The indirect analysis required by the polar cap technique is avoided, and the difficul- ties from internal filtration, while serious, are not nearly so great as in the irradiation of the sperm within the body of the adult fly. In the cul- tures grown from seeds produced by the use of the irradiated pollen (which we may, for convenience, refer to as the Fi cultures), each plant provides the material for testing the effects of the treatment on one irradiated gamete. Dominant effects of chromosome or gene changes induced by the treatment may be observed in the Fi plants, and each F2 culture pro- duced by self-fertilization of one of these plants shows segregation for any haplo-viable recessive alteration induced in the gamete tested. The results reported by Noethling and Stubbe (1934) clearly demon- strated the effectiveness of ultraviolet in inducing mutations. These were detected in segregating F2 populations of Aniirrhinum. A similar increase in the frecjuency of point mutations was found in maize by Stadler and Sprague (193(m), together with evidence of certain chromosomal effects of the treatment and further indications of differ- ences in the genetic action of ultraviolet radiation and X rays. The mutations identified were only those affecting seed and seedling charac- ters. Progenies representing various doses of unfiltered ultraviolet radia- tion yielded 31 mutations from 830 gametes tested, and control progenies yielded 6 mutations from 557 gametes. This increase in mutation rate, while clearly significant, is not large. But among the 31 mutants detected (of which 9 would have been expected without irradiation), there were two cases in which 2 unlinked mutants occurred in a single F2 progeny, and one case w^ith 3 unlinked mutants in a single progeny. These represent cases of two or three presumably unre- GENETIC AND CYTOLOGICAL EFFECTS 259 lated mutants in a single treated gamete. In addition, three of the other mutants occurred in the progeny of plants segregating for defective pol- len. The independent inheritance of the mutants, in these and several similar cases, has been shown by Sprague (1942). The degree of coinci- dence is far beyond that expected by chance, if all the tested gametes received an equal dose. But the treated gametes must receive quite unequal doses, for the sperm nuclei are eccentrically located in the spheroidal pollen grains, and the loss by internal filtration must vary widelj^ with the casual orientation of the individual grains. If this is the explanation of the coincidences observed, the frequency of mutation in the most effectively exposed pollen grains must be very high. In later experiments with more effective ultraviolet treatments, mutation rates of about 20 per cent have been reported (Stadler, 1941b). These are sub- ject to the same limitation by casual orientation of the pollen grains treated, and the results suggest that the effect of ultraviolet radiation on mutation frequency in maize, in individuals that are effectively treated, may be well beyond that produced by X rays. A technical advantage of the maize material is the availability of many endosperm characters of known inheritance. In matings with appro- priate marker genes, the loss of the effect of dominant alleles present in the male parent may be detected at once by phenotypic changes in the endosperms of the seeds produced. Linked endosperm characters, deter- mined by genes located on one arm of one of the chromosomes, permit the detection of deficiencies in this region. Ultraviolet treatment applied to the pollen greatly increased the fre- quency of endosperm deficiencies, as detected by loss of the linked factors C and Wx. The frequency of loss of other dominant genes for endosperm characters {A, Pr, Su) was similarly increased. There are no linked genes for endosperm characters suitable for determining whether A, Pr, and Su losses represent deficiencies. The fact that ultraviolet-induced loss of C and Wx is usually coincidental indicates that the endosperm effect is due usually, if not always, to deficiency rather than to gene muta- tion. Since no genetic analysis can be made, the identification of an individual case as a recessive mutation rather than a deficiency could not be positively established. The cases are referred to as "endosperm deficiencies," with the reservation that there may be included among them an unknown proportion of losses of dominant characters as a result of gene mutation. The endosperm deficiencies resulting from ultraviolet treatment of pol- len included a large proportion of fractionals, in contrast to those resulting from X-ray treatment of pollen, which are largely deficiencies affecting the entire endosperm. The occurrence of deficiency in the Fi plants was not determined by means of marker genes or cytological examination in this experiment. 260 RADIATION BIOLOGY The ahsonco of gross deficiency or translocation could Ix' assumed it' all the Fi plants were free from segregation for defective pollen. It was found, however, that, among about lOOO Fi jjlants examined, almost 4 per cent, showed segregation for defective pollen, ascrihahle to deficiency or to mutations affecting pdjlcn dcxclopment. Later studies showed that mutations affecting pollen development are a frecjuent result of ultraviolet treatment, and lh;it there are also cytologically demonstrable deficiencies induced by the treatment. No translocations were found among the Fi plants characterized by defective pollen segregation. In later studies, however, translocations were found, though in very low freciuency. The rarity of translocations was given special study because of their very high frequency in compara- ble X-ray progenies. Since the frequency of deficiency was lower under the ultraviolet doses used than under the X-ray doses commonly applied, it is possible that the difference in effect on translocation is incidental to dosage. If translocations result from chromosome breakage followed by reattachment of broken ends in new combinations, the rarity of transloca- tion following ultraviolet treatment might be due to the smaller number of iireaks produced by the ultraviolet dose applied. A further trial was made (Stadler and Sprague, 1937) in which a maximal dose of ultraviolet was compared with a rather low dose of X rays, these doses being approxi- mately equal in total frequency of induced deficiencies for the endosperm genes A and Pr. The frequency of translocation was determined for each treatment by direct cytological examination of about 100 unselected plants of the Fi progeny. Only one translocation was found in the ultra- violet progeny, while 44 per cent of the plants of the X-ray progeny showed translocations, several of them two or more independent translocations. Since it w^as subsequently found that the frecjuency of deficiencies in endosperm and embryo is very different under ultraviolet treatment, this comparison represents doses very unequal in frequency of induced defi- ciencies in the embryos. It therefore does not test the possibility that the rarity of translocations under ultraviolet treatment may be due to the smaller number of chromosome breaks produced. It is clear, however, that, for doses equal in frequency of induced mutation, the frequency of induced translocation is much greater with X rays than with ultraviolet radiation. Preliminary comparisons of w^ave-length effectiveness were made in the early maize experiments (Stadler and Sprague, 193Gb, c), using the radia- tion from a commercial mercury-vapor arc with three filters of mercuric chloride solution of varying concentration, and using also the radiation from a commercial mercury discharge tube. The relative effectiveness of the various wave lengths was inferred from the frequency of induced endosperm deficiencies, in terms of the spectral distribution of energy in the filtered radiation applied. The indications regarding wave-length GENETIC AND CYTOLOGIC AL EFFECTS 261 dependence were quite different from those found in the experiments of Noethling and Stubbe (1936) with monochromatic irradiation of Antir- rhinum pollen. The results indicated (1) that wave lengths 313 m^ and longer were relatively ineffective, (2) that wave length 302 m^ was geneti- cally effective but less effective than wave lengths 297 m/x and shorter, and (3) that wave length 254 m^u was much more effective than 297 mju. Subsequent studies with monochromatic radiation, reviewed in a later section, confirmed these indications. The maize data are for the fre- quency of endosperm deficiency, while the Antirrhinum data are for the frequency of point mutations. However, tests of mutation rate in the F2 progenies indicated the same spectral relations for mutations as for endosperm deficiency, as far as could be determined from filtered radia- tions (Stadler, 1941a). The frequency of induced embryo abortion was much lower with the longer ultraviolet wave lengths than with the shorter. Comparing doses approximately equal in frequency of induced endosperm deficiencies, the frequency of induced embryo abortion was about nine times as high for the discharge-tube radiation (chiefly wave length 254 m^u) as for the filtered radiation (chiefly wave lengths 297 mn and longer). The frequency of endosperm deficiencies induced by ultraviolet treat- ment of the pollen is very much higher than the frequency of deficiencies affecting the embryos of the same seeds (Stadler, 1941a). Both values can only be estimated, but the discrepancy is too great to be accounted vor by any possible error in the estimates. With maximal doses of the longer wave lengths, the frec^uency of endo- sperm deficiencies marked by A, Pr, and Su sometimes exceeds 40 per cent. Since these marker genes can detect only a part of the deficiencies occurring in 3 of the 20 chromosome arms, there are presumably several hundred endosperm deficiencies per hundred seeds. These represent the deficiencies (and perhaps gene mutations) realized under the conditions of endosperm development, from alterations induced in one of the sperm nuclei of the treated pollen grains. The embryos of the same seeds may be checked to provide a maximal estimate of the frec^uency of deficiencies realized under the conditions of embryo development, from alterations induced in the other sperm nuclei. By this check, every sperm nucleus in the tested population of treated pollen grains may be accounted for. Assuming that every aborted embryo, every plant which failed to yield a pollen specimen, and every plant which showed segregation of defective pollen represents an induced deficiency, the maximum estimate of defi- ciencies in the gametes tested by embryo constitution is only about 30 per cent. A similar comparison may be made for endosperm deficiencies and embryo deficiencies marked by specific genes affecting both endosperm and plant characters. Endosperm deficiencies of .4 are very common, 202 KAUIATION H1()L(KJY occurring at the doses here considered with fre(iueiicies as high as 20 per cent. Deficieney of A in the Fi seedlings is rare; a series of progenies grown from seeds which inchuled H)S endosperm deficiencies for A yielded only five -1 deficiencies in the Fi plants. Approximately three-fourths of the endosperm deficiencies are frac- lionals, commonly affecting about half of the endosperm. Deficiencies similarly affecting half of the pro-embryo would presumably be present in the resulting Fi plants in only about half of the alTected cases, and this proportion might be reduced by competitive development of the defective and nondefective sectors. But the proportion of fractionals, as shown by the endosperm deficiencies, cannot account for the discrepancy; among the seeds which yielded the five A deficiencies mentioned in the preceding paragraph, there were more than 100 with nonfractional A deficiencies in the endosperm. This pronounced disproportion between the frequency of deficiencies in the endosperm and the embryo does not occur wuth X-ray treatment. Ill cultures marked by specific genes affecting both endosperm and plant characters, deficiencies in the Fi plants are somewhat less frequent than in the endosperms, but not more so than might possibly be accounted for by reduced survival. Aside from possible differences in the exposure to ultraviolet of the two sperms in the treated pollen grain, a possible cause of the wide disparity is contrasting behavior of chromosome l)reaks in endosperm and embryo development. McClintock's study (1941) of the effects of mechanically broken chromosomes suggests that some alterations induced in the two sperms, though similar in character and in frequency, might have quite disproportionate effects upon the frequency of detectable changes in the embryos and endosperms of the resulting seeds. This possibility has l)een investigated by Schultz (1951), using a ring chromosome {Dp 3a) carrying the gene A''. The pollen treated was that of a Dp 3a stock homozygous for a deficiency at the A locus, a-X3, a type in which all functioning pollen carries the duplication (Stadler and Roman, 1948). Pollinations on ears of aa constitution show the loss of A'' in endosperm and embryo tissue by the absence of anthocyanin pigment. With untreated pollen a small percentage of deficiencies occurs in both endosperm and embryo, owing to losses of the ring in late microspore or early endosperm or embryo divisions. The sporadic loss of the ring in subse(iuent mitoses results in variegation in the remaining endosperms and plants. The result of a break in the ring chromosome differs from that of a break in a rod chromosome chiefly in the effects of restitution. If the break may be followed alternatively by restoration t)f the linear order between the original chromatids or crosswise between the sister chroma- tids, the effect in a rod chromosome would be undetectable in both cases. GENETIC AND CYTOLOGICAL EFFECTS 2C3 But, in the ring chromosome, reproduction must take place in a single plane; otherwise the daughters either will be interlocked or will form a double ring with two centromeres. "Crosswise" restitution in the ring chromosome is therefore likely to result in loss of the ring. Schultz (1951) found that ultraviolet treatment very greatly increased the frequency of loss of the ring, both in the endosperm and in the embryo. The frequency of deficiencies for A'' among the Fi plants was about as high as the frequency of entire (i.e., nonfractional) endosperm deficiencies. This indicates that the low frequency of broken chromosomes found in plants from irradiated pollen, as compared with the endosperm, is largely due to a higher rate of restitution in the plant. Another interesting result of the study was the absence of any evidence of the production of a rod chromosome from the ring. The type of break which would ordinarily result in a stable terminal deficiency should, if it occurred in a ring chromosome, convert the ring to a rod. This would be detectable as a nondeficient nonvariegated plant. Among more than a thousand seedlings in progenies from ultraviolet-treated pollen, no such plant was found, although the number of A* deficiencies presumably induced by the treatments was almost 200. This result is not in conflict with the observed occurrence of terminal deficiencies in other studies. The frequency of deficiencies for a given locus as observed in the Fi plants is extremely low, and the primary breaks or potential breaks which occur in a rod chromosome proximal to the locus concerned may be much more numerous than those which would occur within the small ring. Presumably the proportion of potential breaks resulting in deficiencies realized in the embryo may be well below one in 200. The results with the ring chromosome show that the frequency of poten- tial breaks induced by ultraviolet radiation is extraordinarily high, and that in the embryo all but an extremely small proportion of these are fol- lowed by restitution. The types of chromosomal alterations found in the deficient plants that make up this small proportion may be quite mislead- ing as to the primary chromosomal effects of the radiation. The frequency of deficiencies detectable in the endosperm following ultraviolet treatment is comparable with that found following X-ray treatment. The two agents differ widely in the relative frequency of deficiencies affecting the endosperm as a whole and deficiencies affecting only a part of the endosperm ("fractionals"). With ultraviolet, about 75 per cent of the endosperm deficiencies observed are fractionals, and the fraction showing the deficiency is most frequently about one-half of the endosperm, as estimated by the surface area. The deficient sectors vary widely in form and in relative size, sometimes covering only a small frac- tion of the surface and sometimes covering the entire surface except for a small fraction. The frequency distribution of these fractions of varying size is approximately normal about the modal class of )^. Presumably 2()4 RADIATION BIOLOGY they represent the distribution of tissue resulting from chjinoe variations in cleveh)pment, when one cell in the two-celled pro-endosperm is deficient. With X rays, the endosperm deficiencies affect the entire endosperm in most cases. There is a substantial minority of fractionals, but these in general are strikingly different in pattern from those observed with ultra- violet. In most of them, the deficient portion co\'ers the entire surface of the endosperm except for one or more islands of tissue, usually amounting in total to only a small fraction of the entire area. These cases in the X-ray material cannot be regarded as merely the extremes of a range of patterns resulting from random variations in development of the half- deficient endosperm; there is no corresponding frecjuency of half-and-half mosaics and their variants. The distribution of tissue in the fractionals, as observed in the mature endosperms, suggests that in the ultraviolet cases the nondeficient tissue is the sector derived from one cell of the two- celled pro-endosperm, but in the X-ray cases it is the sector derived from only one or a few cells at a more advanced stage of the pro-endosperm. The distribution of tissue is as would be expected if a chromosome frag- ment could occasionally escape elimination through one or more cell divisions, and then be restored to normal mitotic distribution. The elucidation of the breakage-fusion-bridge cycle in mechanically broken chromosomes by McClintock (1941) suggests plausible hypotheti- cal mechanisms by which such endosperm patterns could be produced, though the sequelae of mechanical l)reakage do not parallel those of either X-ray or ultraviolet alteration of the chromosome. A comprehensive study of the chromosomal effects of ultraviolet and of X rays, as shown by the mosaic patterns in the maize endosperm, is being made by Faberge (1951). Using the endosperm marker genes /, Sh, Bz, and Wx, all located in a single chromosome arm, a variety of chromosomal effects may be recognized, including rings, dicentric translocations, and inversions, if accompanied by a breakage-fusion-bridge cycle. Ultra- violet treatment produces all these aberrations in large numbers, as does X-ray treatment. Cytological studies of the chromosomal effects of ultraviolet in maize have been made by Singleton (1939), Singleton and Clark (1940), and De Boer (1945). The accounts of these studies have been published only in abstract form. Singleton (1939) examined Fi plants identified by the loss of dominant characters present in the treated male parent and found cytologically demonstrable deficiencies of the corresponding chromosome regions in four plants. In another series, cytological examination of Fi plants identified by segregation for defective pollen showed deficiencies in several cases, including one plant with deficiencies for parts of two chromosomes. All the deficiencies observed appeared to be terminal. No translocations were found. Singleton and Clark (1940) found, among 16 Fi plants with segregation for defective pollen, 8 with observable GENETIC AND CYTOLOGICAL EFFECTS 265 deficiencies and 8 without. In addition there was one "dehciency trans- location, . . . three-armed, the plant being deficient for parts of chromo- somes 1 and 10." De Boer (unpublished) found several similar cases of deficiency and deficiency translocation. Although all the deficiencies appeared to be terminal, the distinction between terminal and nonterminal deficiencies is not convincing in maize without critical cytological material. Nonhomologous pairing may give a known interstitial deficiency the appearance of a terminal deficiency. De Boer (19-15) has presented evidence of ultraviolet-induced terminal deficiency free from this difficulty. The gene bz is a plant color gene located on the short arm of chromosome 9, and stocks of maize are avail- able bearing a terminal knob on this arm. Ultraviolet- and X-ray- induced deficiencies of Bz, in a stock with the terminal knob, were exam- ined cytologically, the criterion of terminal deficiency being loss of the terminal segment including the knob. Nonhomologous pairing of an interstitial deficiency could result in a terminal unpaired region of the untreated chromosome, but would be recognizable by the presence of the knob, or a portion of the knob, on the deficient chromosome. Among six Bz deficiencies in the ultraviolet series, four showed terminal deficiency of the short arm of chromosome 9. The other two were deficiency translo- cations. Among nine Bz deficiencies in the X-ray series, none was a terminal deficiency. These alterations included one interstitial defi- ciency, three ring-9 configurations, and five deficiency translocations. Straub (1941), in a study of somatic metaphase chromosomes, has shown that ultraviolet-induced translocations occur with appreciable fre- quency in Gasteria. From some 1800 embryos obtained from the fertili- zation of untreated eggs by exposed sperm, 210 were selected for cytologi- cal study because their weak development suggested that they might possess chromosomal abnormalities. Of these, 75 showed chromosomal changes as contrasted to 1 from 300 control embryos. Four of the embryos were chimeras showing some cells with normal and some with altered chromosomes. Straub considered these to be similar to the frac- tional endosperm deficiencies induced by ultraviolet in maize. In the remaining 71 embryos, 72 apparently terminal deficiencies were detected in the long arms of the four G chromosomes, which could be recognized by their conspicuous satellites. The breaks giving rise to these deficien- cies were largely concentrated in the neighborhood of the centromere. Of the remaining cytological abnormalities, five were translocations, includ- ing one of an undefined but probably reciprocal type, two which were defined as "isochromosomes," one dicentric chromosome, and one ring chromosome. Barton (1954) has compared the chromosomal effects of X-ray and ultraviolet treatments in the tomato {Ly coper sicum esculentum Mill.). In this plant each of the 12 chromosomes at pachytene shows a densely 200 RADIATION UIOLUGY staining chromatic n'nioii on cither .side of the chromomere and a Hghtly staiiiiiifj; achromatic rc}>ioii (list ally. Each chromosome arm terminates in a sinfile well-delined chromomere. This circum.stance is very favorable for the detection of terminal deiiciencies, especially those in which the break is in the achromatic region. Barton found a nnicli higher ratio of deficiencies to translocations with ultraviolet than with X rays. The two translocations found with ultra- violet were deficiency translocations. Mutations also showed a much higher ratio to translocations with ultraviolet. The deficiencies found with both ultraviolet and X-ray treatment included both terminal and interstitial deficiencies. Chromosome breakage was highly localized in the chromatic regions; however, the terminal deficiencies (observed in the ultraviolet series included one in which the break was in the achromatic region. The various contrasts in genetic effects which ha\e been mentioned indicate that the chromosome breaks induced by ultraviolet radiation are of a (lualitatively different kind from those induced by X rays. Accumu- lating evideiice from many sources has indicated that the mutations induced by X rays are in many cases, if not in all, extragenic alterations incidental to chromosome breakage. The indication that the chromo- somal effects of the ultraviolet are of a different kind encourages the hope that the induced mutations also may be (lualitatively different. This possibility may be investigated effectively only by the critical study of the mutation of specific genes, for there are no general criteria by which mutations induced at miscellaneous loci may be distinguished from the possible effects of known extragenic phenomena. AVith certain selected loci it may be possible to develop special criteria for the identifi- cation of gene mutations and for the recognition of alterations which, in experiments on the general mutation rate, would .simulate gene mutation. A comparison of X-ray and ultraviolet mutations of the gene .4 in maize (Stadler and Roman, 1943, 1948) indicates that the mutations induced by the two agents may be qualitatively different. Among about 400 alterations affecting A, induced by X rays, and a much smaller num- ber induced by ultraviolet, those most nearly approaching the typical genetic behavior of gene mutations were selected for detailed study and comparison. Among the X-ray alterations, only two were normal plants free from segregating pollen defects. A third haplo-viable X-ray altera- tion, showing segregating for subnormal but not aborted pollen, was included for comparison. Among the ultraviolet alterations, normal plants with normal pollen were more frequent. Three cases with a pheno- type and one with intermediate phenotype (.4") were included in the comparisons. The three X-ray mutants were characterized in varying degree by reduced viability in haplopha.se and by reduction in the frequency of GENETIC AND CYTOLOGICAL EFFECTS 207 crossing over, and were found to be inviable as homozygotes or com- pounds. These are attributes suggestix-e of deficiency, and all three mutants wei'e identified as deficiencies by f2;enetic evidence showing that the induced alteration in each case involved loss of the effects not only of A but of additional genes affecting chlorophjdl de\'elopment and viability. The four ultraviolet mutants gave no indication of deficiency by any of the criteria mentioned. The evidence for Drosophila comparing the genetic effects of X-ray and ultraviolet radiation contrasts somewhat in its general implications with that from plants. It would not be surprising to find actual contrasts, but the evidence from both sources is still too scanty to force the assump- tion of any basic difference in the nature of the chromosomal or genie alterations induced. The contrasting indications are briefly noted as follows : 1. Gross chromosomal rearrangements. Asshownby Altenburg (1934) and by Muller and Mackenzie (1939), there is no appreciable frequency of gross chromosomal rearrangements in ultraviolet progenies which yield an abundance of sex-linked lethals. Demerec et al. (1942) found a single translocation in the progeny of ultraviolet-treated flies, but this single case cannot be considered evidence of an effect of the treatment. The absence of induced translocations in the Drosophila cultures tested does not represent a conflict in the evidence, for there is no necessary implica- tion that the radiation is unable to induce translocation in low frequency. The Drosophila evidence shows that the ratio of induced translocations to induced sex-linked lethals is far lower with ultraviolet than with X rays. The maize evidence also shows that the ratio of translocations to muta- tions is far lower than with X rays, and shows further that translocations are induced in very low frequency. A search for terminal deficiencies of the X chromosome, identifiable by genetic markers, was made by Mackenzie and Muller (1940) and by McQuate (1950), and none was found. The occurrence of ultraviolet- induced terminal deficiencies in maize was shown cytologically by De Boer (1945). 2. Nature of the induced mutations. Although genetic experiments to detect minute rearrangements, by Mackenzie and Muller (1940), indi- cated that they were absent or very rare in ultraviolet progenies, the direct cytological study of induced lethals by Slizynski (1942) clearly showed that short deletions are included in this class. The frequency of cytologi- cally detectable deletions among the sex-linked lethals was not much lower among the ultraviolet than among the X-ray cases. They occur also, in considerable frequency, among sex-linked lethals arising in untreated material. The implication from the evidence in maize is that the ultraviolet mutants are distinctlv different from the X-rav mutants in that the 268 RADIATION mOT-OOY former show no evidence of deliciency, while the hitter Iik ludc dearly detectable deficiencies. The evidence of deficiency here is genetic rather than cytolot!;ical. The only critical evidence is from the induced muta- tions of A studied by Stadler and Roman (1948), and here the number of cases is too small to imply the absence or extreme rarity of short deficiencies among ultraviolet mutations. The fact that no interstitial deficiencies have been found in cytological studies of ultraviolet progenies in maize cannot be considered evidence of their extreme rarity, for here again the only critical evidence, that of De Boer (1945) on Bz deficiencies, relates to a relatively small number of cases. It is dear that ultraviolet radiation induces terminal deficiencies in maize, but it is possible that it may induce interstitial deficiencies also, and that some of these may be included among the alterations genetically identified as mutations. It should be noted also that the alterations identified as mutations in Drosophila and in maize may not be analogous classes. The sex-linked lethals spontaneously occurring in Drosophila appear to be qualitatively identical with sex-linked lethals induced by X rays, and the ultraviolet lethals are not dearly distinguishable from either class. In maize, the evidence of difference in the type of mutation induced by the two agents comes from studies at a specific locus, at which the X-ray mutants are found to be distinctly different from the spontaneous mutants. Here the ultraviolet mutants are found to be dearly distinct from the X-ray mutants and similar to the spontaneous mutants. DIRECT EFFECTS OF ULTRAVIOLET RADIATION ON CHROMOSOMES The pollen-tube technique, as employed with Tradescantia pollen, per- mits a study of the direct effects of ultraviolet on chromosomes before inviable aberrations can be eliminated. As discussed earlier, the tech- nique has certain inherent limitations in that only those aberrations realized by the onset of metaphase can be recognized; any which would form at later stages of cell division, or during the process of fertilization, would escape detection. The chromosomes in the generative nucleus are effectively double to both X rays and ultraviolet, and the aberrations induced are consequently of the chromatid types. In the course of these studies, over 50,000 chromosomes have been examined, and approximately 700 terminal deletions have been identified (Swanson, 1940, 1942, 1943). With the exception of occasional iso- chromatid deletions or chromatid translocations, which were no more fre- quent than in untreated nuclei, the aberrations were all terminal. No interstitial deficiencies have been identified with certainty, but it is realized that the method of analysis is such as to preclude their positive identification. The great majority of the deficiencies involved the break- GENETIC AND CYTOLOGICAL EFFECTS 269 age of only one of the two sister chromatids, although infrequently half- chromatid deficiencies were observed (Swanson, 1947). The frequency of terminal deficiencies increases linearly with increasing dosage if the time of exposure after the pollen grains have germinated is 15 30 60 240 120 EXPOSURE, sec Fig. 7-1. The relation of chromatid deficiencies (terminal deletions) to the dosage of ultraviolet (wave length 254 m/u) in the pollen tube chromosomes of Tradescantia. Radiation given at a distance of 20 cm, at an intensity of approximately 10^ ergs/ mm^/60 sec, and at 2 hr after germination. (Sivanson, 1942.) 5 10 TIME AFTER GERMINATION, hr Fig. 7-2. The relation of chromatid deficiencies (terminal deletions) induced by ultra- violet (wave length 254 lUfx) in the pollen tube chromosomes of Tradescantia to succes- sive prophase stages following germination. Dosage approximates 2 X 10' ergs/ mm^'/eO sec. (Swanson, 1943.) kept constant (Fig. 7-1). With increasing condensation of the chromo- somes as they pass through prophase development, the frequency of deficiencies induced by any given dose declines, however, suggesting that internal changes taking place within the chromosome play a role in deter- mining its susceptibility to breakage by ultraviolet (Fig. 7-2). A ques- 270 RADIATION HIOLOGY lion may Ix' raised concerning the relali\c resistance of the chromosomes to breakage at the 0- and 1 -In- periods. At these time periods the genera- tive nuclei have not, as a rule, passed from the pollen grains into the pollen tubes, and some uncertainty exists as to the degree of absorption of ultra- \iolet by the heavily pigmented pollen wall. At the 2-hr period, however, the nuclei are in the pollen tube, where absorption by overlying materials is at a minimum. After the 10-hr period the fretiuency of induced aberrations does not exceed that found in untreated nuclei. This period corresponds roughly to late prophase. In addition to the terminal deficiencies induced by ultraviolet in the pollen tube chromosomes of Tradescantia, there is also found a type of aberration which, for want of a more definite term, has been called an " achromatic lesion " (Swanson, 19-40). This type of aberration is induced by X rays also. The lesions extend completely or partially across the chromatid in the form of a nonstainable gap. Their fre(iuency increases with increasing dosage. Whether they represent incompletely separated deficiencies, interstitial losses of chromatin, or merely separated coils within the chromosome is not known. Since many of them extend only a part of the way across the diameter of the chromatid, a large subjective error would be involved in any determination of frequency, and for this reason they have been omitted in the tabulated data. The nature of the ultraviolet-induced deficiencies in the pollen tube chromosomes of Tradescantia suggests that, structurally at least, they are comparable to the fractional endosperm deficiencies in maize even though the changes are induced in dissimilar types of nuclei. Each involves the loss of a portion of a chromatid. No aberrations were found in Trades- cantia, however, which involved both chromatids, and which would corre- spond to the entire endosperm deficiencies. Whether this difference can be ascribed to differences in the nuclei studied, to their different states of chromosome condensation, or to some unknown factor cannot be answered at present. ]Muller (1941) suggests that the preponderance of fractional endosperm deficiencies in maize treated with ultraviolet may result from a mutational process initiated in a single-stranded chromosome but delayed in completion until the chromosome has doubled, the effect being restricted ordinarily to only one of the two chromatids. This hypothesis appears unnecessary in light of the Tradescantia data. Since half- chromatid aberrations are found occasionally in treated pollen tube chromosomes, the chromosomes must have at least four strands, and chromatid deficiencies must therefore involve the fracture of t\vo half- chromatids at the same locus. If the chromosomes of the sperm cells of maize pollen have only two strands, the loss of both chromatids by simul- taneous breakage to give rise to entire endosperm deficiencies becomes understandable. P'igure 7-3 illustrates the effectiveness of X rays (370 r) in inducing GENETIC AND CVTOLOGICAL EFFECTS 271 aberrations in the pollen tube chromosomes of Tradescantia at successive prophase periods. A comparison can, therefore, be made with the ultra- \iolet data obtained under similar circumstances (Fig. 7-2). Isochroma- tid deficiencies and chromatid translocations are readily induced by X rays, and the close parallelism of the terminal deficiency and translo- cation curves supports the generally accepted hypothesis that a transloca- tion owes its origin to the illegitimate fusion of the broken ends of two independently induced deficiencies. It would appear that the lack of translocations in the pollen tube following exposure to ultraviolet cannot 0 2 4 6 8 10 TIME AFTER GERMINATION, hr Fig. 7-3. Relation of chromatid aberrations induced by X rays (370 r) in the pollen tube chromosomes of Tradescantia to successive prophase stages following germination. Curve I, terminal deficiencies; II, translocations; III, isochromatid deficiencies. (Swanson, 1943.) be ascribed to a lack of deficiencies. This is more clearly indicated in Table 7-2, where the frequencies of terminal deficiencies induced by the two types of radiation are similar, but those of translocations are not. The great majority of broken ends of chromosomes induced by ultra- violet clearly do not possess the capacity for subsequent reunion. This may result from a more rapid healing of the broken ends, or it may stem from a lack of maneuverability of broken ends imposed by the surrounding chromosomal matrix. The matrix seems not to be disrupted b}^ ultra- violet, and it may, following heavy doses, actually become more promi- nent in appearance (Swanson, 1942, 1943). The terminal deficiencies produced by both radiations in the pollen tube chromosomes of Tradescantia are indistinguishable in microscopic appear- ance, but there is good evidence here, as in maize endosperm, for believing 272 UADIATION HIOLOGY that they are quiihtativrly (UlTcicnt l)oth as to their nature and as to their mode of orij^in. \\'h(Mi ultra \iolet is used in combination with X rays as a pretreatment, the frecjuency of terminal deficiencies is no greater than that Tabi.k 7-2. Kkequencies of Chromatid Aberrations Induced in the Pollen Tube Chromosomes of Tradescantia by X Rays and Ulthavioi.ht (RwMiisoii, MM2.) Percentage aberrations per chromosome Treatment Chromatid deletions Isochroinatid deletions Translocations Total chromosomes Ultraviolet (2537 A) . . X ray (123 r) 10 15 10.00 0 2.7 0 4.4 030 180(i expected from the ultraviolet treatment alone (Table 7-3). It would appear that the circumstances which favor the induction of one kind of terminal deficiency actually suppress the appearance of the other kind. Table 7-3. Frequencies of X-ray-induced Chromatid Aberrations as Influenced by Pretreatment with Ultraviolet (Swanson, 1944.) Percentage aberrations per chromosome Treatment Chromatid deletions Isochroinatid deletions Translocations Total chromo- somes Ob- served Ex- pected Ob- served Ex- pected Ob- served Ex- pected Ultraviolet (2537 A) . . . . X ray (246 r) Ultraviolet -|- X ray . . . 3.05 3.51 3.01 6.56 1.05 0.35 1.05 1.89 0.35 1.89 3540 5304 2256 The X-ray-induced aberrations involved in illegitimate fusion, i.e., iso- chromatid deficiencies and translocations, are similarly suppressed. Since a like reduction in frequency of X-ray-induced al)errations is encoun- tered when ultraviolet is employed as a posttreatment, it seems reasonable to assume that the action of ultraviolet is not to prevent the initiation of X-ray-induced breaks, but rather to lessen their probability of realization. Kaufmann and Hollaender (1946) have demonstrated that a combination of the two radiations has a similar depressing effect on gross chromosomal aberrations in Drosophila, whereas Schultz (1951) has shown that the effects of X rays in maize, as judged by the freciuency of gcrmloss seeds, are completely inhibited by a posttreatment with wave length 297 m^t at the same time that the ultraviolet effects remain uninfluenced by X rays. GENETIC AND CYTOLOGICAL EFFECTS 273 Since the ring-chromosome studies in maize also revealed that breakage by ultraviolet is frequent, a phenomenon obscured in rod chromosomes by restitution, it has been assumed that ultraviolet must have, in addition, a marked influence on the matrices of the chromosomes. Coagulation of the matrices by ultraviolet would thus not only prevent the realization of rearrangements from X-ray-induced breaks but also from those breaks which it itself induces, providing in this manner a mechanism which would lead to an apparent qualitative difference in behavior of the X-ray- and ultraviolet-induced breaks. SPECTRAL RELATIONS Significant comparison of the relative effectiveness of different wave lengths can be made only on the basis of rather precise estimates of the amount of energy reaching the site of the mutagenic reaction. Among higher organisms suited to the genetic analysis of the induced alterations, this is a serious difficulty, and all wave-length comparisons must be interpreted with due regard for the approximations involved in estimating the dose actually applied to the chromosomes whose reactions are determined. With Dr-osophila, using the technique of irradiation of the adult fly, the comparison of effectiveness of different wave lengths is not feasible. Mackenzie and Muller (1940) estimate that about 99.9 per cent of the ultraviolet energy is absorbed before the radiation reaches the germ plasm. Even slight differences in the relative loss for different wave lengths could make tremendous differences in their relative intensity at the site of genetic action. With the polar cap technique the absorption loss is very much less, but the technical requirements for the identification of the individual mutation make the method unsuitable for experiments on the scale required for wave-length comparisons. With seed plants adapted to genetic analysis, irradiation of the pollen presents some opportunity for the comparison of wave-length effective- ness. Extensive data on the effects of monochromatic radiations have been reported for Antirrhinum and for maize. But even within the single pollen grain, internal filtration results in large and variable differences in the penetration of different wave lengths to the site of the nucleus. The maize pollen grain is approximately spherical, with a diameter of about 93 n. Uber (1939) measured ultraviolet transmission, at different wave lengths, for the pollen grain wall and pollen grain contents of maize. The results indicated that, with equal incident energy at wave lengths 297 and 265 m^u, for example, the dose penetrating to a point 16 fx beneath the wall is three times as large for the longer wave length as for the shorter, and at a depth of 32 n it is about fifteen times as large. In most of the pollen grains the sperm nuclei are located within this depth range. But, 274 RADIATION HIOLOGY since the pollen, when treated, is oriented at random with reference to the radiation source, the sperm nuclei in most of the pollen grains treated are at a greater depth, and the filtration losses and ineciualities of filtration loss are mucli greater. Obviously, under such conditions any corrections for dosage must be at best very rough approximations. In the liverwort Sphacrocarpus donncllii, conditions for the comparison of wave-length effectiveness are incomparal)ly better, for the radiation may be applied to the spermatozoid, which is an almost naked nucleus about 0.5 n in thickness. Knapp and Schreiber (1939; see also Knapp et ai, 1939) have compared the effects of monochromatic radiations in this organism. For direct cytological comparison of chromosomal effects, the pollen- tube technique used by Swanson (1940) with Trade scantia is also well suited to the study of w^ave-length effectiveness, with minimal interference from internal filtration of the radiation. Studies of this kind have been exploratory only (Swanson, 1942), but they indicate that considerable wave-length differences are to be expected. Dosage Effect. The comparison of wave-length effects requires a care- ful consideration of the dosage effect for two reasons: (1) for evaluating the error in approximating equal dosage with the wave lengths compared, because of the varying internal filtration already discussed, and (2) for determining the actual form of the dosage curve, as a basis for interpreting the differences found \vith the wave lengths compared. The effect of variation in internal filtration among the individuals treated is to flatten the dosage curve for specific effects. The population treated consists of individuals varying in the proportion of the incident dose that will be received by the gamete nucleus. For example, in a population of maize pollen grains as described earlier, some may be so oriented that the gamete nucleus is reached by radiation that has pene- trated through 1() /i of overlying material, while in others the gamete nucleus can be reached only by radiation that has penetrated through 80 /i of overlying material. The first unit of dosage may produce the effect in any of the pollen grains, and its hits will tend to occur most frequently in the most favorably oriented ones. Added units of dosage may produce additional effects only in the unaffected individuals remaining, which offer a lower probability of hits because of the lowered dose reaching the gamete nucleus in these pollen grains. The flattening of the dosage curve resulting from this factor should occur at all ultraviolet wave lengths, Init should be more pronounced at the shorter wave lengths since these show higher absorption in the pollen grain contents. Using observed values for the position of the miclei within the pollen grain and for the trans- mission losses in jiollen wall and contents, the expected form of the dosage curve at wave lengths 254, 297, and 302 m/x was in fairly good agreement with that observed (Stadler and Uber, 1942). GENETIC AND CYTOLOGICAL EFFECTS 275 In experiments with the discharge-tube radiation (largely wave length 254 m^), it was possible to show the relation of internal filtration to the dosage curve directly (Table 7-4). A given dose may be applied with Table 7-4. Frequency of Endosperm Deficiencies from a GrvEN Dose When Applied to Both Sides of Pollen as Well as to One Side Only (Wave Length 254 M/j.) (Stadler and Uber, 1942.) Frequency, Dose per cent One unit (one side) 18.9 Two units (one side) 24 . 9 Two units (one from each side) 35.4 equal effect from either above or below the layer of pollen. When the dose is doubled by applying a second unit of dose from the same direction, the added frecjuency of induced effects is considerably less than that from the first, and thus the yield from 2 units is considerably less than double the yield from 1. But if, instead, the second unit of dose is applied from the opposite side, the added yield of induced deficiencies is as great as that from the first unit, and thus the yield from 2 units of dose is double the yield from 1. When the measure of radiation effect is not a specific result (e.g., a given deficiency, death of the irradiated individual) but rather an indefi- nite group of results, any number of which may be observed in the single treated individual (e.g., mutations at miscellaneous loci), the result expected from internal filtration is not a flattening of the dosage curve. Instead it is a tendency toward coincidence of independent effects in the single treated gamete, such as was noted in the maize experiments men- tioned in an earlier section of this review (see also Meyer et al., 1950). A flattening of the dosage curve for mutation frequency would be expected as a result of variations in internal filtration only if the accumulation of mutants and other radiation effects is a factor in eliminating individuals from the population tested. In treated populations in which there are large variations in exposure to radiation injury, gross distortion of the dosage curve ma\^ occur. For example, if the treated population were a mass of pollen grains more than one layer deep, the lower layers would be almost wholly shielded from the radiation. With a sufficiently heavy dose, the pollen grains of the top layer might be largely eliminated from the population tested, and the fre- quency of genetic effects in the surviving population would be materially lower than that found with lighter doses. The correlation between genetic effects and killing would be a spurious correlation, but any correla- tion of genetic effects with elimination among the individuals treated would tend to flatten or reverse the dosage curve. The dosage data for maize pollen treatments are therefore of interest 27G RADIATION BIOLOGY chiofly ill relation to the error involved in wave-length comparisons, rather Ihaii to the nature of the reaction of the chromosomes to increasing dose. The results of dosage trials at wave lengths 254, 297, and 302 mn are shown in Table 7-5. Tablk 7-5. l{ia,.\TioN of Do.sk to FHWiiENrY OK Endosperm Deficiency (Stndlcr ;in(l t'l.cr, I'.tt2.) Dose, Wave length, Kiulospenn deficiencies, X lO^ergs/nnn" m^t per cent ± S.E. 1.0 254 3.0 ±0.6 2.0 254 5.7 ±1.1 4.0 254 8.2 ±1.6 8.1 254 10.7 ±1.8 17.1 254 16.8 ± 2.6 4.0 297 2.6 ± 0.7 7.9 297 10.7 + 1.1 15.7 297 18.0 ± 2.2 88.3 297 32.6 ± 2.3 15.6 302 2.9 ± 0.7 32.0 802 9.4 ± 1.0 63.8 302 24.8 ± 1.8 132.0 302 36 . 1 ± 2.7 The relatively low yield of endosperm deficiencies at the lowest doses tested for wave lengths 297 and 302 suggests the possibility of a multiple- hit curve for the dosage relation. This possibility was suggested also by the results of treatments at nine wave lengths in the range 238-293 m/z, comparing the frequency of endosperm deficiencies from doses of approxi- mately 0.5 X 10^ ergs/mm- with that from doses of approximately 2X10^ ergs/mm-. In every case the yield from the lower dose was less than one-fourth of that from the higher dose. Since these treatments were not seriated for the control of daily variation, a separate trial was made with wave length 2G5 m/x, using doses of 0.25, 0.50, 1.0, and 2.0 X 10^ ergs/mm'-. The frequency of induced endosperm deficiencies was again disproportionately low at the lower doses, but the deviation from linearity was not statistically significant. Indications of nonlinearity of ultraviolet effect on the frequency of mutations at low doses have also been found in lower organisms. The data of Emmons and Ilollaender (1939, Table 2; see also Hollaender and Emmons, 1941) on induced mutation frequencies in Trichophyton suggest that low doses are disproportionately less effective than higher doses, although again the departure from linearity is not always obvious or consistent. Similar results have been obtained in AspfrgiUns (Swanson GENETIC AND CYTOLOGICAL EFFECTS 277 et ai, 1948, Table 2) and Neurospora (Hollaender et ai, 1945). The statements of Lea (1946) and Catcheside (1948) to the effect that a linear proportionality holds up to the peak of the ultraviolet mutation curve therefore cannot be accepted without question. The need for more precise measurements of mutation effects at low doses is evident, since the interpretation of data on the comparative effects of different wave lengths must depend in part upon the shape of the dosage curve. The striking drop in mutation frequency observed at very high doses of ultraviolet in fungi (Emmons and Hollaender, 1939) has not been found in maize. This apparently is not related to factors of internal filtration, })ut rather to the selective elimination of individuals from the population. A somewhat similar drop in frequency has been reported in Drosophila. The study by Reuss (1935), in which ventral abdominal exposures of adult males w^as first employed, provided the first quantitative data on the effects of increasing increments of ultraviolet on the frequency of recessive lethal, semilethal, and visible mutations in Drosophila. Expo- sures of 15, 22.5, and 30 min were used, and a leveling in the dosage rela- tions was evident, but the data are inconclusive because of the small num- l>ers of flies observed. Those obtained by Sell-Beleites and Catsch (1942), following similar methods of exposure, are more striking. In two different experiments (Table 7-6), the mutation frequency rose to a peak Table 7-6. Types and Frequencies of Mutations in the X Chromosome OF Drosophila melanogaster Induced in Spermatozoa by Ventral Exposure of the Abdomen to Ultraviolet Radiation (Sell-Beleites and Catsch, 1942.) Duration of exposure, min Total no. of flies Lethals Visibles Mutations, per cent ± S.E. Expt. I 6 2431 26 0 0.92 ± 0.19 12 1195 22 1 1.77 ± 0.38 24 963 3 0 0.16 ± 0.13 Expt. II 5 2478 8 0 0.17 + 0.08 10 2002 14 1 0.60 ±0.17 15 1443 12 4 0.96 +0.26 20 1464 6 0 0.26 ± 0.13 with increasing increments of ultraviolet and then dropped abruptly as added increments were given. Sell-Beleites and Catsch concluded that they were dealing with a greatly disturbed "one-hit" curve, and that the decrease in mutation frequency at high doses results from increasing sterility induced by the penetrating radiation. Many of the irradiated flies were completely sterile or yielded no mutations of any kind. The percentage of sterility 278 KADI \'I'I<>N HIOLOCY increased witli increasing dosage and resnlted presumably from physio- l()gic;il damage unassociated with the mutation process. Once a maxi- mum mutagenic elTect was obtained in those indivi(hiais in which liic ra(hation had successfully penetrated, added increments of ultraviolet would be likely to contribute more rapidly to sterility than to the induc- tion of additional mutations. A drop in mutation frequency would there- fore be expected since the fertile flies with a high degree of filtration and a low frequency of mutations would contribute a disproportionately greater number of offspring to the next generation. Attempts to determine dosage relation in Drosophila by the polar cap techniciue have been reported. Altenburg ct al. (1950) indicated that a linearity of efTect obtains at low doses, but that a leveling of the curve is rapidly achieved, after which large increments of dose are relatively inef- fective in raising the frequency of mutation. Meyer ct al. (1950) also found that a clustering of mutations occurs in cells or chromosomes favorably oriented with respect to the radiation. A study of 1 1 chromo- some II lethals showed that 10 of these chromosomes carried additional mutations. Also, increases in dosage caused death to an increasing num- ber of pole cells, as evidenced by the greater proportion of Fi offspring carrying a particular mutation. Perhaps the best evidence oii the relation of ultraviolet effects to dosage, from the standpoint of the avoidance of internal filtration difficulties, is that from Tradcscaniia pollen-tube treatments. Chromatid deficiencies here provide the criterion of effect (Swanson, 1942). The data indicate that the relation is linear. Figure 7-1 illustrates the curve obtained with wave length 254 m/n. Wave-length Dependence Studies. The earliest studies of the differen- tial genetic effectiveness of various wave lengths of monochromatic light on the germinal material of higher plants were made by Noethling and Rtubbe (1934, 1936; see also Stubbe and Noethling, 1936). The dry pollen of Antirrhinum majus was treated, and the detection of induced mutations was made by observing the segregations in F2 populations. The control populations had a comparatively high frequency of spon- taneous mutations (1.6 per cent). From a study of Fo offspring from some 3000 pollen grains treated with wave lengths 265, 297, 302, and 313 mn, the authors were able to show that each wave length was capable of increasing significantly the frequency of mutations, the highest rate obtained being approximately four times the control frequency. Several doses were compared at wave lengths 265 and 297 m^u. At wave length 265 m/x, doses of 2 X 10'^ ergs/mm' failed to raise the muta- tion frequency significantly, and the rise with increasing doses was slow. At doses of 2.7 X 10' ergs/mm^ the frequency w^as twice that of the controls, while doses of 14.6 X 10' and 50.3 X 10' ergs/mm''^ increased it onlv to three times that of the control. The use of wave length 297 m^ GENETIC AND CYTOLOGICAL EFFECTS 279 in a wide range of doses (1.8 X lOMJo.O X lO'' ergs/mm^), gave signifi- cant increases over the control, but no significant differences between doses. A dose of 65 X 10^ ergs/mm- at wave length 302 m/x and one of 120 X 10^ ergs/mm- at wave length 313 m/x quadrupled the spontaneous frequency. From these results the conclusion was drawn that the peak of genetic effectiveness was in the neighborhood of 300 mfx. Supporting this hypothesis was the demonstration that a suspension of Antirrhinum pollen in 30 per cent alcohol had a maximum of absorption around 300 vay.. Comparable data by Stadler and Uber (1938) for the frequency of endosperm deficiencies induced by monochromatic radiations also showed effects in large numbers for all wave lengths tested in the range 235-302 mM- Longer wave lengths had no appreciable effect. The frequency of germless seeds was increased markedly by radiation of wave lengths 280 van and shorter, but not by longer wave lengths, even at much higher doses. The tabular data on which these conclusions were based were later published (Stadler and Uber, 1942, Table I), together with the results of additional experiments on relative wave-length effectiveness. The frequency of induced effects was higher for wave length 297 m/x than for 2G5 m^i, as in the experiments of Noethling and Stubbe. But, though wave length 297 m/x significantly excelled 265 van at the higher doses, the relation was clearly reversed at the lower doses. Both wave lengths were tested at doses of approximately 1, 2, 3, 6, and 12 X 10' ergs/mm^ While the longer wave length was about twice as effective as the shorter in comparisons made at the heaviest dose, it showed no effect at the lowest dose, and only about one-fourth of the wave length 265 m^u effect at the second dose. The results as a whole showed that there were wide differences in the dosage relations at the different wave lengths tested, and that a study of the dosage relation and of internal filtration was needed before the relative effectiveness of the wave lengths could be estimated. The results of these studies were summarized in the preceding section. The most reliable indications of relative effectiveness come from com- parisons made at low doses. However, at the lowest doses filtration losses are still effective and require correction. The frequency of endo- sperm deficiencies produced in an experimental comparison of seven wave lengths, all at a dose of 2 X 10-^ ergs/mm^, seriated for the control of daily fluctuations, is shown in Table 7-7. At this dose, the endosperm deficiencies induced by wave lengths 265 and 254 m^ix are about 3 times as frec^uent as those induced by wave length 297 m^i and about 15 times as frequent as those induced by wave length 302 m^u. When the dose is increased to about 8 X 10' ergs/mm', the differences are much less pronounced and in some cases hardly signifi- cant. The evidence on internal filtration, subject to the approximations which have been mentioned, indicates that wave lengths 265 and 254 in/u 280 UADIATION BIOLOGY are prohalily more than 10 times as effective as wave length 207 m^ and probably more than 100 times as efTective as wave length 802 in/i. in terms of ('(|ual int(Misity at the surface of the sperm inicleus. Tablk 7-7. Tnio Fbequency of Endosperm Deficiencies in Maize as a Function ok Wave Length at Two Different Doses (Stadlor and Tber, 1942.) Endosperm deficiencie.s, per cent ± S.E. Wave length, niM At 2 X 10* ergs/mm* At 8 X 10* ergs/mm* 248 9.9 ± 1.0 22.9 ± 2 :i 254 15.5 ± 1.:^ 23.2 + 2 2 2(i5 11.6 + 1.0 15.2 ± 12 270 7.8 ± 1.6 20.9 ± 19 280 9.3 ± 0.9 23 . 7 ± 1 5 289 6.2 ± 0.8 18.5 ± 1.5 297 4.7 ±0.5 11.4 ± 1.2 In the comparison of wave-length effects in Sphaerocarpns, complication from internal filtration is at a minimum, for the spermatozoid irradiated consists almost entirely of nuclear material. Knapp ei al. (1939) com- pared the effectiveness of six wave lengths in inducing genetic alterations in Sphacrocarpus donncllii. The spermatozoids were irradiated in water suspension, and the sporogonia produced by fertilization of untreated Table 7-8. The Frequency of Induced Mutations in Sphaerocarptis as a Function of Wave Length, Dosage 2 X 10' ergs/mm* (From Knapp ei al., 1939.) Wave length No. of sporogonia No. of Percentage m^u analyzed mutations mutations 254 61 17 27.8 265 53 22 41.8 280 64 14 21.9 297 52 3 5.8 302 71 4 5.6 313 65 0 0.0 Control 64 0 0.0 female gametophytes were tested by analysis of the spore tetrads. Mendelizing mutations were identified by the production of two normal and two mutant plants; lethal "mutations" (i.e., all genetic alterations w'ith haplo-lethal efTect), by the production of only two instead of four plants by the spore tetrad. By the tetrad analysis of 50 to 75 sporogonia representing each treatment, it was possible to show sharp differences in GENETIC AND CYTOLOGIC AL EFFECTS 281 the effectiveness of some of the wave lengths applied in inducing such alterations. Radiation injury was in general parallel to genetic effective- ness. The data are shown in Table 7-8. These data represent the most significant evidence now at hand, bear- ing upon the ac^tion spectrum of the ultraviolet in producing genetic effects. It is interesting to note that wave length 2()5 m/x is about seven times as effective as wave lengths 297 and 302 m^, and that the two latter wave lengths are not appreciably different in effectiveness. The effective- ness of wave length 254 m^t is about two-thirds that of 265 m/x though the significance of this difference is perhaps questionable. The effectiveness of wave length 280 mn is about half that of 265 m^. From the resem- blance between these values and the absorption spectrum of nucleic acid, the authors conclude that absorption by nucleic acid is of essential sig- nificance in determining the genetic effects of ultraviolet radiation. REFERENCES Altenburg, E. (1928) The limit of radiation frequency effective in producing muta- tions. Am. Naturalist, 62: 540-545. (1930) The effect of ultraviolet radiation on mutation. Anat. Record, 47: 383. (1931) Genetic effects of ultraviolet radiation. Anat. Record, 51: 108-109. (1933) The production of mutations by ultra . iolet light. Science, 78: 587. (1934) The artificial production of mutations by ultraviolet light. Am. Naturalist, 68: 491-507. Altenburg, L., E. Altenburg, H. U. Meyer, and H. J. Muller (1950) The lack of proportionality of mutations recovered to dosage of ultraviolet administered to the polar cap of Drosophila. Genetics, 35: 95. Barton, D. W. (1954) Comparative effects of X-ray and ultraviolet radiation on the differentiated chromosomes of the tomato. Cytologia, in press, liishop, C. J. (1949) Pollen tube culture on a lactose medium. Stain TechnoL, 24: 9-12. Catcheside, D. G. (1948) Genetic effects of radiation. Advances in Genet., 2: 271-358. Clark, C. (1948) A flotation method for the collection of insect eggs. Drosophila Information Service, Carnegie Inst. Wash., Cold Spring Harbor, N. Y., 22: 79. De Boer, K. O. (1945) Comparison of ultraviolet and X-ray deficiencies. Maize Genetics Coop. News Letter, Cornell Univ. Pp. 43-44. Demerec, M., A. Hollaender, M. B. Houlahan, and M. Bishop (1942) Effect of monochromatic ultraviolet radiation on Drosophila melanogaster. Genetics, 27: 139-140. Durand, E., A. Hollaender, and M. B. Houlahan (1941) Ultraviolet absorption spectrum of the abdominal wall of Drosophila melanogaster. J. Heredity, 32: 51-56. Emmons, C. W., and A. Hollaender (1939) The action of ultraviolet radiation on dermatophytes. II. Mutations induced in cultures of dermatophytes by expo- sure of spores to monochromatic ultraviolet radiation. Am. J. Botany, 26: 467-475. Faberge, A. C. (1951) Ultraviolet-induced chromosome aberrations in maize. Genetics, 36: 549-550. 282 HADIATKJ.N liKM.OCiY Cipigy, R. (1031) Actii)ii do I'liltraviolct sur 1p polo Rorrninal dans I'ocuf do Dmsophilu mclanogunlrr. Hov. sui.sse zool., iW: 1S7. Gottsohowski, CJ. (11*37) Kiinstlioho Hofruclitunn Ix'i DmsophUn. Naturwissoii- schaften, 25: 050. Guyenot, K. (1914) Action dos rayons nltraviolct sur Drnsniihilu aniptlophild, I-cnv. Hull. sci. France et Bolg., 5: HiO 1 (•.'•. llollaondor, A., and C. W. Emmons (1941) Wave-length dependence of mutation production in the ultraviolet with special emphasis on fungi. Cold Spring Harbor Symposia Quant. Hiol., 9: 179-185. HollacndiT, A., 11 U. San.some, E. Zimmer, and M. Domorec (1945) Quantitative irradiation experiments with Neurospora crassa. II. Ultraviolet irradiation. Am. J. Botany, 32: 22() 235. Kaufnuinn, B. P., and .V. Hollaonder (1946) Modification of tiie frequency of cliromosonuil rearrangements induced by X rays in Drosophila. II. Use of ultraviolet radiation. Genetics, 31: 368-376. Knapp, E. (1938) Mutatioiisau.slo.sung durch ultraviolottos Uchi boi dom Lobermos Sphaerocarpus donndlii Aust. Z. indukt. Abstamm.- u. Verorbungslohro, 74: 54-69. Knapp, E., A. Reuss, O. Risse, and H. Schreiber (1939) Quantitative Analyse der mutationsauslosonden Wirkung monochromatischen U.V.-I Jchtes. Xaturwis- .senschaften, 27: 304. Knapp, E., and H. Schreiber (1939) Quantitative Analyse der mutationsauslosonden Wirkung monochromatischen U.V.-Lichtes in Spormatozoiden von Sphaero- carpus. Proc. 7th Intern. Genetics Congr., Edinburgh. Pp. 175-176. Lea, D. E. (1946) Actions of radiations on living cells. Cambridge University Press, London {also The Macmillan Company, New York, 1947). McClintock, B. (1941) The stability of broken ends of chromosomes in Zea mays. Genetics, 26: 234-282. Mackenzie, K. (1941) Mutation and lethal effects of ultraviolet radiation. Proc. Roy. Soc. Edinburgh, 61: 67-77. Mackenzie, K., and H. J. Muller (1940) Mutation effects of idtra-violet light in Drosophila. Proc. Roy. Soc. London, B129: 491-517. McQuate, J. T. (1950) Chromosome loss occasioned by ultraviolet treatment of Drosophila spermatozoa. Genetics, 35: 680-681. Meyer, H. U., M. Edmondson, L. Altenburg, and H. J. Muller (1950) Studies on mutations induced by ultraviolet in the polar cap of Drosophila. Genetics, 35: 123-124. Muller, H. J. (1928) The problem of genie modification. Verhandl. V. intern. Kongr. Vorerbungsw. (Berlin, 1927). Z. indukt. Abstamm.- u. Vororbungslohre, Suppl. 1. Pp. 234-260. (1941) Resume and perspectives of the symposium on genes and chromo- somes. Cold Spring Harbor Symposia Quant. Biol., 9: 290-308. Muller, H. J., and K. Mackenzie (1939) Discriminatory effect of ultraviolet rays on mutation in Drosophila. Nature, 153: 83-84. Noethling, W., and H. Stubbe (1934) Untersuchungen iiber experimen telle Aus- losung von Mutationen bei Antirrhinum majus. V. Die .\usl(')sung von Gon- mutationon nach Bestrahhmg reifer mannlicher Gonen iiiit Licht. Z. indukt. Abstamm.- u. Vererbungslehre, 67: 152-172. (1936) Neuere botanische Untersuchungen iiber die Beziehung von Gen- mutabilitiit zur Quantitiit und Qualitat kurzwelliger Strahlung. Verhandl. 3d Intern. Kongr. Lichtforsch., Wiesbaden. Pp. 238-246. Promptov, A. N. (1932) The effect of short ultraviolet rays on the appearance of hereditary variations in Drosophila melanogaster. J. Genetics, 26: 59-74. GENETIC AND CYTOLOGICAL EFFECTS 283 Reuss, A. (1935) ttbcr die Auslosung von Mutationen durch Bestralihiiif^ crwach- sener Drosophila-Mannchen mit ultraviolcttem Licht. Z. iiidukt. Abstamm.- u. Vererbungslehre, 70: 523-525. Schidtz, J. (1951) The effect of ultra-violet radiation on a ring chromosome in Zea mays. Proc. Natl. Acad. Sci. U.S., 37: 590-600. Sell-Beleites, I., and A. Catsch (1942) Mutationauslosung durch ultraviolettes Licht bei Drosophila. I. Dosieversuche mit filtriertem Ultraviolett. Z. indukt. Abstamm.- u. Vererbungslehre, 80: 551-557. Singleton, W. R. (1939) Cytological observations on deficiencies produced by treat- ing maize pollen with ultraviolet light. Genetics, 24: 109. Singleton, W. R., and F. J. Clark (1940) Cytological effects of treating maize pollen with ultraviolet light. Genetics, 25: 136. Slizynski, B. M. (1942) Deficiency effects of ultra-violet light in Drosophila melano- gaster. Proc. Roy. Soc. Edinburgh, B61: 297-315. Sprague, G. F. (1942) Transmission tests of maize mutants induced by ultraviolet radiation. Iowa State .\gr. Expt. Sta. Research Bull., 292. Stadler, L. J. (1941a) Genetic studies with ultraviolet radiation. Proc. 7th Intern. Congr. Genet. (Edinburgh, 1939), J. Genet., Suppl. Pp. 269-276. (1941b) The comparison of ultraviolet and X-ray effects on mutation. Cold Spring Harbor Symposia Quant. Biol., 9: 168-177. Stadler, L. J., and H. Roman (1943) The genetic nature of X-ray- and ultraviolet- induced mutations affecting the gene A in maize. Genetics, 28: 91. ^ (1948) The effect of X rays upon mutation of the gene A in maize. Genetics, 33: 273-303. Stadler, L. J., and G. F. Sprague (1936a) Genetic effects of ultraviolet radiation in maize. I. Unfiltered radiation. Proc. Natl. Acad. Sci. U.S., 22: 572-578. (1936b) Genetic effects of ultra-violet radiation in maize. II. Filtered radiations. Proc. Natl. Acad. Sci. U.S., 22: 579-583. (1936c) Genetic effects of ultra-violet radiation in maize. III. Effects of nearly monochromatic ' 2537, and comparison of effects of X-ray and ultra-violet treatment. Proc. Natl. Acad. Sci. U.S., 22: 584-591. (1937) Contrasts in the genetic effects of ultraviolet radiation and X rays. Science, 85: 57-58. Stadler, L. J., and F. AI. Uber (1938) Preliminary data on genetic effects of mono- chromatic ultraviolet radiation in maize. Genetics, 23: 171. (1942) Genetic effects of ultraviolet radiation in maize. IV. Comparison of monochromatic radiations. Genetics, 27: 84-118. Straub, J. (1941) Chromosomenmutationen nach U.V.-Bestrahlung. Naturwissen- schaften, 29: 13-15. Stubbe, H. (1930) Untersuchungen liber experimentelle Auslosung von Mutationen bei Antirrhinum majiis. I. Rontgenstrahlen, ultraviolettes Licht, Temperatur- schocks und Zentrifugierungen. Z. indukt. Abstamm.- u. Vererbungslehre, 56: 1-38. Stubbe, H., and W. Noethling (1936) Untersuchungen iiber experimentelle Aus- losung von Mutationen bei Antirrhinum majus. VI. Die Auslosung von Gen- mutationen durch kurzwelliges Ultraviolet. Z. indukt. Abstamm.- u. Ver- erbungslehre, 72: 378-386. Swanson, C. P. (1940) A comparison of chromosonuil aberratioii.s induced by X-ray and ultraviolet radiation. Proc. Natl. .\cad. Sci. U.S., 26: 36()-373. (1942) The effects of ultraviolet and .\-ray treatniciit on the pollen 1iil)(> chromosomes of Trudescatitia. Genetics, 27: 491 503. — — (1943) Differential sensitivity of prophase pollen tube chromosomes to X rays and ultraviolet radiation. J. Gen. Physiol., 26: 485-494. 284 UADIA'PION HIOI.OCY (1944) X-ray and ultraviolet studios on polh-n tube chroniosomos. I. The effect of ultraviolet (2537 A) on X-ray-induced chromosomal al)errati()ns. Genetics, 29: 01-G8. (1947) X-ray and ultraviolet studies on pollen tube chromosomes. II. Tiic q\iadripartite structure of the prophase chromosomes of Tradencantia. I'roc. Xatl. Acad. 8ci. U.S., ;W: 229-232. Swanson, C. P., A. Hollaender, and B. N. Kaufmann (1948) Modification of the X-ray- and ultraviolet-induced mutation rate in Aspergillus terreus hy pretreat- ment with near-infrared radiation. Genetics, 83: 429-437. Uber, F. M. (1939) L'ltraviolet spectrophotometry of Zea mays pollen with the quartz microscope. Am. J. Botany, 26: 799-807. Manuscript received by the editor July 5, 1951 CHAPTER 8 The Effects of Radiation on Protozoa and the Eggs of Invertebrates Other than Insects* R. F. Kimball Biology Division, Oak Ridge National Laboratory Oak Ridge, Tennessee Introduction. Lethal effects: Kind of death — Sensitivity — Recovery — Substances present in the medium during irradiation — Effect on the medium. Retardation of cell division: Recovery — Sensitivity — Localization. Inherited effects. Miscellaneous effects: Activation of eggs — Excystment of protozoa — Motility and behavior of protozoa — Sensiti- zation to heat — Miscellaneous microscopically visible changes — -Various physiological, biophysical, and biochemical effects. References. INTRODUCTION From the point of view of a biologist interested in the effects of radia- tion there is a certain unity between investigations with protozoa and with the eggs of marine invertebrates — in both instances, one is deahng with single cells in liquid medium; in both, the methods employed have tended to emphasize the cell as an individual rather than as a member of a popu- lation. So, while the connection between investigations with the two kinds of material has sometimes been slight, it seems proper to consider them together. The review could have been organized in many ways. The actual choice, a consideration of each kind of effect separately, declares the point of view to be that of an experimental biologist rather than a biochemist or biophysicist. It seems important to stress that such frequently employed radiobiological criteria as death and division delay may not be the same in all materials. Actual occurrence of death of cells may be observed, not just the end result, such as failure to produce daughter cells in large num- bers. Division delay may be examined in detail rather than as an over- all effect on the number of cells or mitoses in a population. From such studies, it is clear that a variety of phenomena are concerned. When investigations on many organisms have been made, it may be possible to recognize certain phenomena which are common to all. Until this is * Manuscript prepared and work at Oak Ridge performed under Contract No. W-7405-eng-26 for the Atomic Energy Commission. 285 '2R{\ KADI \ TlON HlOl.OfiY (loiic, any attempt to treat death, division delay, etc., as having the same cause in all cells is likely to he mislcadinji;. 'This rc\ie\\ has l)e(Mi limited, for the most pari, to papers appearing from the year H).'^') through the year 1950. A few earlier pieces of work have been included when they seemed especially important for the topics discussed, or when (hey were early papers in a series which continued beyond 1935. A number of new lines of investigation have developed since this manuscript was submitted. It has not been feasible to revise the manuscript to include them. References to most of this work may be found in fliese (1953). Wichterman (1953). and in Xudear Science Abstracts. LETHAL EFFECTS Kind of Death. Many investigations have been concerned with the lethal effects of radiation. However, the nature of the death is certainly not the same in different cases. A variety of possible causes of death will be discussed in the following paragraphs. First, there is death which occurs during or shortly after irradiation. Numerous investigators have observed such death and described in more or less detail the accompanying phenomena, such as loss of motility, vacuolization and coagulation of the cytoplasm, and other changes. At least a part of the investigations recorded in Tables 8-2 and 3 involve phenomena preliminary to death. This type of death may occur very rapidly, as shown by the observa- tions of Rentschler and Giese (1941) and Harvey (1942), with intense flashes of ultraviolet. Some organisms, notably the ciliates, disintegrate within a few seconds. In other cases, some minutes or even an hour or so may elapse between irradiation and disintegration. The doses of ionizing radiation required to bring about this tj^pe of death are very high. Dognon and Piffault (1931a) estimate the imme- diate lethal dose for Paramecium to be about 500,000 r. Halberstaedter (1938) showed that a dose of more than 100,000 r of X rays was needed to immobilize Trypanosoma gambiense. Halberstaedter and Back (1942) report loss of motility and cytolysis of the colonial flagellate Pandorina morum after doses of 300,000-000,000 r. The very high doses required for immediate killing have sometimes been considered characteristic of the protozoa. However, Scott (1937) in his review points out that imme- diate, as contrasted to delayed, death requires doses of similar magnitude in other organisms. Giese and Leighton (1935a) report on the immobilization and vesicula- tion of Paramecium, by monochromatic ultraviolet. In both cases 2804 A appears to be more effective than other wave lengths. However, calcu- lations of quantum efficiency, based on measurement of absorption of paramecia. suggest that 2654, 2804, and 3025 A were about equally effec- PROTOZOA AND INVERTEBRATE EGGS 287 tive, whereas 2537 A was less effective. McAulay and Taylor (1939) investigated the death (by bursting) of Paramecium as a result of exposure to monochromatic ultraviolet. They found that all wave lengths up to 3000 A were quite effective but that effectiveness was much less at longer wave lengths. It may be doubted that their techniques were sufficient to detect differences in effectiveness among the shorter wave lengths. Shettles (1938), using the flagellate Peranema trichophorum, observed death after 2 hr exposure to 2537 A ultraviolet but no death after 8.5 hr exposure to 2650 A. Likewise, no death was found after comparable exposures to longer wave lengths. The intensities of the different wave lengths are said to have been eciualized by use of a photometer. Thus no general conclusions are possible about wave-length dependence of immediate death after ultraviolet. It is probable that immediate death is due to extensive damage to the cellular material. This is suggested by both the rapidity with which it can occur and the very large doses of ionizing radiation necessary to bring it about. It might be surmised from the microscopic observations that coagulation of the protoplasm or damage to the cell membrane, or per- haps both, are involved, but no really conclusive evidence is available. Possibly, different kinds of damage lead to death in different cells under different circumstances of irradiation. It is rather generally accepted (Lea, 1947) that irradiated cells may be able to survive until they divide, at which time death is caused by loss of parts of chromosomes as a result of chromosome aberrations induced by the radiation. Such a mechanism is suggested by Hoi week and Lacas- sagne's observation (1931a, b) of death at division in the flagellate Polytoma. At certain doses of a. particles, many of the cells remained normal until division and then disintegrated. Using the hit theory, they calculate that a two-particle event is involved with a target diameter of 2.3 ju, corresponding roughly to the area presented to the particles by the pericaryosomal space in the nucleus, the region in which, presumably, the chromosomes are located. Similarly, Halberstaedter and Back (1942) report that Pandorina colonies exposed to between 3000 and 300,000 r remain normal until cell division, when cytolysis takes place. This was so regardless of whether old colonies that underwent cell division shortly after irradiation or young colonies that did not undergo cell division for some time were used. When the test of survival is the ability of the cell to multiply and pro- duce a culture, death due to mutations or chromosome aberrations must be considered as a possible explanation, although other causes cannot be excluded. Two cases showing the ability of free-living flagellates to produce cultures after X irradiation may be mentioned. Ralston (1939) distinguishes immediate from delayed death and reports the median lethal dose foi- delaved death in Dunaliella salina as between 10,000 and 13,000 r. 288 RADIATION HIOLOGY S('h()enl)oni (1949) reports the median lethal dose for Astasia longa to be between 20,000 and 40,000 r. There have been a iiunil)er of investigations of the killing of parasitic protozoa by radiation; and in most of these the criterion was the abiUty to infect the host after irradiation in vitro. Thns chromosomal changes may have been invohed. Ilaberstaedter (1938) fonnd that a dose of 12,000 r of X rays to Trypanosoma gatnbiense, in vitro, was sufficient to prevent the infection of mice, whereas doses of more than 100, 000 )• were necessary to produce any obvious changes in the motility of the trypanosomes. Other investigations with parasitic protozoa are summarized in Table 8-1. There are a few cases of delayed death in which a genetic explanation is more difficult. Holweck and Lacassagne's case (1931a, b) with Polytoma in which death occurred after division has alicady been mentioned, and the interpretation has been suggested that chromosomal aberrations were involved. Using a particles, they distinguished other kinds of effects as follows: (1) the cells remain motile and grow in size but finally lyse without division; (2) the cells are immobilized but grow, yet they finally lyse without division; (3) the cells become immobile, fail to grow, and lyse without division. These workers (see also Lacassagne, 1934a, b; Holweck, 1934) made sensitive volume calculations for the first and the last two effects and found for the first a volume approximately the equiva- lent of the centrosome, whereas for the last two, the volume was approxi- mately that of the kinetosomes. These rather remarkable identifications presumably would offer an explanation for the failure to divide and for the immobilization, but would not in themselves account for the eventual death. Holweck and Lacassagne (1931b) suggest that death is due to the suppression of reproduction and of motihty. However, Polytorna cells are probably haploid and so lethal mutational changes could be invoked as an explanation on the assumption that a mutation can express itself in the cell in which it arises. Nevertheless, the possibility must be borne in mind that this is a case of delayed death due to causes other than muta- tional change. In the ciliate protozoan, Paramecium, a nongenetic explanation for delayed death appears by far the most likely one. Sonneborn (1947) has shown that the macronucleus in this ciliate contains many sets of genes. It is therefore improbable that loss of chromosomal material or gene mutation in either the macronucleus or micronucleus would have much effect on ciliates multiplying vegetatively. In order to allow for such an effect the unlikely assumption would have to be made that a given muta- tion or deficient chromosome is dominant over many normal genes or chromosomes. Alternatively, the radiation would have to be assumed to cause sufficiently extensive damage to the chromosomes to lead to death even with many sets. For this reason, Paramecium and other ciliates might be expected to be PROTOZOA AND INVERTEBRATE EGGS 289 relatively well buffered against radiation damage and, in a sense, this is true. The continued reproduction of the flagellates is prevented by a few tens of thousands of roentgens of X rays, but hundreds of thousands are needed to prevent the vegetative multiplication of ciliates. Back (1939) found for Paramecium caudatum that a dose about two-thirds the immediate lethal dose, i.e., about 300,000 r, led to eventual death. The animals survived for several weeks but decreased in size, and finally died. Lacassagne (1934b), working with Glaucoma scintillans, had previously reported death after several days without division. However, he found that some increase in size occurred. Thus delayed death does occur in these organisms but it occurs without division. Kimball and Gaither (1951) have been able to distinguish at least two kinds of delayed death in Paramecium aurelia following exposure to ultra- violet. With higher doses, some of the animals survive a day or more without division, but eventually die. Long periods of the kind reported by Back (1939) for X rays were not observed for death without any divi- sion. However, at slightly lower doses, the animals pass through two or three divisions rather slowly (in 2 or 3 days) but then cease dividing entirely for several days. Some animals finally recover the normal divi- sion rate after remaining undivided for periods as long as three weeks, but others die during this "cessation" period. All animals, whether they eventually recover or die, become very small and thin during this cessa- tion of division, and apparently all come very near death. From the decrease in size of the animals, it seems probable that the delayed death of Paramecium involves an effect on the synthetic processes within the cell. Death could well result when the cell, no longer capable of making new material, comes to the end of its resources. In the case of ultraviolet, several divisions ordinarily elapse before the synthetic processes come to a halt, while with X rays. Back's study (1939) suggests that this is not so. Also, recovery has been found for the ultraviolet effect but no recovery has been reported for X rays. Whether these differences are real or only apparent remains for future investigation. In any case, it seems hardly possible that death during the cessation period after ultra- violet irradiation is due to mutational changes, since recovery can occur even in the sister animals of those which die. It also seems improbable that enough genetic damage could be done either by X rays or ultraviolet to cause the delayed death without division, although perhaps such an explanation is just possible with the large doses of X rays employed. Nonetheless, death due to mutational changes is known for Paramecium aurelia. Animals given doses of 10,000 r or less divide normally as long as they multiply vegetatively, but when they undergo the self-fertilization process of autogamy many of the exautogamous clones are inviable. This phenomenon will be discussed in more detail in the section on inherited effects. 21)0 UADIKIION HIOl-OCY The killing of nematode cj^gs hy ladiaiion may involve, in addition to (l(>ath of (-('lis, rather diverse processes, such as abnormal development of embryos and failure of the younp; worms to hatch. Several invest ij^at ions with ultraviolet will be reviewed here. The eggs of Asrari.s have been, in the past, favorite subjects for radiobiological research with X rays but most of this work can be found summarized in Duggar (193G) and in the tables of Dognon and Biancani (1948). Wright and McAlister (1934) examined eggs of Toxocara canis and Toxascaris leonina for cmbryonation after exposure to 3()o0, 3130, 3022, 29()7, 2804, and 2050 A monochromatic ultraviolet. At the lowest dose used (()840 ergs/mm-) effects were found only with the two shortest wave lengths. Effects were found with 3022 A at a dose of 274,000 ergs/mm', but even 1,370,000 ergs/mm^ of the two longest wave lengths had no effect. These results are in agreement with those of Jones and HoUaender (1944) with .4 scans lumhricoides. They found that energies of about 6,000,000 8,000,000 ergs/mmr of ultraviolet of the wave-length band 3500-4900 A were needed to prevent cmbryonation of a large part of the eggs. Slightly higher energies were needed to prevent the hatching of the eggs of the pinworm Enterobius vermicularis. These very long wave lengths do have a lethal effect but only at very high doses. In an earlier communication HoUaender ci al. (1940) show that the action spectrum for the prevention of hatching of Enterobius eggs has a small peak at 2804 A and then rises rapidly to the shortest wave length tested, 2280 A. This action spectrum is similar to the absorption spectra of some proteins and lipids. HoUaender and coworkers suggest, as possible modes of action, hardening of an outside protein layer of the shell, change in composition of the lipoid membrane, damage to the embryo, or production of toxic sub- stances within the egg. The last is considered unlikely at the energies used. In summarizing this section on kinds of death, it would appear that at least the following categories of cell death can be recognized: (1) death within a maximum of a few hours after irradiation, (2) death after con- siderable periods of time but without division, (3) death at or shortly after the first division, (4) death after several divisions, and (5) death fol- lowing sexual processes. It seems quite possible that both (3) and (5) are the result of gene mutations and chromosomal aberrations and so it is not surprising to find that they occur in detectable amounts even at quite low doses. The very high doses characteristic for (1), together with the immediate changes involved, suggest extensive damage to cellular mate- rials. The death in both (2) and (4) may involve disturbances in the synthetic processes of the cell which finally lead to death when the resources of the cell are exhausted. In some instances, mutational changes can be excluded as a probable explanation of (2) and (4). When PROTOZOA AND INVERTEHRATE EGGS 291 the ability of the cells to produce cultures, or infections in the case of parasitic forms, is investigated, death will be due, obviously, to the most sensitive of these processes occurring within the part of the life cycle investigated. Sensitivity. The sensitivity of individual cells of the same species to the lethal effects of radiation varies considerably. Halberstaedter and Back (1942) found that all cells in a colony of Pandorina died at the same dose although different colonies required different doses. Secondly, they found, by giving repeated increments of 100,000 r and examining for immediate death after each increment, that the colonies of a small clone (16 colonies) all died at the same, or approximately the same dose; but colonies of different clones died at quite different doses. In this way some clones were found which died at 300,000 r and others which died at 600,000 r. Back and Halberstaedter (1945) demonstrated a somewhat similar phenomenon with Paramecium caudatum. When groups of about eight paramecia from cultures derived from a single animal by at least six divisions were tested, it was found that the eight might die at quite different doses. However, the four to eight animals derived from one, by two or three divisions, were found to die almost invariably at the same dose, although different groups died at quite different doses. Therefore, the sensitivity is the same for closely related individuals but not for the more distantly related ones. This investigation of Back and Halberstaedter is of much interest, for it suggests that minor variations between cells may lead to rather marked changes in sensitivity. The evidence also suggests that these minor variations may be maintained over a few cell divisions. It is improbable that the similarity between products of a single Paramecium could be due to the products being in the same part of the fission cycle, for the numbers given in the tables suggest that Back and Halberstaedter (1945) often used groups in which some of the animals had divided once more than the others. Different stages in the life cycle of a given species may be of different sensitivity. Tang and Gaw (1937) find that older cultures of Paramecium bursaria are more susceptible to the immediate lethal effects of ultraviolet than younger ones. Brown et al. (1933) report that cysts of the ciliate Ewplotes taylori are killed by approximately 400,000 r of X rays, whereas the motile form requires approximately 460,000 r. The criterion in this case was the ability to survive 48 hr. However, they suggest that this difference may have been due to the different media in which the cysts and motile forms were kept. Bennison and Coatney (1945) found that 8000 r of X rays prevented infection of chicks by a suspension of sporo- zoites oi Plasmodium gallinaccum, while 20,000 r was required to prevent infections by suspensions of trophozoites. Here, as in the work with •292 RADIATION HIOLor.Y Kiiplotcs, the possibility of an effect of the medium during irradiation must l)e considered. Packard (1924) reported what appears to have been a major difference in sensitivity to radium (principal effect said to have been due to slow /3 particles) between two species of ciliates. The lethal dose for Paramecium was 3 hr exposure to the source while that for the hypotrichous ciliate Styloni/chia was 15 hr exposure. Since Paramecium. is very resistant to ionizing radiation, this result is (}uite surprising. Packard attributes the difference to the lesser permeability of Stijlonijchia. Unfortunately, there has been no other work with Stylonychia; however, the related hypotrich, Euplotes, is killed at approximately the same dose as Paramecium (Brown et ai, 1933). More recently, Wichterman (1948a, b) has reported results which suggest a small difference in sus- ceptibility to X rays between Paramecium hursaria and P. calhinsi. He has slated that some P. hursaria survived doses between 400, 000 and (iOO.OOO r, whereas all P. calkinsi were killed by 400,000 r. More striking differences have been reported in the lethal doses of ultra- violet. Rather extensive comparisons between different species and strains of protozoa, primarily ciliates, are to be found in the works of Giese and Leighton (1953b) for long wave lengths, of Giese (1938b) for shorter wave lengths, and of Harvey (1942) for intense flashes of ultra- violet. Giese (1946b) has made similar comparisons for the eggs and sperm of a number of marine invertebrates. Shalimov (1935) reported that eggs of Ascaris equorum and Enterobius vermicularis were killed by ultraviolet in 5 min, whereas those of Sirongylus equinus were killed in only 3 min. Wright and McAlister (1934) found Toxascaris eggs to be more readily affected by monochromatic light than were the eggs of Toxocara. They suggest that this may be due to differences in the absorption of the shell. This emphasizes the difficulties which are inher- ent in interpreting differences in sensitivity to ultraviolet. Differences in absorption in the outer layers of cytoplasm or egg shells and differences in the action spectrum for superficially similar effects may be involved, as well as more su})tle differences in the biological organization of the organ- isms being compared. Recovery. The possibility that recovery may occur from changes which ordinarily lead to death has been investigated by use of fractionated doses. The method assumes that the effect is not due to a single "hit." If this is true and recovery does occur, then fractionated doses with sufficiently long rest periods in between the fractions should have less effect than the same total dose given as a single exposure in a brief period of time. Such a decreased effectiveness of fractionated doses of X rays has been reported by Crowther (1926) for the ciliate Colpidium colpoda and by Back (1939) for Paramecium caudatum. Growther (1926) found that one dose given in about 20 min produced death, whereas a dose one and a half times greater was required when given as three exposures at 2-hr intervals. It PROTOZOA AND INVKRTKBRATE EGGS 293 can be estimated from Crowther's data that the doses used were of the order of 10* r. Back (1939) reported that about half the immediate lethal dose given every day for 3 to 4 successive days produced the same effect as two-thirds the immediate lethal dose given in a single exposure; i.e., the paramecia survived for some time without division, but eventually died. Since the final dose was several times the single exposure dose for immediate death, it can be concluded that fractionated exposures were less effective than single exposures. Quite different results were reported by Berner (1942) for immediate killing of Paramecium caudatum. He found that doses of X rays given in fractions, one fraction every 24 to 48 hr, were considerably more effective than doses given at more frequent intervals. He presented evidence that X irradiation decreased the min- eral content as shown by ashed preparations and that recovery from this decrease was just complete in 48 hr. He believed that at this time the animals were more susceptible to X rays because their reserves had been depleted by the recovery process. The results of his investigations with ashed preparations and with death were quite variable, indicating a need for further investigation before these conclusions can be fully established. Nonetheless, the idea that a recovery process may lead to a temporary increase in sensitivity to radiation is an important one. Halberstaedter and Back (1942) found that fractionation into several parts with 1 or more days between had no noticeable effect on the action of X rays in Payidorina morum, either on immediate death or death after division. Halberstaedter and Luntz (1929) had previously found a simi- lar lack of effect of fractionation of the dose of radium rays on the related species Eudorina elegans. As just pointed out, death after division in Pandorina might be due to chromosomal aberrations. If a large propor- tion of the total were attributable to one-hit aberrations, the failure to find an effect of fractionation would be expected. However, the lack of an effect of fractionation on immediate death is surprising, especially since doses of 300,000 r and greater are necessary to bring it about. On the other hand, Forssberg (1933) found a marked effect of the intensity of X rays on killing and division delay in the single-cell algae, Chlorclla vulgaris, Scenedesmus hasiliensis, and Mesotaenium caldariorum. The effectiveness increased with intensity, and reached a maximum at about 1600 r/min. Halberstaedter and Back (1942) used intensities of 9000 r/min and greater. Thus it is possible that this intensity was too high to allow discovery of an effect of fractionation. Substances Present in the Medium during Irradiation. There have been a number of reports of the combined action of radiation and substances of one sort or another added to the medium. Dognon and Pift'ault (1931c) reported that the lethal dose of X rays for Paramecium was distinctly decreased in the presence of several dyes and toxic salts, e.g., potassium cyanide or iodide. Preliminary irradiation of the compounds had no 294 RADIATION BIOLOGY clTccl l>ut irradiated paramenia added to the compounds died more rapidly tliaii the controls. Resorcinol and sodium hypo.sulfite protected against the combined action of radiation ;iii(l these compounds. These authors believed that the death was due to the easier penetration of toxic sub- stances broufiiht about by a radiation-induced increase in the permeability of the membrane. lilack (llK^ti) lias studied the effects of a numl)er of salts on the cytolysis of .1 mocha proteus by ultraviolet irradiation. Koehring (1940) has shown that the ameba, Chaos chaos, is more readily killed by a combination of neutral red and the radiation from radon than by either alone. Bohn (1941) found that paramecia in various salt solu- tions, dyes, etc., survived quite normally in the dark but were killed in a few hours on exposure to visible light. It is possible to consider all these effects as due to increased permeability to injurious substances, but it should be emphasized that there is no complete agreement that permea- bility is changed by irradiation (see Table 8-4). Levin and Piifault (1934a, b, c) have found that Paramecium aurelia placed in suspensions of lecithin or of cholesterol become resistant to the immediate killing action of X rays. Thus, after exposure for 3 days to a mixture of 1 part of lecithin emulsion to (^00 parts of culture fluid, a dose of X rays three and a half times the normal was needed to kill the animals. It is, of course, of interest that the substances concerned are considered to be important constituents of the cell membrane. However, it must be kept in mind that animals kept in emulsions of this sort for some days ma}^ change their nutritive condition. Giese and Heath (1948) have shown the importance of the nutritive condition for sensitivity to X rays. Like- wise, cholesterol and lecithin may have a protective action of the sort found by Evans et at. (1942) for sea-urchin sperm. Halberstaedter and Back (1943) found that pretreatment of Para- mecium caudatum with sublethal concentrations of colchicine for 2 days lowered the resistance to X rays. In controls, the dose required to produce immediate death of 50 per cent of the animals lay between 250,000 and 300,000 r, while in the colchicine-treated animals it was between 100,000 and 150,000 r. They found no effect of colchicine on resistance to arsenic or ultraviolet. No explanation is offered for these findings. Effect on the Medium. There is considerable evidence that radiation may act indirectly on cells by way of an effect upon the medium surround- ing them. To what extent, then, can these effects on the medium account for the total effect of radiation upon the cell? It seems obvious that this is not a matter of mutually exclusive alternatives. Rather, it is a ques- tion of the relati\'e impoi'taiice, under the conditions employed, of dif- ferent mechanisms by which the radiation effect could be brought about. All discussion of effects on the medium will be inchided in this section even though other than lethal effects are involved. I'llOTOZOA AND INVERTEBRATE EGGS 295 Taylor et al. (1933) found that an irradiated tap- water extract of com- mercial yeast killed Colpidiinn rampi/lutn when added after irradiation. They were able to demonstrate the presence of hydrogen peroxide in th^ irradiated water in concentrations sufficient to kill, and concluded that this substance probably played a major role in the death of irradiated protozoa, although they added that production of other toxic agents by irradiation of the yeast medium was not improbable. Since that time, several workers have reported that their media were not rendered toxic to unirradiated paramecia by doses of X rays sufficient to kill directly irradiated organisms (Piffault, 1939; Back and Halberstaedter, 1945; Giese and Heath, 1948; Wichterman, 1948a). However, Piffault (1939) reported that medium exposed to a dose about four times as great as that necessary to produce death of irradiated animals was toxic and gave posi- tive tests for peroxide. Giese and Heath (1948) reported that medium irradiated with 1,000,000 r was not toxic, while only 560,000 r, directly to the animals, led to complete death within 75 min. It would seem then that the lethal effect of the radiation cannot be ascribed to stable poison- ous substances produced in the medium. Obviously, an unstable poison of short half* life is not excluded, since an appreciable time had to elapse between the end of irradiation and the addition of cells to the medium. Mention may also be made here of a report by Heilbrunn and Young (1935) which states that eggs of sea urchins irradiated in the presence of minced ovarian tissue are more affected by X rays (cleavage delay) than eggs irradiated free of such materials. They believe that the irradiated ovarian material produced poisonous substances. This is in line with Loofbourow's finding (1948) of similar injurious substances from yeast and other organisms. Heilbrunn and Young were unable to demonstrate the production of such substances by organs other than ovaries. The problem of lethal substances in the medium has been carefully investigated with sea-urchin sperm by two groups of workers (Evans et al., 1942; Evans, 1947; Barron et al., 1949a, b). Evans et al. (1942) showed that the effect of X rays on Arbacia sperm as measured by the percentage of fertilized eggs was markedly influenced by dilution of the sperm and by the addition of various protective substances to the medium in which irradiation was carried out. The more dilute the sperm suspension during irradiation, the more effective was a given dose of X rays. A wide variety of substances, such as egg albumin, gelatin, and egg water protected against X rays if present during the irradiation. No effect of protective substances was found on cleavage delay by irradiated sperm. Tests for hydrogen peroxide suggested that too little was formed to account for the effects. The investigators accepted Fricke's activated water as an expla- nation of the effects. On the other hand, Evans (1947) comes to the conclusion that hydrogen 20() K ADI XriON lilOLoGY peroxide can ai'couiil for at least a part of the effects at very high doses. He shows that both the percentage of eggs fertilized and the time of first ciea\'age were affected by treating sperm with hydrogen peroxide; in about the concentration in which it is found in heavily irradiated sea water. Contrary to the conclusion of Evans ct al. (1942), cleavage delay is there- fore not necessarily a direct effect. Evans (1947) believed that the effect of hydrogen peroxide was slow, so that by irradiating a dilute suspension of sperm and removing the sperm ([uickly to fresh medium all or almost all this effect could be eliminated. The peroxide effect appears to be dif- ferent from the "activated water" effect in a number of respects such as the effect on cleavage as well as on fertilizing power of the sperm. Conse- quently, both these mechanisms of indirect action through the medium have to be taken into account. Barron et al., (1949a, b) have also studied this problem with Arbacia sperm by using respiration of the sperm to measure the radiation effect. Diluted sperm (1 -.200) showed that X rays, even at doses as low as 100 r, caused a measurable inhibition of respiration. These investigators pointed out that hydrogen peroxide in low concentration increased respiration and so the effect at low doses could not be caused by this sub- stance. Furthermore, they found that sea water exposed to 100,000 or 200,000 r had a marked inhibitory effect on sperm respiration, and that the addition of catalase to the water before addition of sperm had no effect. Finally, they were not able to demonstrate any hydrogen peroxide in sea water exposed to 200,000 r although such water inhiljited respiration by about 60 per cent. They believe that stable organic peroxides which may be formed in sea water can account for this and other cases in which irradiated fluids have an effect. Attention should be drawn here to the finding of Stone and his coworkers (Wyss et al., 1950) that mutagenic substances are formed by ultraviolet irradiation of culture medium. In summary, there is a good deal of evidence that stable substances which produce biological effects can be formed in the medium as a result of irradiation. However, these substances do not appear to be formed in sufficient concentration to account to any large extent for such effects as death of paramecia, since medium treated with a dose which would have been lethal to the animals is not in itseff lethal. Evans et al. (1942) have presented evidence for the formation of very unstable substances in the medium; and such substances may be responsible for at least part of the effects produced. This group was unable to find evidence that cleavage delay was affected by such unstable substances. It can be concluded that stable and unstable substances produced in the medium all play a role but that direct effects in the cells are probably also involved. The rela- tive importance of these diverse pathways of action of the radiation m^ay not be the same for different effects. PROTOZOA AND INVERTEBRATE EGGS 297 RETARDATION OF CELL DIVISION Giese (1947a) has reviewed in detail much of the work on the effects of radiation on cell division, and Hevesy (1945) has presented a review from a rather different point of view. Nevertheless, it seems desirable to sum- marize the major work on fission delay in the protozoa and cleavage delay in invertebrate eggs and to expand, somewhat, particular topics upon which the reviewer wishes to express opinions. It has been fully estab- lished by many investigators that ultraviolet and ionizing radiations, in sufficient dosage, retard cell division. In some cases at least, this retarda- tion may last for several divisions; but, unless death intervenes, recovery of the normal rate occurs sooner or later. Perhaps this recovery is one of the most interesting features of the effect. There are a few cases in which visible light has been reported to retard division. Most of these are the result of photodynamic action (Blum, 1941, may be consulted for a review of this phenomenon). Tennent (1942) mentions delay in cleavage in the sea urchin by visible light in the presence of several photodynamic dyes. Giese (1946a) reports delay in cell division in Paramecium caudatum in the presence of eosin and in the ciliate Blepharisma, w^hich contains a naturally occurring photodynamic pigment. However, Zhalkovsky (1938) claims a reduction of cell division in Paramecium caudatum by visible light in the absence of a photodynamic dye. The delay was said to be more marked in direct than in reflected light. Phelps (1946) reports that the division rate of cultures of the colorless Tetrahymena geleii was lowered by exposure to sunlight. This has since been shown to result from destruction of necessary substances in the medium (Phelps, 1949). Perhaps a somewhat similar interpretation would be possible for Zhalkovsky's results. There have been a number of purported cases of acceleration of division by small doses of radiation. Giese (1947a) reviews these cases and comes to the conclusion that most of the evidence is of questionable significance. However, he apparently accepts several reports, mainly from the older literature, of acceleration by ionizing radiation. In all cases, the effects are small, and careful statistical analysis has not been made. Moreover, there would seem to be considerable inherent difficulty in being sure that there are no systematic differences between the controls and the experi- mentals other than in the exposure to radiation. Further investigation seems necessary before accepting stimulation of division by low doses of radiation as a real phenomenon. Although division delay is an extremely common result of irradiation, it is not universal. Halberstaedter and Luntz (1929) and Halberstaedter and Back (1942) were unable to find division delay in Eudorina or Pandorina at any sublethal dose of radium rays or X rays. Recovery. As far as the reviewer is aware, there is no adequate evidence 298 RADIATION HIOLOGY that division ck'hiy by nidiatiou ever lasts for more than a few divisions, provided the cells survive at all. The eases in microorganisms in which lasting reduction in rate of multiplication have been found are probably the results of genetic changes quite independeni of the original retarda- tion of division. The time course of reccnery may vary greatly in ditferent cases. In some cases, retardation may last for several divisions before complete recovery occurs; in some, recovery may be complete, or nearly so, by the first division, while in others, there seem to be stages during which no recovery occurs. The studies have been concerned mainly with (lualita- tive and ciuantitative descriptions of the time course of recovery with only a small amount of attention being devoted to attempts to influence recovery experimentally. The most complete ([uantitative study of division delay has been car- ried out with echinoderm eggs and sperm, chiefly those of Arbacia. Most of the investigations with ionizing radiations have been concerned with the first cleavage only. However, Miwa et al. (1939a) irradiated unfertilized eggs of Pseudocentrotus depressus with /3 particles from radon and recorded the time to both the first and the second cleavage. The data suggest to the reviewer that the interval between the first and second cleavages may be slightly longer than normal at higher doses although the the authors say that ''there is little or no delay in the . . . second divi- sion" (see Fig. 8-la). Yamashita et al. (1939) exposed fertilized eggs of this same sea urchin to X, y, and 0 rays and stated that they could find no evidence that irradiation during most of the period before the first division had any marked effect on later cleavages. However, Blum . . . Loos (1949) mention in an abstract that they have obtained the same results with X rays and with ultraviolet radiation in fertilized Arbacia eggs. Presumably, this includes delay in cleavages later than the first. The method used by Blum and his coworkers (see Blum and Price, 1950) is probably better designed to detect small difi'erences in clea^'age times than were those of previous investigators. It therefore seems probable that the effects of ionizing radiations last for more than one cleavage but are, in most experiments, of small importance in intervals beyond the first interval following the treatment. The recovery of sea urchin eggs from cleavage delay by ionizing radia- tions has been investigated by two groups of workers, a Japanese group (Miwa, Mori, and Yamashita) and Ilenshaw and his collaborators. Henshaw's results have been interpreted theoretically by Lea (1938a, b, 1947). Ilenshaw (1932, 194()c) and Miwa d al. (1939a) found that the longer the period between irradiation of eggs and insemination with unirradiated sperm, the less the effect. In other words, recovery occuncd between irradiation and insemination. Irradiation of sperm also brings about PROTOZOA AND INVERTEBRATE EGGS 299 cleavage delay, l)ut there is no recovery if sperm are kept for a time l)efore they are used for insemination (Henshaw, 1940a; Miwa et al., 1939a; Mori ct al., 1989). The cleavage delay produced by irradiated sperm can be shown to increase as a linear function of the dose. According to Lea (1947), the data of Henshaw (1940a) show an increase of 25 min in cleavage delay for each doubling of the dose. Lea (1938a, b) shows that the recovery in eggs can be adequately repre- sented by an exponential decay of the original effect with time according to the expression e~'^^, where / is the time after irradiation and T is a con- stant. The value of T was calculated to be 35 min for the fertilized egg and 104 min for the unfertilized egg. For Arbacia, Henshaw (1940b) has shown that there is little if any delay in stages before the prophase of the first cleavage. Most of the delay is in the prophase, with minor delays in the later stages of mitosis. Yamashita ct al. (1940) find major delays in the late nuclear fusion and prophase stages for Pseudocentrotus and Str'ongylocentrotus. Henshaw and Cohen (1940) irradiated eggs at different times after fertilization and found that the effect produced by a given dose increased for the first 10-15 min and then declined, so that by the end of prophase there was little if any effect on the time of the first cleavage. The decline was not quite regular, there being a small secondary increase in sensitivity at about 25 min (early prophase). Henshaw and Cohen (1940) show that there is good agreement between the first peak in sensitivity and changes in viscosity and permeability, but point out that recovery in the egg pro- nucleus up to the time of fusion might also be involved. Lea (1947) also ■ suggests that the early increase in sensitivity is due to a recovery process which he believes can continue up to prophase. Thus the time for recovery decreases as the time after insemination increases. The later drop in sensitivity may be due to irreversible changes leading to division which cannot be affected by radiation; but, as pointed out by Henshaw and Cohen (1940), this explanation fails to account for the secondary peak. Henshaw (1940d) has shown that low temperature (0°C) decreases the rate of recovery. Mori d al. (1939) found no effect of dilution of sperm immediately after irradiation, and conclude that failure of the sperm to recover is not due to something produced in the medium by the radiation. Recovery of the sort reported for Arbacia apparently does not occur for all invertebrate eggs. Henshaw ct al. (1933) treated Cumingia and Arbacia eggs simultaneously with X rays. The Arbacia eggs showed recovery but the Cumingia eggs did not. It should be noted that recov- ery, in the sense that the egg developed successfully, did occur. The Cumingia eggs simply showed the same delay in first cleavage whether the dose of X rays was given at low intensity over a long time or at high intensity for a brief period. Cook (1939) has also reported no recovery for Ascaris cquorum eggs exposed to X rays and then kept at 5°C for 300 RADIATION BIOLOGY periods of time ranfj;iiiK up to six (a) o H Z o o u. o UJ a 3 2 0-1 2-3 3-4 INTERVAL (O) n DENDRASTER-CHASE- UV ■ URECHIS -CHASE - UV 0 STRONGYLOCENTROTUS-GIESE 2804 A, 1244/ergs/mm^ Dl PSEUDOCENTROTUS- MIWA et al., BETA RAYS (b) ■ P. AURELIA-KIMBALL a GAITHER 2650 A, lOOO/ergs/mm^ □ R CAUDATUM -GIESE 2650 A, 2000/ergs/mm Fig. 8-1. Bar diagrams to show the rela- tive importance of delay in various divi- sion intervals after irradiation. The data from the various authors v/as recal- culated as time for the division interval in question and this time was expressed as a nudtiple of the control time for the same interval, (a) Data (Chase, 1938; Miwa et al., 1939a; Giese, 1938c) for the first three cleavages of various marine eggs. The eggs were irnuliated sliortly before insemination, and 0-1 is the inter- val between insemination and the first cleavage. (6) Data for two different species of Paramecium. The interval 0-1 is between irradiation and the first division thereafter. (Giese, 1945b; Kim- ball and Gaither, unpublished.) months. The e{i;frs kept in the cold .sho\v(!d the .siinu; dciUiy in the first fe\v di\isions as those allowed to devek)p immediately at 25*'C. Ho\vever, another effect, produc- tion of ahnormal embryos, showed recovery during the period in the cold. Evans (lOoO) has confirmed these results hut has found a some- what different situation with Arbacia eggs. Arbacia eggs irradi- ated \vith low-intensity X rays divide without further delay when the irradiation ceases, which sug- gests that recovery and inhibition occur at nearly equal rates. How- ever, the effects on later embryonic development are more pronounced after longer exposures, indicating that the rates of recovery for the two effects are quite different and that recovery of division delay is more rapid. There have been a number of re- ports that marine eggs exposed to ultraviolet show delay in cleavages later than the first one after treat- ment. Chase (1937, 1938), using a quartz mercury arc and the eggs of the marine worm Urechis caupo and the sand dollar Dendraster excen- tricus, found that several successive divisions were affected when radia- tion was given before fertilization. Not all of Chase's data demonstrate recovery but observations were ex- tended over only the first few cleavages. Giese (1938c), using monochromatic ultraviolet, found the same thing for Strongijloccnt- rotus purpuratus. Sample cases from Chase and Giese are shown in Fig. 8-la. However, Giese (1939b) irradiated the sperm of PROTOZOA AND INVERTEBRATE EGGS 301 S. purpuratus and obtained retardation of first cleavage with, at most, a very slight effect on later divisions. Marshak (1949b) reports that sperm exposed to 2537 A ultraviolet delay the first cleavage but have no appre- ciable effect upon the second. He also reports that there is less delay in division if the sperm are irradiated shortly before insemination than if they are irradiated ^-1^ hr before. Perhaps this increase of the effect with time between irradiation and insemination was due to some sub- stance produced in the medium. Blum and Price (1950) report a detailed study of recovery in Arbacia eggs irradiated with ultraviolet from a mercury arc. In most cases, the 80- 60- \. 40- 20- NORMAL CLEAVAGE INTERVAL i — r— 50 — I — 100 — 1 150 0 (6) NORMAL CLEAVAGE INTERVAL L — 1 — 50 100 150 TIME (MIN) FROM IRRADIATION TO CLEAVAGE 0,« I st TO 2nd CLEAVAGE A,A2ndT0 3rd cleavage □ .■3rdT0 4th cleavage Fig. 8-2. Graphs, modified from Blum and Price (1950), to show the recovery of Arbacia eggs from the effects of ultraviolet irradiation. Eggs were irradiated at various times and the time in minutes between a given pair of cleavages was plotted against the time from irradiation to the cleavage beginning the interval in question. The open and solid symbols represent different experiments in which radiation was given at different times. The points are approximately the center of distribution of a whole series of points given by Blum and Price (1950). (a) Irradiation after the first cleavage. (6) Irradiation h^efore the first cleavage. eggs were irradiated after fertilization, often after the first cleavage. Eggs irradiated early in a cleavage interval showed a maximum effect on the duration of that interval, while those irradiated late in an interval showed no effect until the succeeding intervals. By plotting the length of the interval against time from irradiation to the cleavage beginning the interval, a smooth curve showing recovery from the effect was obtained (see Fig. 8-2). The smooth form of this curve and its extension over more than one division interval suggests that the recovery process was inde- pendent of the occurrence of cleavage. Recovery was also demonstrated to occur in eggs irradiated before cleavage. Blum . . . Loos (1949) state in an abstract that X-irradiated eggs behave in the same way as those exposed to ultraviolet, but they give no details. The results of Blum and Price (1950) are in agreement with those of 302 UADIATION lUULOGY Ilonshaw and Cohen (1940) in that sensitivity decreases as the time after fertilization increases. However, Ilenshaw and Cohen found a small secondary increase in sensitivity in the early prophase stage. Gross (1950) found that Chartoptcrus vg^s exposed to ultraviolet showed some- what the same sensitivity relations as did X-irradiated Arhacia eggs. 'Hie eggs were (juite sensitive for the first 30 min, after which the sensi- tivity decreased hut increased again in the period from 40 to 50 min. The simplest explanation of the results of Blum and Price (1950) would be that there is a period after which division is irrevocably determined. However, the results of the other two investigators are not so easily explained, and it seems probable that rather complex factors are involved in the.se changes in sensiti\'ity. These reports make it clear that sponluncous recovery from the effects of ratliation occurs in the sea urchin. For ultraviolet, it appears estab- lished that this recovery is a gradual process extending over more than one division. Both Henshaw and Lea treat the X-ray data as thougli recovery were complete by the time division occurs, but Henshaw and his coworkers do not present evidence on later cleavages. In view of this and the statement of Blum . . . Loos (1949), that X- and ultraviolet- irradiated material showed the same behavior, it seems possible that in both cases the recovery process is independent of the occurrence of cleavage. If this is so, as suggested also in a brief statement in Blum et al. (1951), then radiation must affect something which controls the rate at which division occurs instead of simply destroying some material which must be restored to its original amount before any cleavage can take place. The separation between cell division and recovery is much more marked in the cihate protozoa. Giese (1939a, 1945b) has shown clearly for Paramecium caudatum that ultraviolet retards several divisions following the irradiation (see Fig. 8-lb). Giese and Reed (1940) have shown the same thing in somewhat less detail for several species of Paramecium. (Jiese (194(ia) has made similar findings for tfie retardation of division by visible light in Paramecium exposed to eosin, and in Blepharisma. Kim- ball et al. (1952) find the following pattern for P. awre/m exposed to mono- chromatic ultraviolet (see Fig. 8-16). The same pattern has been found for wave lengths 2378, 2537, 2()50, and 2804 A. The first division follow- ing irradiation is usually markedly delayed. The next division is also delayed, but less so. Either the third or fourth interval is often extremely long, lasting in some cases for two weeks or more. Finally, recovery of the normal rate is usually complete by the sixth division. It seems possi- ble that at least thn^e prcx'cs.ses should be recognized: (1) retardation of the first division after irradiation, (2) a long but not permanent cessation of division, usually setting in after two or three divisions have occurred, and (3) a relatively small increase in all other division intervals through about the sixth. The relative magnitude of these various effects appears I'UOTUZOA AND INVEKTEHRATE EGGS 30.3 to (*hanji,v with dose, the second process becoming relatively more impor- tant as the (lose increases. However, even at quite low doses, elTects lasting through about six divisions can be recognized. Ivccovery from lower and higher doses appears to require about the same number of divisions. The effect of X rays on cell division in the ciliates has been investigated to only a small extent, mainly because the doses necessary to produce an appreciable delay are of the order of a hundred thousand roentgen units. Perhaps it is this feature more than any other which emphasizes the great radioresistance of protozoa, for most other cells are retarded by much smaller doses. Back (1939) reports that the X-ray dose for death within 2 hr in P. caudatum lies between 400,000 and 600,000 r. A dose between two-thirds and five-sixths the lethal dose leads to a permanent cessation of division. About half the lethal dose leads to a retardation of the first division of some 36 to 48 hr after which the normal rate is restored, appar- ently with no effect on divisions later than the first. Giese and Heath (1948) report an effect only on the first division at lower doses but effects on later divisions at higher doses. Powers and Shefner (1950) report that 650,000 r reduces by half the rate at which irradiated P. aurclia reaches the first division, but effects on later divisions are not mentioned. Kim- ball et al. (1952) found an effect of X rays on divisions later than the first in P. aurelia, but the effect was much smaller, relative to delay in the first division, than that for ultraviolet. Thus the results ior Paramecium and the sea urchin seem quite different. A guess might be made that cleavage delay in the sea urchin corresponds more nearly to the delay in the first division in Paramecium, while the other delays in this organism have no counterpart in the sea urchin. However, it is not quite certain that the latter is true. In Paramecium, the four products of the first two divisions of one treated animal may have cessation periods of rather different duration (Kimball et al, 1952). In the sea urchin, a similar occurrence would lead to abnormal cleavage, and perhaps development would finally stop. A number of workers have reported abnormal cleavages following irradiation of invertebrate eggs (see Giese, 1949, for review). Mention may be made here of the investigations of Robertson (1935a, b) with the flagellate Bodo caudatus. She found that continuous exposure to 7 rays from radium led, at first, to a decrease in rate of cell division, but later a partial recovery toward the normal rate occurred even though the irradiation continued. Meanwhile, the flagellates became larger than normal in size. Following cessation of irradiation, they multiplied more rapidly for a time before the normal rate was restored. The partial resistance to radiation did not persist. Sensitivity. There has been a rather miscellaneous group of investiga- tions on the effects of various factors on retardation of cell division by ;j()4 |{ ADl A'PIOX IIIOLOGY radiation. Alpatov and Xastiukova (iy34(') found that the effect of ultraviolet on Paramrriiim was intensified by exposure to slifi;htly unfa- \-oral>le temperatures after irradiation, the least effect hein^ evident when the animals were irradiated at temperatures near the middle of tlu; \ital range. They suggest tliat these changes in sensitivity may he related to protoplasmic viscosity. In another paper (1934h) they report that sodium sulfate and electrical stimulation, both of which increased the viscosity, decreased the effect of ultraviolet while potassium thiocyanate and mild narcosis, which decreased the viscosity, increased the sensitivity to ultraviolet. On the other hand, Wilbur and Recknegel (1943) have shown that treatment of Arbacia eggs with potassium citrate (0.35 M) only slightly decreased the retardation of cleavage by X rays, whereas addition of calcium or magnesium to the sea water had no effect at all. All these treatments affected the viscosity of the egg. These investiga- tors also report that doses of X rays (30,000 r) which markedly affected the rate of cleavage had no detectable effect on viscosity. They con- cluded that changes in ionic composition and viscosity cannot be impor- tant factors in division delay by X rays. Zirkle (1936) has shown that a high carbon dioxide content in the atmosphere at the time of irradiation increases the sensitivity of Paramechim to the division-retarding effects of X rays. Hutchings (1948) reported that Arbacia eggs suffer cleavage delay when briefly exposed to 36°C^ 10 min after insemination. She found that the cleavage delays produced by this temperature and by 2537 A ultraviolet were additive, or nearly so, when the eggs w^ere exposed to the high temperature either before or after the irradiation. The phenomenon of photoreactivation has been studied for clea\'age delay in sea urchin eggs exposed to ultraviolet by Blum . . . Robinson (1949); Blum, Loos, and Robinson (1950); Blum, Robinson, and Loos (1950); Marshak (1949a, b); and Wells and Giese (1950). It has also been found for di\'ision delay in ultraviolet-irradiated P. aurclia by Kim- ball and Gaither (1951). The subject will not be discussed further here, since it will be reviewed in detail by Dulbecco in Chap. 12 of this volume. Differences in sensitivity between various strains and species have been reported by several investigators. Alpatov and Nastiukova (1934a) found distinct differences in sensitivity to ultraviolet between P. caudatum and P. bursaria. Giese and Reed (1940) made an extensive study of different species and stocks of Paramecium and found considera- ble difference in their sensitivity to the division delay produced by ultra- violet. They also found that starved pai'amecia were more susceptible than well-fed ones. Giese (1946b) reported a wide range in sensitivity to ultraviolet in the eggs and sperm of different marine invertebrates. The echinoderms, whose eggs have indeterminate cleavage, are mature when shed, and cleave radially, showed a consideral)le difference in sensitivity between egg and sperm, and cleaved abnormally only when given high PROTOZOA AND INVERTEBRATE EGGS 80") doses. 'I'he other organisms were from various phyla whose eggs have determinate cleavage, are immature when shed, and cleave spirally. They showed little difference in sensitivity between the egg and sperm and showed irregular cleavage at low doses. Henshaw et al. (1933) report marked differences in sensitivity to cleavage delay by X rays between Chaetoptenis, Nereis, Cumingia, and Arhacia eggs. Localization. A considerable amount of evidence in regard to the part of the cell which is responsible for radiation-induced cleavage delay has been accumulated. It rather strongly suggests a nuclear effect for the sea urchin but is not so clear for the protozoa. Of course, the delay need not be due to the same causes in such different types of cells, although such a unifying hypothesis would be attractive. Thus the details of the mitotic process in the ciliate protozoa are quite different from those in the sea urchin. Moreover, in the eggs, there is no growth in size between cell divisions, whereas in ciliates growth occurs, and is, perhaps, essential for later divisions. In the sea urchin, it has been repeatedly shown that irradiation of either the sperm or the unfertilized egg can bring about cleavage delay. Among reports on this subject may be mentioned those of Henshaw and Francis (193()) and Henshaw (1940a, b) on X-irradiated Arhacia; Marshak (19-l:9b) and Blum, Robinson, and Loos (1950) on Arhacia exposed to ultraviolet; Giese (1939b, c; 1946b) on a variety of marine invertebrates exposed to ultraviolet. The two gametes obviously contribute quite dif- ferently to the zygote. Thus the sperm contributes the male pronucleus and the centrosome which functions in the first cleavage (Henshaw and Francis, 1936). The egg contributes the female pronucleus and the bulk of the c^ytoplasm. As Henshaw and Francis (1936) point out, the delay produced by irradiation of the unfertilized egg indicates that injury to the centrosome is not involved since this gamete does not contribute a func- tional centrosome. The one portion of the zygote to which both gametes are known to contribute is the nucleus. Therefore, the simplest conclu- sion would be that the effect is on this structure. This conclusion is not absolutely demonstrated by such evidence since it is possible that the sperm contributes cytoplasmic elements which, though small in bulk, are important in division. Nonetheless, the very fact that irradiation of either gamete produces delay certainly suggests a nuclear effect. This conclusion is not affected by the difference in sensiti^'ity between egg and sperm, for the nuclei in the two gametes are in quite different physical states and are subject to different amounts of shielding in the case of ultraviolet. Supporting evidence for a nuclear site of the injury is furnished by experiments with eggs fragmented by centrifugation. Henshaw (1938) has shown that X irradiation of either the whole Arhacia egg or the nucleated half results in cleavage delay but X irradiation has no effect on 30(i UAUlvriON lUOLC^GY eiuu'leute halves subscMiiuMitly fertilized with unirrudiated .sperm. Blum, R()l)iiis()ii, and Loos (1950, 19ol) carried out similar experiments with ultraviolet-irradiated Arharia ejiigs and found the same results. They also demonstratetl that other combinations, such as irradiated sperm with unirradiated enucleate halves of egs^, result in dela3^ Their conclusion was that the locus of the primary injury must he in the nucleus. Harding; and Thomas (1949, 1950) found that centrifuged Arbacia eggs irradiated unilaterally with ultraviolet through the fat cap were more affected than were those irradiated through the pigmented end. They draw no final conclusions from these results, but a nuclear effect seems to be fa\-ored since the nucleus would be displaced toward the fat cap. Marshak (1949b) suggests that the relative inefficiency of ultraviolet for the egg as compared to the sperm favors a nuclear effect. Otherwise, the high pro- portion of ultraviolet absorbed in the cytoplasm should make the egg more, not less, sensitive. Thus most of the evidence clearly favors a nuclear effect. However, there is certain evidence which is not in full agreement. Giese (1939b, 1947a) has shown that the action spectrum for delay by ultraviolet-irradi- ated sperm resembles the absorption spectrum for nucleoprotein, whereas that for ultraviolet-irradiated eggs resembles the absorption spectrum for certain other proteins (Fig. 8-3). Giese (1939b, 1947a) discusses various explanations among which is the possibility that the effect is partially cytoplasmic in the case of the egg but entirely nuclear for the sperm. However, in the egg, the primary absorption might be in the cytoplasm with secondary effects on the nucleus or it might be by proteins, other than luicleoprotein's, in the nucleus. Since the nuclei in the tw'o gametes are not in the same state, such differences in importance between nucleic acid and protein absorption would be possible. This emphasizes that action spectra cannot be used to reach a clear decision between a nuclear and a cytoplasmic site of radiation injury since nucleic acids and several kinds of proteins are present in both. Blum and Price (1950) believe that the fact that recovery from ultra- violet-induced delay is independent of the occurrence of cleavage suggests a cytoplasmic locus, since the nucleus undergoes major changes at the time of cleavage. However, Blum, Robinson, and Loos (1950, 1951) pre- sent e\'idence that the primary absorption of the ultraviolet is in the luicleus. On the basis of their belief that the sperm cannot be photore- activated before fertilization, they conclude that recovery is a cytoplasmic process. The argument that cytoplasmic rather than niu'lear processes are suggested by a recovery independent of cleavage appears weak since the cytoplasm at cleavage may well undergo changes (luite as profoiuid as those in the nucleus. The evidence that sperm are not subject to photo- reactivation has been called in cjuestion by the finding of Wells and Giese (1950) of some photoreactivation of Stromiijloccntrotus sperm. Blum, ( PROTOZOA AND INVERTEBRATE EGGS 30: RoV)in.son, and Loos (1951 ) do not l)elieve that this applies to Arbacia. Thns there are compelling reasons for thinking that cleavage delay is dne to nuclear damage. The evidence that recovery depends on cytoplasmic events is suggestive but not very strong. The situation for the protozoa is not so clear. Hohveck and Lacassagne (1931a, b) found that one of the efTects which occurred in the flagellate Polytoma uvella when it was exposed to a particles was a cessation of divi- sion accompanied by an increase in cell size. The cells failed to recover from this effect and finally died, so it is not clear that the effect should be classified with division delay. Hohveck and Lacassagne do not give detailed data but state that the effect was due to a single-particle event 100 80- 60 >- o 40 20- 2400 2600 2800 WAVE LENGTH. A 3000 1 3200 Fig. 8-3. Action spectra for retardation of cleavage in the sea urchin for irradiated sperm and irradiated eggs, replotted from Giese (1947a). Solid circle = sperm irradiated. Open circle = egg irradiated. and that sensitive volume calculations suggested a body the size of the centrosome. Certainly, it would be expected that injury to the centro- some might lead to difficulties with division; but in the absence of detailed data it is hard to judge how compelling is the evidence for this identification. Using Amoeba proteus, Mazia and Hirshfield (1951) have found evidence for both nuclear and cytoplasmic effects on division delay by ultraviolet The nucleated halves of bisected amebae are more sensitive to division 308 T{ADI\TT<)N HIOLOCY delay by the nidiation than are whole amehae. This cannot be inter- preted as the result of shieldinp; of the nucleus by the larger amount of cytoplasm in whole amebae, for both whole and half amel)ae si)read over the substrate so that they are of approximately the same thickness. Mazia and Ilirshfield (H)51) sug{i;est that the increased sensitivity reflects an effect of the cytoplasm on recovery processes. They also find evidence for a cytoplasmic effect of the radiation in the fact that irradiated enucleate halves die more rapidly than the unirradiated halves. lOOn 90- 80- 70 o 60- 50 40- 30- 20- 10 2400 — I 3000 2600 2800 WAVE LENGTH, A Fig. 8-4. Action spectra for retardation of cell division in Paramecium, modified from Giese (1945bj. Solid circle = time to third division — starved. Open circle = time to recovery — well-fed. For Paramecium, the evidence as to localization of the effect is not ade- quate. Giese (1945b) has found an ultraviolet action spectrum for retardation of the third division in starved P. caudatum similar to the absorption spectrum for nucleoproteins (Fig. 8-4). A similar action spectrum was found for recovery of the normal rate in \vell-fed animals. However, the action spectrum for time to the third division in well- fed animals is rather nondescript, having a very slight maximum at 2804 A (Fig. 8-5). Kimball et al. (1952) have been able to confirm, although with differences in detail, the nondescript action spectrum for well-fed P. aurelia (Fig. 8-5) but have been unable to demonstrate a nucleoprotein- like spectrum for recovery of the normal rate. Giese (1947a) concludes that "the immediate effect is upon the cytoplasm but the more lasting effect is upon the nucleus." It does not seem to the reviewer that this PROTOZOA AND INVIOUTEBRATE EGGS 309 conclusion is justified. As has been pointed out in a preceding paragraph, a nucleoprotein-type action spectrum does not, by itself, indicate that the nucleus is involved. Effects upon cytoplasmic nucleic acids are just as possible. Moreover, the duration of an effect through a number of divi- sions before recovery does not necessarily mean that the effect is nuclear. Long-lasting cytoplasmic effects are also possible. It can only be con- cluded that there is no critical evidence on the localization of the changes leading to division delay in Paramecium. 100-1 80- 60- 40- 20- 2200 2400 2600 WAVE LENGTH. A. 2800 3000 Fig. 8-5. Action spectra for retardation of cell division in well-fed paramecia, modified from Giese (1945b), and from Kimball, Geckler, and Gaither (1952). Open circle = time to third division (Giese) — Paramecium, well-fed. Solid circle = time to sLxth division (Kimball, Geckler, and Gaither) — Paramecium, well-fed. INHERITED EFFECTS There have been scattered reports of mutation induction in inverte- brates, other than insects, but such studies add nothing basically new to the studies of mutation which are to be considered elsewhere in this series. This section will be concerned almost entirely with the protozoa. For this group a rather different point of view from that in classical mutation investigations must be taken where inherited effects are concerned. Work reviewed by Sonneborn (1947, 1949) has made it clear that watch must be kept for kinds of inherited differences which are not dependent on 310 HAOIATION niOLOGY clirt'ciciices ill the genos for tlioir maiiitciiaiico. In tlio protozoa, iiih(!ril- ancc ill iiii<'a}2;(>s of cells niultiplyiiif? vegctatixciy ;is \v(»ll as iiihcrit.ancc over the sexual processes can l)e investigated. Thus, in addition to the classical mutation approach, (jther lines of investifi;ation may prove fruit- ful. Actually, the inxestigations availal)le for this review ha\'(> added only scattered bits of information along these lines. To the reviewer's knowledge, MacDougall (1929, 1931) was the first to report the induction by radiation of inherited changes in protozoa. She exposed mass cultures of the ciliate Chilodonella uncinatus to ultraviolet from a (luartz merciiiv aic for brief periods on several successive days. In some cultures abnormal animals appeared. A few of the abnormalities proved to be inherited for many generations of asexual reproduction, and in some cases through conjugation. These included apj)arent tetraploid and triploid forms as well as others exhil)iting only changes in form and size. Since the mutant forms appeared in only a few of the irradiated cultures, the conclusion that they were induced by the radiation can hardly l)e considered established, especially since the number of control cultures is not given, but the aim of this woik was to obtain mutant forms, not to investigate their origin. Alottram (1941, 1942) exposed cultures of an amicronucleate Col- pidinm (said in a footnote to belong to this genus though called Para- mecium in most of the text) to daily doses of ultraviolet or y radiation (also to low and high temperatvn-es and to carcinogenic hydrocarbons). After 4 to 62 days, a few abnormal animals were found. The doses of ultraviolet are not given. The doses of y rays ranged from 800 to 12,1()0 r. The abnormals continued to produce abnormal descendants though not necessarily of the same type. Somewhat similar changes were produced by ultraviolet irradiation of Glaucoma sctosa. Investiga- tions with Paramecium caudatum and Aspidisca sp. are also reported, but it is not stated that radiation was employed. Mottram (1942) suggests that changes in viscosity are invoUed, that the changes are cytoplasmic, not nuclear, and that they are similar to those involved in carcinogenesis in higher forms. The arguments Mot- tram gives for cytoplasmic and not nuclear change are inconclusive. Nonetheless, it is hard to see how chromosomal changes could be involved in an amicronucleate Colpidium. However, further investigation is recjuired of both the origin and inheritance of these changes. It is espe- cially necessary that the experiments permit a quantitative study of the origin of the abnormalities followed by a careful genetic analysis of the inheritance. Spencer and Calnan (1945), working with P. multimicronucleatum, report a long-term deleterious effect of continuous sublethal exposures to radium, to a number of dyes, and to methylcholanthrene. The animals were grown in mass culture with continuous exposure to the agent. PROTOZOA AND INVERTEBRATE EGGS 311 Transfers were made every 10-12 days. The division rate was about one per day. Although the exposed cultures continued to multiply through many transfers, they eventually died out while control cultures survived. Thus the cultures exposed to radium succumbed at the 190th transfer while 16 control series were still aHve after more than 20(i transfers. It would appear that the effects of sublethal exposure to radium and the other agents were accumulati^•e o\'er the course of many generations of cell division. However, no genetic analysis of the material was made, and the method allows autogamy, and so gene recombination, to occur. Various complex processes of selection of both the paramecia and their accompanying bacteria are also possible. An interpretation in terms of mechanism seems impossible without further analysis. Schaeffer (1946) reported an inherited change in size induced in the giant multinuclear ameba Chaos chaos. Some of the amebae broke into fragments following X irradiation. The largest fragments developed into clones of normal size and the smallest died. However, some of the medium-sized fragments grew into clones whose average volume was about 60 per cent of the parent clone. One such clone was maintained for four years. When this small clone was exposed to X rays, clones were obtained which were still smaller. These latter clones had been main- tained for three months at the time of the report. Schaeffer does not propose a mechanism to explain these results. In the light of the multi- nuclear condition of this species, it seems difficult to suppose that gene mutations or chromosomal aberrations were involved. If fragmentation into medium-sized pieces is really a necessary first step, interesting specu- lations concerning the determination of size in such multinucleate proto- plasmic masses might be made. However, no data on the frequency with which the change has occurred are given, so that it is difficult to evaluate the apparent correlation l)etween the size of the fragment and the occur- rence of the variant. The self-reproducing cytoplasmic particle, kappa, of P. aurelia has been shown to be inactivated by X rays (Freer, 1948, 1950), by nitrogen mustard (Geckler, 1949), and by 2537 A ultraviolet (Kimball, 1950). Freer (1950) finds that the curve of the logarithm of the number of par- ticles against dose is not quite linear and suggests several sources of diffi- culty which might explain the departure, since he believes that the inac- tivation of kappa is basically a single-event phenomenon. Depending on the interpretation, the true inactivation dose (37 per cent dose) is con- sidered to lie between 3400 and 4000 r or at approximately 10,000 r. Sensitive volumes calculated on this basis are in reasonable agreement with the size of the particles which can be observed under the microscope. Freer reports that microscopic examination showed that it takes some 2 to 3 days in the absence of cell division for the visible kappa particles to disappear following X irradiation. 312 RADIATION 1U()I- of mating type and autogamy has not been fully established for this species. Thus some other pathway of action of the X rays is possible. A series of reports on genetic changes in /^ aitrclia induced by 0, ultra- violet, and X radiation and by nitrogen mustard ha\-e been made by Geckler, Kimball, and Powers and their coworkers. The method used by all these workers was basically the same and depended on the fact that autogamy makes the animals completely homozygous. The paramecia were exposed to the radiation, and a number of the exposed animals were isolated. After a period of vegetative multiplication autogamy was induced, and a number of autogamous animals were isolated from the progeny of each treated animal. Each autogamous animal was allowed to multiply for a period of several days and was then checked for survival (Powers) or for survival and amount of growth (Kimball, Geckler). The percentage surx^iving with normal growth can be taken as a measure of the effect. When ionizing radiations were used, it was found that at doses much too low to have immediately detectable effects either on survival or rate of division of vegetative animals, many of the autogamous clones were not viable or, if viable, divided more slowly than usual. The dose range used has been from about 300 to about 20,000 r ; above the latter dose almost all the exautogamous clones were affected. Such observations have been reported by Kimball (19-l:9a, b) for /3 particles from P'^' outside the culture medium and for X rays, by Powers (1948) for P'*- and for a mixture of Sr^^ Sr^", and Y^" in the medium, and by Powers and Shefner (1948, 1950) for X rays. Geckler (1950) reported similar findings for nitrogen mustard; Powers and Shefner (1950) and Powers and Raper (1950) reported on doses of X rays and nitrogen mustard which were sufficiently high to have a distinct immediate effect on the animals in addition to the effect after autogamy. Kimball and Gaither (1951) report that doses of 2650 A ultra\iolet, which an^ sufficient to produce a detectable effect after autog- amy, also cause a temporary retardation of the first few cell divisions following irradiation. The simplest interpretation for effects which do not appear until after autogamy is that they are due to gene mutations or chromosomal aberra- tions in the micronuclei. Sonneborn has shown that autogamy results in the formation of a completely homozygous synkaryon from which the new PROTOZOA AND INVERTEBRATE EGGS 313 macroniiclei and the micromicloi of tlie exautogamous clone are derived. Sonnehorn also has given evidence that the macronucleus is a compound structure in which each gene and chromosome are represented many times. The evidence is reviewed by Soinieborn (1947). Under the cir- cumstances, mutations or chromosomal aberrations would not be expected to express themselves immediately following irradiation but only after homozygosity of both the macro- and micronuclei had been brought about by autogamy. On this basis and from the results of several breeding experiments, Kimball (1949b) came to the conclusion that most of the death and low rate of multiplication in the exautogamous progeny of irradiated animals were the results of gene mutations or chromosomal aberrations. Kimball (1949a) showed that a given total dose of 0 particles divided into several small daily fractions, with the animals undergoing several cell divisions between each fraction, was as effective as the same dose given in a single exposure of a half-hour's duration or less. This was taken to mean that the mutational changes must have been gene mutations or one-break chromosomal aberrations rather than two-break aberrations. However, the dosage curve was more nearly typical of a "multiple hit" than a "one- hit" curve. For this reason, Kimball (1949a) suggested that most of the non-normal exautogamous clones were the result of the combined action of a number of mutant genes with individual effects too small to be detected. Powers and Shefner (1948) using X rays and Geckler (1950) using nitrogen mustard have both presented further e^'idence from breeding experiments for the genie or at least the micronuclear basis of the post- autogamous effect. However, Geckler (1950) reported on a number of findings which can be explained in terms of micronuclear inheritance only with great difficulty if at all. Kimball (1949b) reported one case of inheritance which did not conform to expectations. Another phenomenon, not at present explained, is that reported by Powers and Shefner (1950) for very high doses of X rays and by Powers and Raper (1950) for nitrogen mustard. In both cases, they found that death after autogamy rose to a maximum as the dose increased, then declined somewhat. With X rays, there was a secondary rise at very high doses. The reviewer believes that there is strong evidence that radiations and nitrogen mustard induce mutations in the micronuclei of P. aurclia and that these mutations express themselves in death and low rate of multi- plication of exautogamous clones. Ho\yever, there is rather convincing evidence that this is not the whole story and that other phenomena may also play a significant role. Further experiments to define this situation more thoroughly are needed. Most of the work with Paramecium has concerned itself more with the ,'il I UADIATION liUJLOUY iKiturc of the iiiliciilcd chaiifJics which are prochiccd tliaii with the mech- anism !)>• which they are pnxhiced. 'I'he work of Powers (I'.)IS) with raihoactix-e isotojx's in the mechuin is an exception, lie lonnd that tor eijual activities, as measnred by an air ionization chamher and vibrating; reed electrometer, P^' was four to six times as effective in producing death after autogamy as a mixture of Sr«", Sr«", and ^''"'. It would Ix; expected tliat the phosphorus would be eoneentrated in the nucleus but not the strontium and yttrium. Rubin (1948) computed from Powers' data the expected increase of specific ionization due to the concentration of phos- phorus in the nucleus and came to the conclusion that it could not account for the total difference in effect which Powers found. He concluded that some other factor must be involved and was inclined to believe that it was the transmutation phenomenon, i.e., the result of the radioactive disin- tegration of phosphorus atoms incorporated in the molecules of the chro- mosomes. A number of approximations of necessity enter the calcula- tions so that it would seem well to withhold (inai judgement until further investigations of this matter have been made. In summary, nuclear mutations have been induced in Paramecium aurelia and are subject to ciuantitative study. In addition, several ill- defined inheritable changes have been found after irradiation of various protozoa. Further advances in understanding these changes must depend on obtaining them in a situation in which definitive genetic analysis is possible. MISCELLANEOUS EFFECTS Activation of Eggs. Loeb (1914) discovered that unfertilized eggs of Arhacia and Chartopierus could be stimulated to l^egin parthenogenetic development by exposure to ultraviolet from a ([uartz mercury vapor lamp. Giese (1949) has recently reviewed the subject but a brief dis- cussion of it seems desirable here. The activation of the sea urchin egg has been studied by several investi- gators. This egg is mature when shed and activation is indicated by membrane elevation and cleavage. The later cleavages in activated eggs may be quite irregular, with spindle abnormalities and fragments of chromatic material on the spindle (Nebel et al, 1937). Hollaender (1938) has shown that wave lengths of 2()50 A and longer have very little effect on the whole Arhacia egg. The curve of effectiveness rises sharply around 2400 A and is still rising at the shortest wave length used, 22()0 A (Fig. 8-0). This type of curve is a rather generalized one, resembling the absorption curves for certain proteins and for lipids. Giese (1949) believes that it may be the result of absorption in the lipids of the cell membrane. It would appear that the curve for different eggs may not be the same. Giese ( 1 938d) found no activation by 2537 A ultraviolet, in the doses used, of the eggs of the sea urchin Sfrongyloccntrotus, but he (1939c) PROTOZOA AND INVERTEBRATE EGGS 3io lOOn readily activated eggs of the marine worm Urechis caupo with this same radiation. Harvey and HoUaender (1938) fractionated the Arbacia egg by ceiitrifu- gation into white (nucleated) and red (nonnucleated) halves and separated the latter into yolk and pigment (luarters. Activation was obtained with some differences in detail when either half or either of the two quarters were exposed to ultraviolet of 2480 A or below. Activation was also obtained for the red half and its two quarters with doses of the band of wave lengths 2650-3000 A, which were ineffective with the whole egg or the white half. It can be concluded that the nucleus does not play an im- portant role in activation by ultraviolet. While there seem to be differences in detail in the activation of different parts of the egg, it is not at all unlikely that changes in the surface of the eggs are involved. This is further indicated by the studies of Reed (1943) and Spikes (1944) which have shown localized effects on membrane formation as the result of unilateral irradiation of sea urchin eggs. Similarly, Tchakotine (1935a, b) has shown localized changes in the surface followed by activation phenomena as a result of localized irradiation of the Pholas egg. The investigations of Heilbrunn and Wilbur (1937) and Wilbur (1939) on the effects of calcium and magnesium on ultraviolet activation of the Nereis egg also suggest a surface phenomenon. Heilbrunn and Wilbur (1937) have shown that citrate inhibits the activation ; they suggest that this is due to removal of cal- cium from the cortex, so preventing Heilbrunn's views, is responsible for •— 2200 2400 WAVE LENGTH, A 2600 Fig. 8-6. Action spectrum for activation of Arbacia eggs, from HoUaender (1938). the calcium release which, on the activation. Alsup (1941) found that photodynamic activation of Nereis eggs by visible light in the presence of eosin or rose bengal was likewise inhibited by citrate. Wilbur (1939) has shown that magnesium inhibits activa- tion by small doses of ultraviolet but the inhibition is overcome by larger doses. Calcium antagonized this action of magnesium. It was suggested that magnesium acts in the same manner as calcium but less efficiently. Excystment of Protozoa. The process of excystment in the ciliate pro- 316 UADIATION HI()I,(»(;V tozoan Colpoda steinii is under excelleiil experimental control. Exposure to a special medium results in complete excystment, startinji; at alxjut 2 hr after exposure and endinji" in less than I hr from its inception. Taylor el al. (193()) huve shown tiiat a dose of 88, 100 i- of X rays fj;i\'(>n at the rate of 1280 r/'sec within the first (iO min after exposure of the ciliates to the excystment medium increases the time to 50 per cent excystment to about 420 mill. The same dose given 120 min after exposure to the excystment medium has very little effect. From the form of the curves of percentage excj'sted against time, it is concluded that, in the period between GO and 120 min, there is a mixture of sensitive and resistant cysts and some in a transitional state between sensitive and resistant. The cysts in this transitional state are apparently moi'e easily prevented from excysting by the X rays than are cysts in the; other two states. Giese (1938a, 1941, 1945a), using the same techniques, has shown thai monochromatic ultraviolet also increases the time to excystment. The action spectrum has been determined for wave lengths from 2537 to 3()()() A. There is a small peak at 2804 A suggestive of the absorption spectrum of certain proteins. The dose of 2654 A ultraviolet to double excystment time is approximately ()00 ergs/mm-. MoiUiti) and Beha.'ior of Protozoa. There have been a number of obser- vations on the behavior and motility of protozoa during or immediately following irradiation, (liese and Leighton (1935a; Giese, 1938b, 1945a) have presented a series of quantitative observations on the effect of ultra- violet on a variety of ciliates. In particular, Giese (1938b) gives com- parative data for 50 per cent rotation on the long axis and 50 per cent immobilization for Tetrahymena glaucomiformis, Colpidium colpoda, Stylonychia curvata, Paramecium bursaria, P. aurdia, P. caudatiim, and P. multimicronucleatum , Blepharisma undulans, Spirostomum ambiguum, Bursaria truncateUa, and Fabrea salina. Giese (1945a), using monochro- matic ultraviolet, has shown that the action spectrum for immobilization and for ciliary reversal in Paramecium has a peak at 2804 A, suggesting that it is similar to the absorption spectra for certain proteins (Fig. 8-7). A variety of other studies on motility and behavior are summarized in Table 8-2. Immobilization has been observed often and in many cases is probably a sign of impending death. However, this is not necessarily so. The reviewer (unpublished) has observed complete immobilization of P. aurelia by 2250 A ultraviolet at a dose (1000 ergs/mm'- incident on the quartz container) which not only is not lethal but has only a very small effect on the time to the first division after irradiation. Wichterman (1948a, b) has reported in some detail the effect of X rays on the mating reaction in P. bursaria and P. calkinsi. Doses in the range between 100,000 and 700,000 r lessen or prexeiit the mating reaction and the pair formation which usually follows from it. Apparently, pair for- PROTOZOA AND INVERTEBRATE EGGS 317 rnation is somewhat more easily affected than is the mating reaction itself. At nonlethal doses, recovery from these effects seems to be possible. Paramecium calkinsi is affected by somewhat smaller doses than P. bursaria. Sensitization to Heat. Bo vie and Klein (1919) first reported that para- mecia could be made more sensitive to heat by exposure to ultraviolet. Giese and his coworkers have made a detailed investigation of this phe- nomenon. Giese and Grossman (1945a) may be consulted for reports of work with other organisms. The time to death at a single lethal temperature, 42°C, was used as a measure of the resistance to heat. The possibility that the minimum lethal temperature was changed was not tested. Giese and Grossman IOOt 80H >• o 2 60- UJ o 40- 20- 2400 2600 2800 WAVE LENGTH, a 3000 3200 A CILIARY reversal"! # IMMOBILIZATION J STARVED O IMMOBILIZATION- FED Fig. 8-7. Action spectra for immobilization and ciliary reversal in Parawectuw, from Giese (1945a). (194oa) investigated the action spectrum for heat sensitization of Para- mecium. They found that 2483 A, the shortest wave length used, was much more effective than 2537 A or still longer wave lengths. There was a small secondary peak at 2804 A. The dose to reduce the time to death at 42°C to half its control value can be calculated to be about 400 ergs/mm- for 2483 A and about 1800 ergs/mm^ for 2804 A. They also found that recovery from the effect occurred. For all wave lengths other than 2654 A the course of recovery was much the same, and it was complete in about 4 days. Animals exposed to 2654 A recovered much more slowly and recovery was only about two-thirds complete by 4 days. Giese and Grossman suggest that this means that more than one material is involved and that the slow recovery from 2654 A is due to a larger component of effect on nu(;leoproteins than at the other wave lengths. Giese and Grossman (1945b) have shown that visible light in conjunc- tion with a photodynamic dye can increase the sensitivity of Paramecium 318 RADIATION BIOLOGY lo heat. Gipso (194r)a) has shown that viRil)l(' lijj;ht hy itself can do so Tor the ciliatc liU phitrismn, wliicli contnins u natural pliolodynjimic pifiiiKMit . Ciicsc ami Heath ( M> IS) dcnioiistrated that paramocia can be sensitized to heat by sublethal doses of X rays. They emphasize that dosage calcu- lations with the thill window 1ul)e employed are subject to much ((uestion l)ut give 50,000 r/min as an approximate rate. On this basis, it can be estimated from their data that do.ses between lOO.OOO and 300,000 r are necessary, depending on the nutiitive state of the animals, to reduce the time for death at 42°C" to half. Well-fed, rai)idly dividing animals are more sensitive than starved ones. This finding contrasts with Giese and Reed's finding (1940) that well-fed paramecia are more resistant to the division-retarding eifects of ultraviolet than are starved ones. Thus one cannot speak of a general resistance to radiation but only resistance to specific effects of specific radiations. Recovery from heat sensitization was shown to occur when the paramecia were fed after irradiation but not when they were starved. Giese and Heath (1948) conclude that recovery must involve the synthesis of new materials bj'' the cell. Giese and Grossman (1945b) .suggest that radiation partially denatures proteins. Exposure to heat is then supposed to complete the denatura- tion and so lead to death more rapidly than in animals not exposed to radiation. Giese (1947b) found evidence for sensitization of nucleo- proteins to heat by ultraviolet. Partially purified nucleoprotein from Strongylocentrotus sperm, dissolved in 2 M sodium chloride, forms threads when poured into dilute sodium chloride. Brief exposures to ultraviolet (mainly 2537 A) followed by a 10-min exposure to 80°G changed the luicleoprotein so that the threads did form. The reverse procedure, exposure to 80°C and then to ultraviolet, had no effect. Miscellaneous Microscopicalli/ Visible Changes. ^Microscopically visible changes in the nucleus of the cell are treated by Garlson (Ghap. 1 1 , volume 1 of this series). Some of the more recent observations of various changes J in both the nucleus and cytoplasm in protozoa and invertebrate eggs are | summarized in Table 8-3. No attempt has been made to include papers in which microscopic observations were merely incidental to other work. As has been pointed out in a previous section, many of these changes may be those that occur in dying cells. Various Physiological, Biophysical, and Biochemical Effects. For the most part, the work on protozoa and invertebrate gametes has not been directed to a study of the enzyme systems affected by the radiation, the colloidal changes in protoplasm, the changes in permeability of mem- branes, and alterations in the chemical composition of the cell. Infor- mation of an indirect sort on some of these matters has been mentioned in preceding sections of this chapter but direct investigations are scarce and are listed briefly in Table 8-4 without discussion in the text. PROTOZOA AND INVERTEBRATE EGGS 3 ID Tablk 8-1. KiLLixn of Parasitic Protozoa by 1{ adiatiox Organism Radiation Criterion Dose Reference Tr[ipano^oma Soft X rays Decreased infective 6,000 r Patel (1936) brucei power Ultra\- inlet Decreased infective power H.S.E. Patel (1936) Trypunosoina X ra3's Visible change 100,000 r Halberstaedtcr gambiense Failure to infect 12,000 r (1938) Triipanosoma X rays No visible change 100,000 r Emmett (1950) cruzi Decreased infective power 10,000 r Plasmodium Ultraviolet" Failure to infect Not given Russell ft al. (jallinaceum (1941) sporozoites I'lasmodium X rays No effect on infection 150-700 r Zain (1943) gaUinaceum endothelial stages Plasmodium gaUinaceum sporozoites X rays Failure to infect 8,000 r Bennisoii and trophozoites X rays Failure to infect Rate of development of the infection 20,000 r 4,000 to 8,000 r Coatney (1945) Plasmodium X rays Failure to infect 5,000 r Bennison and malaria Coatney (1945) trophozoites Plasmodium X rays Failure to infect 16,000 r Rigdon and Rudi- lophurae sell (1945) trophozoites Eimeria tenella Ultraviolet No infection of chicks 2 units Fish (1932) oocysts 100 per cent mortal- ity in vitro 1 unit Eimeria tenella X rays Failure to infect 13,500 r Albanese and oocysts Effect on severity of infection 9,000 r Smetana (1937) Eimeria tenella X raj'S Distinct effect on se- verity of infection Some infection 9,000 r 13,500 r Waxier (1941) Eimeria Ultraviolet Inhibition of in rilro Not given Litwer (1935a, b) perforans development of Eimeria oocysts stiedae A delina X rays Abnormal oocysts 1,000 r Hauschka (1944) deronis Endamoeha Ultraviolet Decrease in luimber Not given Stoll el al. (1945) hisiolylicu excysting cysts Endamoeba X rays Partial inliihition of 60,000 and Sadun et al. his(oh/ficu growth in vitro 120,000 r (1950) trophozoites Infectivity unchanged 120,000 r » Unless otherwise stated in this and other tables " Ultraviolet " moans the radiation from a quartz mercury arc presmnably witli major output at 2537 A. 320 RADIATION BIOLOGY Tabi.k 8-2. Motility and liKiiAVioK ok I'hotozoa Radiation Organism Effect Dose Reference X rays Paramecium Acceleration of movement followed by slow- ing, spinniufi, and iiiiin()l>ili/.ati()ii <5 X 10* r Dognon and Pif- fault (1931a) X rays Paramecium Irregular swint- 1 to 6 X 10' r Back and Hal- caudatum niing, immobiliza- tion berstaedter (1945) X rays Paramecium Acceleration 1 X 10'' r Wichterman bursaria Retardation Immobilization 2 X 10'' r 1 X 10'^ r (1948a) X rays Paramecium calkinsi Retardation 4 X 10' r Wichterman (1948b) X rays Trypanosoma gambiense A few nonmotile 2 X 10' r Halberstaedter (1938) X rays Pandorina Immobilization 3 to () X 10' r Halberstaedter and Back (1942) Neutrons . . . Euglena Clear zone at top of culture (change in motility?) 1250 n Jennings and Garner (1947) y (radium) . . Physarium polycephalum Plasmodium moves more slowly Plasmodium moves away from needle Not given Seifriz (1936) a (polonium) Polytoma uvella Immobilization Not given Holweck and Lacassagne (1931a, b) 2804 A Paramecium 50 per cent immo- 1.1 X 10' Giese and Leigh- ultraviolet multimicro- nucleatum bilization ergs /mm ^ ton (1935a) 2537 A Paramecium 50 per cent rotation 1.8 X 10' Giese and Leigh- ultraviolet multimicro- nucleatum ergs/mm^ ton (1935a) Intense Paramecium Avoiding reaction, Not given Rent.schler and flashes of muUimicro- retardation, rota- Giese (1941) ultraviolet nucleatum tion, immobiliza- tion Ultraviolet. . Paramecium Acceleration of con- Not given Roskin and Shl- ' caudatum tracitile vacuoles, retardation of food vacuole formation shliaeva (1933) Ultraviolet. . Spiroiitomum ambiguum Various effects Not given Shirley and Finley (1949) Ultraviolet . . Amoeba proteus Various effects of several salts Not given Black (1936) Visible plus Trypanosoma Immobilization Not given Levaditi and I)hoto(ly- (jamhiense Prudhomme namic dye and T. evatisi (1945) PROTOZOA AND INVERTEBRATE EGGS 321 Table 8-3. Microscopically Visible Changes Induced by Radiation Radiation •> (radium) 7 (radium) X rays Ultraviolet Ultraviolet 2537, 2654, 2804, 3025 A 2650, 2804 A Ultraviolet Ultraviolet Organism Ultraviolet, microbeam Ultraviolet, microbeam Ultraviolet, in- tense flashes Ultraviolet, intense flashes V'isible light plus photo- dynamic dye Visible light plus photo- dynamic dye Physarium Opalinid eiliates Unto tumidus eggs I'tiio tumidus eggs Amoeba proteus Paramecin m multimi- cronurleatum Paramecium aurelia Kahlia simplex Spirostomum ambiguum Amoeba proteus, Avioeba verrucosa, Paramecium caudalum Paramecium caudatum, Spirostomum avibigu um, A m ph ilep- tus claparedei Paramecium multim.i- cronuclealum Amoeba proteus, Amoeba dubia, Euglena, Volvox, Chilomonas, Stylonychia, Chilodonella, Coleps, Urocentrum, Paramecium bursaria, Epistylis, Stentor, Bursaria, Frontonia Amoeba dubia Effect Protoplasmic stream- ing, viscosity, distri- bution of granules Distribution of mito- chondria and vegeta- tive granules Changes in appearance of nuclei due to coagu- lation and permeabil- ity changes Coagulation within nucleus Form and general ap- pearance Vesiculation Formation of large, vacuolated bodies in the macronucleus Double animal forma- tion Fragmentation of macronucleus, various cytoplasmic changes Localized coagulation in cytoplasm Localized colloidal changes in macronu- cleus Change in shape, vesic- ulation Various changes, mainly fragmentation Reference Lytechinus eggs Blister formation, hyaline areas formed, pseudomombranes formed Variety of nuclear and cytoplasmic changes Seifriz (li)36) Horning (1937) Wottge (1939) Wels (1938) Black (1936) Giese and Leigh- ton (1935a) Kimball (1949c) Kimball and Gaither (1951) Horvath (1947) Shirley and Finley (1949) Tchakotine (1935c) Tchakotine (1936) Rentschler and Giese (1941) Harvey (1942) Hvman and How- land (1940) Tenncnt (1942) 32: |{ SDI \ riON lUOhoCV TaBI-K 8-4. liloniKMICAI. and HiOIMIYSKAI, I'lKKKfTS JOllVct t Organism i liainiii ((uididu Hadiafion 1 0 and 7 Heferencc Increased pcniH-ability at Simon (l<)3y) low tlo.sos I'KK (radium) Dccrcasoil poriiioahility at lii^li (loses Increased permeability of Unto tumid us egg X rays Wottge (1939) nuclear membiaiie ("liaiine in pernieahility . . . . I'uianifcium Ultraviolet Roskin and Shi.shlmeva (1933) No detectable change in Strongiflocentroius lltraviolet Reed (1948) pertiieabilitj' at doses Pgg 2483, 2537, wliich retard cleavage 2654, 2804, 3130 A No detectable change in Arbacia egg X rays Lucke et al. (1951) permeability at doses 10,000 r which retard cleavage Changes in viscosity Amoeba proteus Photodynamic .\lsup (1942) 1 and .4. duhia action No effect on viscosity at Arbacia eggs X rays W'illjur and doses which markedly re- Recknegel (1943) tard cleavage Changes in viscosity Spirostomum Ultraviolet Shirley and Finley (1949) No effect on respiration at Arbacia and X rays Chesley (1934) doses causing early death Chaetopterus eggs No effect on respiration .... Baniea Candida /3 and y Simon (1939) egg (radium) 50 per cent inhibition of Arbacia sperm .\ rays Barron et al. respiration 10,000 to 20 , 000 r (1949a, b) Decreased assimilation of Paramecium Ultraviolet Roskin ^nd lipoid and glycogen Shishlmeva (1933) Changes in the amount of Bodo audatus y (radium) Lawrie and ammonia produced per Robertson (1935) cell Free fat formed in the cyto- Amoeba proteus ITltraviolet Heilbrunn and plasm and .4. dubia Daugherty (1938) Destruction of red pigment Blepharisma Visible light Giese (1938e); Giese and Zeuthen (1949) Change in the mineral con- stituents of protoplasm . . Paramecium X rays Berner (1942) Destruction of the enzymes Arbacia sperm X rays Barron el al. for oxidation of acetate (1949a, b) and succinate More rapid killing by sulf- Paramecium Visible light Calcutt (1950) hydryl inhibitors PROTOZOA AND INVERTEBRATE EGGS 323 REFERENCES Albanese, A. A., and H. 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(1942) The photodynamic action of dyes on the eggs of the sea urchin, Lytechinus variegatus. Carnegie Inst. Wash. Publ., Papers from Tortugas Lab., 35: 1-153. Waxier, S. H. (1941) Immunization against cecal coccidiosis in chickens by the use of X ray attenuated oocysts. J. Am. Vet. Med. Assoc, 99: 481-485. Wells, P. H., and A. C. Giese (1950) Photoreactivation of ultraviolet light injury in gametes of the sea urchin Strongylocentrotus purpuratus. Biol. Bull., 99: 163-172. Wels, P. (1938) Beobachtungen am bestrahlten Zellkern. Naunyn-Schmiedeberg's Arch, exptl. Pathol. Pharmakol., 189: 113-139. Wichterman, R. (1947) Action of X rays on mating types and conjugation of Para- mecium bursaria. Biol. Bull., 93: 201. (1948a) The biological effect of X rays on mating types and conjugation of Paramecium bursaria. Biol. Bull., 94: 113-127. (1948b) Mating types and conjugation of four different races of Paramecium calkinsi and the effects of X rays on the mating reaction. Biol. 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The effects of roentgen, gamma, and beta rays upon fertilized eggs. Gann, 33: 117-121. (1940) The action of ionizing rays on sea urchins. V. The mitotic observa- tions on the effects of roentgen rays upon the unfertilized eggs and sperm. Gann, 34: 239-245. Zain, H., and A. Wolf (1943) Einfluss der Rontgenstrahlen auf die Entwicklung der Endothelstadien der Vogelmalaria {Plasmodium gallinaceum). Deut. Tropen- med. Z., 47: 68-71. Zhalkovsky, B. G. (1938) On the difference of biological action of transmitted and reflected visible light. I. Experiments with Paramecia caudata. Bull. biol. med. exptl. U.R.S.S., 5: 493-495. (Not seen. Abstract in Ber. wiss. Biol., 50: 171 seen.) Zirkle, R. E. (1936) Modifications of radiosensitivity by means of readily pene- trating acids and bases. Am. J. Roentgenol. Radium Therapy, 35: 230-237. Manuscript received by the editor, Xov. 29, 1950 CHAPTER 9 Radiation and Viruses* S. E. LURIA Department of Bacteriology, University of Illinois Urbana, Illinois Introduction. Effect of radiations on virus infectivity: Ionizing radiations — Ultra- violet radiation — Visible light. Differential effect of radiations on various properties of viruses: Nonlethal effects — Separation of properties of inactive virus particles. Irradiated virus in multiple and mixed infection: Interference phenomena — Reactivation phenomena. Intracellular irradiation of viruses: Irradiation of cells infected with exogenous viruses — Radiation and latent viruses. References. 1. INTRODUCTION To few fields of biology have radiation studies contributed as much as they have to virology. The reasons are to be found in the properties of viruses and in the methodology of virus research. A virus can be defined as a submicroscopic entity capable of self-reproduction after exogenous infection of specific Uving cells (Luria, 1950). According to host, viruses may be classified as animal viruses, plant viruses, and bacterial viruses or bacteriophages. In their extracellular state, virus particles have charac- teristic sizes and chemical compositions, a common feature of which is the presence of protein and of nucleic acid of either the ribose or the deoxy- ribose type. Their small size, nucleoprotein content, and ability to reproduce inside cells make them useful models for the study of repro- duction of genetically specific biological units. At the same time they offer unique opportunities for observation both in the resting, extra- cellular state, in which they can be submitted to a variety of treatments, and in the reproductive, intracellular state, in which they behave as com- ponents of an integrated cellular system. Radiations, because of the discrete nature of their action, are more useful than chemicals in affecting free virus in such a way that, upon reintroduction into a host, the host- virus interaction will be abortive or variously modified; the results are very informative as to the biology of viruses. Moreover, it is possible by * This chapter has not been revised since being wyitteji in 1951. Vakiable discussion and bibliography of more recent work are given by Pollard (1953). 333 334 IJADIATION mOLOGY means of radiation to reach a virus within the host cell and to obtain information as to its intracellular properties. Althou}i;h viruses do not constitute a homogeneous group that may a piiori he expected to react to radiation in a uniform way, there does not appear to be any fundamental dilTerence in the he^havior of different \iruses toward ratliation, and most effects can he described under com- mon headings for all viruses. The major differences reflect the different purposes for which radiation experiments with different viruses have been performed. In this chapter various types of radiation effects and appli- cations of radiation analysis will be discussed, different viruses being used as examples rather than each virus being followed separately through the various approaches. It should be the goal of the radiobiologist to inter- pret the effects of radiation on \'iruses in terms of chemical alterations in nucleoproteins and other virus components. At this time, however, the radiation chemistry of large biological molecules is so poorly understood that a strictly chemical approach to the topic is precluded. 2. EFFECT OF RADIATIONS ON VIRUS INFECTIVITY The most thoroughly investigated effect of radiation on viruses has been the loss of infectivity or "inactivation " of free virus particles when exposed to radiation. It must be recalled that "activity" of a virus can be defined as the ability to reproduce and to cause a detectable manifes- tation when introduced by a proper route into a suitable sensitive host. Quantitative studies are made possible by the relative accura(;y of the titration methods for viruses ; the amount of virus in different samples can be compared fairly accurately with a precision that may vary from 5 per cent in some instances to a factor of 2 or more in others. According to a majority of authors the results of virus titration give values proportional to the actual number of individual virus particles, each particle acting as one infectious unit with a probability that may be much lower than unity (Luria, 1940; Lauffer and Price, 1945). The results of titration can be used, however, to compare the active virus content of different samples (e.g., of an irradiated and a control suspension) even if infection requires the summation of the action of large numbers of virus particles rather than the reproduction of one successful particle, provided that a definite rule of proportionality exists between the amount of active virus intro- duced and the number or extent of the host manifestations. This is true, for example, for a method of titration that uses the incubation period or the time of death of an infected animal as related to the infecting dose (Bryan and Beard, 1939; CJard, 1943) rather than the counting of indi- vidual lesions or the dilution end point. One important caution for the radiobiologist is the control of the mode of testing virus activity. This has become particularly important since KAIMATION AND VIRUSES S.S.') the recognition that ba('teriophag;o inactivated l)y radiation can be reac- tivated in its host (Luria, 1947; Dulhecco, IQoOj; siniihir ])henomena may occur for other viruses. In the case of bacterioj^hases, because of the possil)iht3" of reactivation, an inactive virus particle must be defined as one that is unable to parasitize a host cell or that, upon parasitizing a bacterial cell under conditions where no reactivation occurs, fails to give rise to the production of active bacteriophage (see Sect. 3-2). A comparison of the titers of control and irradiated virus suspensions generally provides reproducible inactivation curves. In many cases the rate of inactivation of viruses by irradiation has been found to follow a simple exponential relation according to the equation N - iVoe-^-«, (9-1) where A^o = the titer in the unirradiated control, N = the titer in an irradiated sample, D = the dose of radiation, and k = Si constant, characteristic for a given virus, a given radiation, and sometimes for a given mode of titration. The dose for which N = A^oe~^ is often called the "inactivation dose" and designated as Di/e (Lea, 1946). 2-1. IONIZING RADIATIONS X rays, y rays, a rays, electrons, neutrons, and deuterons have all been used to inactivate virus particles. It is now recognized that the inacti- vation of viruses by ionizing radiations may result from two major cate- gories of effects: indirect and direct. 2- la. Indirect Effects. These effects are mediated by toxic substances produced by radiation in dilute aqueous solutions. The toxic products are responsible for most of the inactivation observed, provided that suffi- cient amounts of protective substances which compete for the toxic products are not present. The current theories on the nature and chemical properties of the toxic substances produced by ionizing radia- tions in water and on the mode of their action are discussed by Dale and by Barron (in Vol. I of this series). As far as viruses are concerned, the indirect effect of X rays was first recognized on papilloma virus (Friede- wald and Anderson, 1940, 1941), then on phage (Luria and Exner, 1941), and on plant viruses (Lea et ai, 1944). The effect manifests itself by a higher inactivation rate of viruses exposed to radiation in water or in saline solutions than when exposed either in crude suspensions containing large amounts of foreign substances or in the dry state. A variety' of substances can act as protective agents. Proteins such as gelatin and egg or serum albumin are effective. Several substances of small molecular 33() UAOIATION mOLOOY woiglit, t'.f!;., tryptopliane, thiourea, and histidiiie, give complete protec- tion of hacteriophafj;e in \ery low concent rations (Latarjet and Ephrati, 1948; Watson, 1952). Ordinary bacteriological media such as beef broth are excellent protecting metlia. (Concentrated \'irus suspensions in nonprotective media are inactivated more slowly than dilute suspensions both because of the presence of impurities and because the virus itself may act as a protective agent. In some cases the inactivation rate does not increase with dilution beyond a certain point; the diluting medium itself may contain a small amount of protective substances. In very dilute suspensions there is also the possi- bility that some of the toxic agents produced by the radiation, such as free H and OH radicals, may recombine before having an opportunity to meet a virus particle and react chemically with it (Lea, 194()). The rate of inactivation by the indirect effect of ionizing radiation varies from case to case. Deviations from the logarithmic relation Eq. (9-1), with the rate increasing with the dose, could be due to a need for cumulative damage of the virus (true multiple-hit effect), to a progressive accumidation of toxic products in the medium, or to destruction of pro- tective substances by radiation itself. With purified tobacco mosaic virus. Lea et al. (1944) reported logarithmic rates of inactivation by X rays even for dilute suspensions of purified virus, but their data are not very satisfactory. For bacteriophages (Alper, 1948; Watson, 1952) the inactivation rate increases with time of exposure. This was interpreted by Alper (1948) as indicating progressive accumulation of toxic products in the medium. Watson (1952), however, was able to show that the increasing rate of inactivation was due almost exclusively to accumulation of damage in the phage particles. A phage sample in buffer received a small dose of X rays; most inactivation was due to indirect effect. The phage was then diluted in a completely protecting medium and later was again diluted in buffer and exposed to the same dose of X rays. The second exposure was much more effective than the first, as expected from the previously determined survival curve, although the second medium had not been irradiated previously. Not all the indirect effect of X rays on viruses is exerted during actual exposure. There are also aftereffects of irradiation, mediated by rela- tively stable toxic products. If either water or buffer solutions are exposed to radiation, and virus is then introduced without appreciable amounts of protective substances, some inactivation will follow. In the case of bacteriophage (Watson, 1952) at least tw^o agents are invoh-ed, one short-lived, detectable only during actual irradiation, and the other a long-lived one, which is quite stable at 5°C and is slowly inactivated at room temperature. Many of the effects of the latter can be duplicated by peroxides. The different nature of the two agents is evident from the different properties of phage inactivated by one or the other of them (see RADIATION AND VIRUSES 337 Sect. 3-2). Protection against both agents is generally afforded by the same substances. 2-1 b. Direct Effect. The inactivation of viruses b}' the indirect effect of ionizing radiations is mediated by chemicals produced in the medium. Its rate is affected by the temperature and by the distribution of dose in time, as e.xpected from considerations of chemical kinetics. Because of its occurrence some questions have been raised as to the very existence of a direct effect exerted by the primary absorption of radiation energy within the physical domain of the virus particles. Yet such a direct effect is certainly present, and its analysis is possibly more revealing than that of the indirect effects, as far as the mechanism of biological effect of radia- tions is concerned. A direct effect of ionizing radiations is defined as a "nonprotectable" effect, i.e., an effect that cannot be eliminated by alter- ations of the medium. If the concentration of protective substances in the medium is increased beyond a certain level or if the virus is irradiated in the dry state, inactivation will proceed at a minimum rate, which cannot be further reduced (Luria and Exner, 1941 ; Lea, 1946). This residual inactivation is a function of only the total radiation dose and is not modified by changes in oxygen tension (Hewitt and Read, 1950), in temperature (Watson, 1950), or in the intensity of the radiation beam (Wollman et al., 1940; Lea, 1946), thus exhibiting all the characteristics of photochemical reactions. The distinction between direct and indirect effects has recently been emphasized, at least in the case of bacteriophage, by the finding that the biological properties of phage particles inactivated by X rays in the presence of an excess of protective substances differ in many respects from those of particles inactivated by various types of indirect effects (Watson, 1952; see Sect. 3-2).^ The direct effect of ionizing radiation on viruses has been analyzed repeatedly in relation to the mechanism of radiation action not only on viruses but on genetic units and cells in general (Lea, 1946). In all well- investigated cases, inactivation of viruses proceeds according to Eq. (9-1). This indicates that one radiation "hit" inactivates a virus particle, i.e., a particle is inactivated by one successful act of absorption, without sum- mation of individual effects. Thus virus inactivation is a good test for further analysis based on the hit theory, and it has been employed widely in testing the validity of the 'Experiments by A. H. Doermann in 1951 (unpubli.shed) show that addition of cysteine or BAL to a suppo.sedly completely protecting medium such as nutrient broth can reduce the rate of bacteriophage inactivation by X rays by as much as a factor of 2. This important discovery, if confirmed, might force a revision of the definition of direct effect. More likely, it may be an indication that the direct efTect, although direct in a geometric sense, i.e., exerted through acts of radiation absorption within the virus particle, is in part mediated through water tiound around or within the virus in a way that permits agents such as cysteine or BAL to interact with the oxidizing products of water decomposition. .338 |{.\l)l A riO.N HKtLOCJY so-called "target theory," accordiiifi; to which the effective hits are those that occur within a .specific physical domain which may coincide with all or parts of the l)iolo.\ iii()L()(;v tion cfticieiicy on ionization density conid result from any goometric (lex i- atioii from the "single spherical target" model/' A study of the inactixat ion of a l)a('terioi)hage hy deutcron beams of diiferent energy (Pollard and Forro, 1!)4'J) sliowed a dependence of effect- iveness on the energy of the beam. This was interpreted at first as being due to a thlTerence in the effective diameter of the ionization column because of ultraviolet emission by excited atoms near the target but out- side it. This was clearly incompatible with the known low (juantum yield for ultraviolet inactivation of phage, and the deuteron data have been reinterpreted (Pollard, 1951) by a method substantially eciuivalent to the associated volume method of Lea (1946) and open to similar criticisms. In conclusion, it may be said that radiation studies on viruses based on the target theory have not yet provided any basic information on the nature and structure of viruses, especially because not enough attention has been paid to the interaction of an irradiated virus with its host. A virus particle is defined as inactive when it has become unable to produce active replicas of itself. This failure of reproduction may be caused either by inability of the virus to attach itself to a host or to a susceptible cell, by inability to penetrate and invade the host, or by inability to carry out any one of the probably numerous steps intervening between infection and production of new active virus in an infected cell.^ Different portions of a virus particle may be functioning in each of these processes. Chemical changes may be produced in a virus particle by radiation or other means without the particle registering as inactive, either because the damaged portion is not essential for reproduction or because that portion, although used in reproduction, may be replaceable by other portions. For example, suppose that a virus particle has a discrete number of different surface areas. A, B, C, . . . , any one of which can act to the exclusion of others as the "receptor" involved both in adsorption of the particle on a suscep- tible cell and in the following penetration, and suppose that, if receptor .1 is utilized for absorption, the same receptor .4 will also control penetra- tion. If receptor A is damaged by a radiation hit in such a way that it " A similar criticism can be made of an attempt (Bonet-Maury, 1948) to interpret the supposedly lower sensitivity of vaccinia virus to X rays than to a particles (an unconfirmed observation; see Lea and Salaman, 1942) by assuming that the virus particle consists of an agglomeration of individual units, each of \vhi(4i nmst be inacti- vated for suppression of infectivity. The a rays supposedly would inactivate all the units in the agglomerate by energy spread. This hypothesi.s would require that each particle break apart into single units before infection ; otherwise, the X-ray inactivation curve would be of the multiple-hit type. There is no evidence for .such a structure of the vaccinia virus particle. * In the case of viruses acting on multicellular organisms, if activity is detected by appearance of multicellular foci of infection or of general reactions, a virus may be considered inactive if it is unable to reproduce sufficiently to overcome the host defen.ses. RADIATION AND VIRUSES 343 can still function in adsorption but not in penetration, reproduction will 1)6 blocked whenever receptor .1 is utilized, and the probability that inactivation results from such a hit is the inverse of the number of areas .1 , B, C, . . . present on the virus surface. Considerations of this type underline the naivete of the target theory in its narrow form, as applied to the analysis of virus inactivation. An interesting result has recently been obtained (Hershey et al., 1951) from a study of the spontaneous inactivation of bacteriophage that con- tains relatively large amounts of radioactive phosphorus. Bacteriophage particles containing up to 100 or more P^^ atoms (out of a total of approxi- mately 500,000 phosphorus atoms in nucleotides) show a definite insta- bility, with a one-hit type of activity decay. On the average, one particle is inactivated for every ten P^' disintegrations. The inactivation is apparently not due to the emission of /3 rays, but to the nuclear event itself. This result suggests either that only 10 per cent of the phosphorus atoms of a phage particle are necessary for infectivity, the others being dispensable, or that, when a phosphorus nucleus disintegrates, there is an average probability 0.10 that this change will result in inactivation. Some authors (see Riehl et al., 1941) have discussed the problem of how a hit in any one point of a large physical volume can produce inactivation of a virus (or mutation of a gene) and have speculated on the po.ssible need and mechanism for energy migration within a large biological mole- cule to a specific site of action. Such an approach has not led very far, however, since little is known about such energy-migration mechanisms. The need to invoke their intervention in virus inactivation is not apparent. 2-2. ULTRAVIOLET RADIATION Viruses have generally been exposed to ultraviolet radiation either in stirred suspensions or in thin layers in order to avoid or equalize the screening effect of impurities. A continuous-flow technique has also been described (Levinson et al., 1944). The possibihty that in such experi- ments some indirect effects of radiations may be observed has often been neglected since the doses of ultraviolet radiation needed for inactivation of viruses do not seem to produce appreciable amounts of toxic substances in water. Toxic products might, however, originate from impurities. For most viruses the proportion of active virus has been reported to decrease exponentially with the dose according to Eq. (9-1), the total dose (intensity times time) being the relevant variable (Hollaender and Duggar, 193G; Price and Gowen, 1937; Taylor et al., 1941; Latarjet and Wahl, 1945; Oster and McLaren, 1950; Fluke and Pollard, 1949). One ciuantum is apparently the effective hit. Recent data on bacteriophage, for which the precise titration method makes it possible to obtain more accurate inactivation curves, indicate deviations from the simple loga- rithmic relation. Some phages (T2, T4, T6) exhibit a slow initial rate of 344 RADIATION UIOLOGY iiiactivation for very siricall doses (about 10 crj^.s X mm-' for 2537 A), soon clian^iinjz; to a lotj;aritlimic rate as the dose increases (Beiizer cl al., 1950). The reason for this heluvvior is obscure; it does not seem to be due to the presence of agf!;re}j;ates of \ irus particles. Other phages (e.g., Tl, T7) show an initial logarithmic rate with a br(!ak to a slower rate for sur- vivals lower than lO^-. The more resistant fraction of virus is not geneti- cally dilTerent; it is possible that it is combined with screening materials. Complications of this kind make calculations of iiiactivation rates and of (juantum yields somewhat (luestionablc. Action spectra have been reported for several viruses (liivers and ( lates, 1028; Sturm et a/., 1932; dates, 1934; HoUaender and Duggar, 1936; IloUaender and Oliphant, 1944). Comparisons were generally made only for incident energies, however, by plotting the inverse of the incident dose re(iuired to produce a constant amount of inactivation versus the wave length. For most viruses the graph resembles the absorption curve of nucleic acids with a minimum at 2400 A, a maximum around 2000 A, and very low effectiveness beyond 3000 A. For some \'iruses, howe\'er, the maximum and minimum at 2()00 and 2400 A, respectively, are much le.ss pronounced than for other viru.ses. There is no clear correlation between total luicleic acid content and type of action spectrum since vaccinia virus and tobacco mosaic virus have approximately the same nucleic acid con- tent (in percentage of dry weight), yet give different action spectra. It has been pointed out that the viruses, whose action spectra are less similar to the absorption spectrum of nucleic acid, siipposedly contain the ribose instead of the deoxyribose type (HoUaender, 1946). Although these results indicate that, at least in most cases, a large pro- portion of the effective radiation is absorbed by the nucleic acid of the \irus particles, they do not indicate the relative effectiveness of quanta absorbed by different virus components. If the part of a radiation absorbed by luicleic acid and that absorbed by other components, e.g., proteins, were equally effective in producing inactivation, the greater absorption coefficient of the nucleic acids for most ultraviolet wave lengths would cause them to appear as the main contributors to the effective absorption whenever they are present in the amounts found in many viruses (5-40 per cent of the dry weight). More information could be gained from action-spectrum studies based on measurements not of incident radiation energy but of actual (juantum yield. Absorption measurements on purified virus preparations are easily feasible, yet surprisingly few data on quantum yield for virus inactivation have been reported. In most cases they are for one wa\(> length only, the 2537 A line of mercury. One difficulty, of course, is that the actual virus content of a preparation, in terms of particles per milliliter, is .seldom accurately known. For tobacco mosaic virus and 2537 .\ the values of 2.6 X 10-^ (Uber, 1941) and 4.3 X 10-'^ (Oster and McLaren. 1950) have been reported for the cinantum yield, the latter \alue being probably more RADIATION AND VIRUSES 345 accurate. For phage T2, the quantum yield (2537 A) is about IQ-" (M. R. Zelle, personal communication), assuming a one-hit mechanism in spite of the deviation from the logarithmic inactivation rate. These (juantum yields are much lower than those reported for several chemical changes in simple organic compounds, including nucleic acid constituents (0.01-0.1). Most absorption takes place in the nucleic acid component of the virus. Unless absorption in this component happens to be much less effective than that in some other components (which is not supported by our knowledge of the action spectra), we are led to suppose that a virus can withstand an appreciable amount of chemical change in its nucleic acid moiety without being inactivated. This is in agreement with the results of experiments on phage inactivation following radioactive decay of its P^' atoms (Hershey et al, 1951; see Sect. 2-1), and the same con- siderations apply to both instances. The ultraviolet sensitivity of several viruses of a certain group, such as bacteriophages, roughly parallels the particle size when the doses are measured in incident energy (Luria and Dulbecco, 1949). This probably reflects in part the greater cross section of larger viruses and suggests that the quantum yields for inactivation may be of the same order. Bacterio- phages T2, T4, and T6 have equal size and morphology, yet T4 is twice as resistant to ultraviolet (2537 A) as T2 or TG. It is not yet clear whether this difference is due to a low^er nucleic acid content or to a lower quantum yield. The radiation resistance of T4 becomes associated with some of the distinctive characteristics of T2 or TO in type-hybrid phages produced by mixed infection (Luria, 1949). This makes it possible to investigate the determination of the ultraviolet sensitivity of a group of viruses by genetic means. 2-3. VISIBLE LIGHT Wahl and collaborators (Wahl, 1946; Wahl and Latarjet, 1947) found that several bacteriophages are inactivated at an appreciable rate when exposed to visible light. The action spectrum has a maximum in the near-ultraviolet and violet regions and a limit of effectiveness in the green region of the spectrum. Yellow and red light are ineffective. This might indicate that the viruses contain a pigment with a maximum of absorption or of photochemical yield for the long ultraviolet radiation. It is unknown whether this pigment plays any role in the photoreactivation phenomenon (see Chap. 12 of this volume). It has also been suggested that the inactivation may be due to a photodynamic action mediated by components of the medium (Dulbecco, personal communication). 3. DIFFERENTIAL EFFECT OF RADIATIONS ON VARIOUS PROPERTIES OF VIRUSES The loss of ability to reproduce is only one of the alterations that may be produced in a virus particle. Since inactivation results from suppres- 346 i; \i)i A ri»).\ HioLOOY sion of any one of the steps needed for sueeossful infection and virus repro- duction, it is often more easily affected than any other recof^ni/ahle prop- erty of the virus particles. Changes produced by radiation, other than inactivation, may he even more interesting than inactivation itself since they may reveal new properties of the virus particles and their dependence on the integrity of specific virus functions. Only very large radiation doses cause actual disintegration of the par- ticles. In this section are considered, first, nonlethal efTects of radiation, i.e., changes recognizable in virus particles that survive irradiation; then a series of changes recognizable^ in iiiacti\-e particles. 3-1. NONLETHAL EFFECTS Nonlethal effects are recognized as alterations in the properties of those virus particles that survive exposure to radiation; some alterations are nonhereditary, others are transmitted to the progeny. Among the former may be mentioned a delay in reproduction of bacterioptiage particles that survive ultraviolet irradiation, as evidenced by an increase in the latent period between infection of a bacterium and its lysis with liberation of new virus (Luria, 1944). The new virus gives a normal growth cycle; the reproductive delay, then, persists for only one cycle of intracellular repro- duction. Another nonlethal effect consists in a slower adsorption by bacteria of phage surviving exposure to X rays under conditions where indirect effects are prevalent (Watson, 1952) ; there is probably a surface alteration of the phage by toxic substances produced by X rays in the medium. A more important group of nonlethal effects of radiation on viruses is the induction of phenotypic mutations, a field as yet insufficiently investi- gated. Exposure of tobacco mosaic virus to X rays has been reported to produce mutations both from wild type to aucuba and back ((lowen, 1941). The data indicate that the probability of inducing a mutation is about one one-thousandth that of inactivating a virus particle. A report is available on mutation induced in tobacco mosaic virus by irradiation of virus-infected leaves (Pfankuch el al., 1940). With bacteriophage T2, Latarjet (1949) has reported that, following ultraviolet irradiation of infected bacteria, there is an increase in the pro- portion of bacteria that liberate phage mutants T2h. 3-2. SEPARATION OF PROPERTIES OF INACTIVE VIRUS PARTICLES The detection of the effect of radiations on different properties of viruses depends on the number of properties recognizable by the limited mode of analysis. With viruses such as bacteriophages and influenza viruses, several properties can be separated by increasing doses of radiation or by different types of radiations. Some groups of properties, however, are always lost simultaneously. When the properties studied represent RADIATION AND VIRUSES 347 recognizable events in the interaction of a virus with the host cell, the simultaneous loss of two properties may be taken as an indication that the corresponding events result from the same step in interaction. Thus, successive steps in host-virus interaction can be traced by the analysis of the residual properties of virus particles exposed to different radiations under different conditions. As an example, this type of analysis as carried out for bacteriophages T2, T4, and T6 active on the common host Escherichia coli B will be described. The major phases of interaction between these viruses and their common host are fairly well known (for reviews, see Delbriick, 1942; Cohen, 1949; Luria, 1950; Benzer et al., 1950). One or more active par- ticles of bacteriophage become adsorbed by the susceptible bacterium; the adsorption is irreversible under the usual environmental conditions. Reproduction of the bacterial cell is stopped, and there is complete sup- pression of the synthesis of the specific components of bacteria, in par- ticular, of bacterial enzymes. There occurs a quick and profound change in the cytologically recognizable nuclear apparatus of the bacterial cell, which in fixed preparations appears to be disrupted and is later replaced by a fine, granular material giving the cytochemical reactions of deoxy- ribonucleate and probably representing the new virus. All synthetic processes in the infected cell are directed toward the synthesis of bacterio- phage components — phage protein and phage nucleic acid — through the activity of preexisting bacterial enzymes. After a rather precisely defined latent period, during which the synthesis of phage components is followed by the appearance of large numbers of new phage particles, the bacterial cell is lysed and releases the new phage into the medium. The outcome of the infection also depends on the number and genetic constitution of the infecting particles. If too many particles are present, there may occur a "lysis from without," apparently resulting from a massive damage to the bacterial surface. This type of lysis takes place without phage reproduction and without disruption of the bacterial nuclei. The T-even bacteriophages, in their wild types, also exhibit a phenomenon of "lysis inhibition," i.e., a delay in lysis if two or more par- ticles infect a bacterium at an interval of several minutes. Let us see what happens if active bacteriophage particles are replaced by particles inactivated by exposure to radiation. Phage particles inactivated by moderate doses of ultraviolet radiation (N/No > e~^^, by extrapolation), if tested under conditions where no reactivation occurs (see Sect. 4-2), are still capable of being adsorbed by bacteria and of killing the bacterial cell (Luria and Delbriick, 1942). Bacterial nuclei are disrupted and bacterial syntheses are suppressed, but no synthesis of phage components takes place. Lysis and liberation of active bacteriophage are absent; even if the infected cells arc artificially l)roken, no active bacteriophage is extracted (Luria and Human, 1950). 348 HAUIATION HIOLOGY Thus ultraviolet irradiation separates the early phases of infection from the later ones; it provides evidence that bacterial killinji; occurs througli the disrupting; action of the infectin}^ pha}>;e and does not re(iuire its rei)ro- (hiction. With other ultraviolet-inactivated phages (e.g., Tl and T7j bacterial infection is followed by an increase of material that reacts cyto- ehemically like deoxyribonucleic acid, but no active phage can be recovered from the bacteria. For X-ray inactivation, it is necessary to distinguish between direct and indirect effects (see Sect. 2-1). Bacteriophage particles inactivated by the direct effects of X rays (Watson, 1950) are normally adsorbed by host bacteria, but their bacteria-killing ability is often lost. The fraction of "killing" particles diminishes logarithmically with the X-ray dose, with a slope approximately one-third the slope of the inactivation curve. The killing particles affect the bacteria in the same way as does ultraviolet- inactivated phage. All adsorbable phage particles, whether killers or not , retain both the "lysis-inhibiting" property and the ability to produce "lysis from without." These effects, then, require only the changes brought about by phage adsorption, without further intromission of the virus particle into the economy of the host cell. Thus the comparison between active phage particles and particles inactivated by ultraviolet and by X rays permits the distinguishing, in the early preproductive phases of host-virus interaction, of two stages — one of "adsorption" and one of "invasion." The latter involves the disruption of that part of the bacterial machinery that impresses on the newly synthesized material the specificity of bacterial protoplasm. Interference phenomena (see Sect. 4-1) reciuire particles capable of invasion; they are produced only by particles that can kill bacteria. Phage particles inactivated by the indirect effect of X rays (exposure in the absence of protective substances; Watson, 1952) exhibit a greatly reduced rate of adsorption onto the host cells, which hinders the analysis of those phage properties that manifest themselves in later stages of the host-virus interaction. This suppression of adsorption results only from exposure to the short-lived toxic agent present during actual irradiation in water. Phage particles inactivated by introduction into a freshly irradi- ated medium give a completely different picture; they are readily adsorbed and retain their killing ability, with all the properties that attend this. No interpretation in chemical terms of the effects of indirect irradiation on various phage properties is available; differential effects of chemical poisons on different parts of virus particles are clearly to be expected. (lenerally, inactive virus particles that are still adsorbable by the bac- terial cells are not physically disintegrated and can still be recognized, e.g., in electron micrographs. Very large doses of ultraviolet radiation disrupt the complex morphological structure of some bacteriophages. Upon dis- ruption, some of the large coli bacteriophages release part of their nucleic RADIATION AND VIRUSES 349 acid (Dulbecco, 1950) and also liberate an agent, smaller than the virus particles and separable by differential centrifiigation, which produces lysis of the susceptible bacteria (Anderson, 1945). This lytic agent may or may not be implicated in the normal lysis of bacteria infected with active phage; the possibility of liberating active principles from virus particles by means of radiation is, at any rate, suggestive of a new approach to virus research. The situation described for the phages of the T group is by no means unicjue; at least for influenza viruses, similar observations have been made (Henle and Henle, 1947). Exposure to ultraviolet for progressively longer periods of time eliminates, one after the other, all the properties of the virus that can be studied. Reproductive ability disappears first, followed by toxicity, which, according to Schlesinger (1950a), is a mani- festation of an abortive infection in cells incapable of supporting full reproduction of the virus. The ability of the virus to interfere with the reproduction of another virus disappears next, followed by the immu- nizing capacity for a susceptible host (which may have to do with both antigenicit}^ and interfering ability). Hemagglutination — that is, the ability to agglutinate red blood cells — is much more resistant and dis- appears only after doses of radiation which probably disrupt the virus particle. Complement-fixing antigens, mainly present in crude virus preparations in the form of small "soluble" antigens, are greatly resistant to irradiation. It is interesting that hemagglutination and complement fixation should be the two most resistant properties of the influenza virus since both of them can be found separated from virus activity in the course of normal growth (Hoyle, 1948; Henle and Henle, 1949) and may be in the form of immature elements of greater ultraviolet resistance. It may be noted that, to an inactive particle of influenza virus, radia- tion can leave both the ability to agglutinate red blood cells and the ability to be eluted from them enzymically, whereas heat, for example, preserves the ability to agglutinate red blood cells but suppresses the enzymatic elution. The separation of infectivity from the antigenic properties of a virus by radiation is of fairly general observation. It has been proved for phages, for plant viruses (which retain enough of their integrity to form the same crystals or paracrystals as their active counterparts; see Bawden, 1950), and for a series of animal viruses. The persistence of serological proper- ties, however, may be limited to the effect of ultraviolet or of X rays acting directly. In the case of papilloma virus, the indirect effect of X rays gives a closer parallelism between the destruction of infectivity and that of complement fixation than does the direct effect of X rays (Friede- wald and Anderson, 1941). Because of the persistence of its antibody-stimulating ability, virus which has been inactivated by radiation, in spite of some observation to 350 RADIATION HIOLOOY the contrary, is now coiisidcred a rathor Kood .source of vacciiie.s (Web.ster and Ca.sals, 1942; Leviiusoii el *//., I'.tll; Milzer et at., 1941; Mil/er and Levinsoii, \\)V.)). As is the rule with inactive viruses, hirf^c; amounts of irradiated virus must be used in vaccination .since there is no increase in antigen l)y niultipHcation of virus in the host. It is po.ssihle that a certain role in the immunity phenomena oh.served with inactive virus vaccines may be played by the interference phenomena discus.sed in the next section. 4. IRRADIATED VIRUS IN MULTIPLE AND MIXED INFECTION l-l. INTERFEREXCE PHENOMENA Under interference phenomena is included a complex jjjroup of phe- nomena involving an alteration in the growth or manifestations of a virus due to the presence in the same hc^st of more virus of the same or anotiier type. The virus particles do not interact among them.selves in vitro, and the interference phenomena are strictly cellular. Only with bacterio- phage, however, have interference phenomena been analyzed at the cellular level (Dell)rack, 1950). Mixed infection of a common host with two unrelated bacteriophages results in mutual exclusion, only one virus type reproducing in any one given cell. The excluded virus may exert a depressor effect on the yield of \vinning virus. These exclusion phenomena are not exerted at adsorp- tion but take place intraceUularly. Related viruses give incomplete exclusion, which becomes less and less evident as the viruses become more closely related; particles of two virus strains differing by one mutation only do not exclude one another. Whenever exclusion fails, the total yield of virus per cell is lower than the sum of the yields that each virus would produce by it.self; the two viruses share the maximum potential yield per cell. With irradiated phages the following rule is fairly well established: Whenever a phage particle, after exposure to ultraviolet or X rays, can still invade and kill the cell, it retains the exclusion power it had when active; particles that are adsorbed but do not kill the host do not produce exclusion (Luria and Delbruck, 1942; Watson, 1950). It is not known whether an ultraviolet- or an X-ray-inactivated virus particle, if excluded, can still exert a depressor effect on the yield of an active, heterologous, excluding virus. Irradiated interfering phage excludes homologous active phage if it reaches the bacterium several minutes earlier; otherwise, exclusion fails, and the yield of active phage is normal (Luria and Dul- becco, 1949). It is evident then that the interfering ability of inactivated phages is related to their ability to kill the bacterial host. If, as seems likely, the latter process results from the virus taking oxor and redirecting the syn- RADIATION AND VIRUSES 351 thetic machinery of the host, interference is probably also a manifestation of the appropriation by one virus of the directive pattern of specificity to the exclusion of another virus. It is interesting in this connection that "lysogenic" bacteria, which carry a phage in a form that does not inter- fere with bacterial life, can be infected and lysed by other, unrelated phages and can liberate them normally. In the case of animal and plant viruses, interference phenomena have generally been studied only in their mass manifestations when a tissue or a whole organism is exposed to two viruses in succession or simultaneously. It is difficult therefore to interpret interference in terms of cellular events. For animal viruses, interference phenomena have been discussed by Henle (1950) and Schlesinger (1950b). Interference may occur between related or unrelated viruses, but not all unrelated viruses interfere with one another, and two viruses can often be shown to multiply in the same cell. Whenever there is interference between two active viruses, it is also observed with virus . ^activated by ultraviolet radiation ; other radiations have hardly been studied in this connection. With influenza viruses in the allantoic cavity of the chick embryo, it has been shown (Henle and Henle, 1943, 1945; Ziegler et al., 1944) that a large amount of a virus, e.g., influenza type A, after inactivation by ultra\iolet radiation can prevent reproduction either of homologous or of heterologous active virus, e.g., influenza type B. This interference was at first attributed to suppression of virus adsorption because the irradiated virus destroyed the virus recep- tors on the allantoic cells, 't is now known, however, that interference may occur with amounts of virus that do not prevent adsorption and also by introduction of the interfering virus after the first virus has been adsorbed (Henle, 1950). In the case of bacteriophage, as well as influenza virus, interference probably takes place at the level of the reproductive process. That a blockade of the synthv^^c machinery is involved, rather than a competition for building blocks, is suggested by the fact the inactive virus, although unable tc reproduce, retains the interfering ability. 4-2. REACTFx vTION PHENOME A Reactivation phenomena hi. oeen reported only with bacteriophages, but the possibility of their occurrence in other viruses should be explored. Bacteriophages exposed to radiation give different activity titers accord- ing to the conditions of titration. Two factors have been found relevant : The number of irradiated particles absorbed per bacterium (Luria, 1947) and the exposure of the infected bacteria to light of certain wave lengths (Dulbecco, 1950). Xo reactivation effect has been observed following treatments of irradiated phage before adsorption to the host bacterium. Reactivation of phages by light ("photoreactivation") is discussed in detail in Chap. 12 of this volume. 4-2a. MuUiplirify Reactivation. In phage titration the phage must be 352 RADIATION UIOI.OOY mixed witli scnsitiNc bacteria. Kor irradiated phase tlie residual titer is minimum when the phaf^e is exposed to such an excess of bacteria that the f2;reat majority of tlie infected l)acteria receive only one phage particle. I'nder these conditions, it is possible to distinguish a fraction of "residual active particles" and one of "inactive particles." The usual inactivation curves are obtained in this manner. For some phages and for some types of radiation, there is an apparent increase in activity under conditions of "multiple infection" of bacteria with phage. This multiplicity reacti- vation has been observed with the coli phages Tl, T2, T4, Tf), and T6 after ultra\iolet irradiation (Luria and Dull)ecco, 1949) and with T2, T4, and T() also after exposure to X rays (Watson, 1950). To participate in reactivation, an irradiated particle must retain its ability to kill the host (see Sect. 3-2). Table 9-2. The Basic Observation in Multiplicity Reactivation of Bacteriophage (Modified from Luria and Dulbecco, 1949, Table 2.) Phage: T6r, 1.5 X 10'" units/ml exposed for 20 sec to ultraviolet germicidal lamp, General Electric Company, 15 watts, at 50 cm; 7 ergs/mm^/sec Bacteria: E. coli B, 2 X 10« colls/ml = B Platings: 0.05 ml of phage dilution plus 0.2 ml of B per plate Total dilution of original Plaque Mix- Dilution of phage phage in count ture Procedure when first mixed suspension (sum of No. with B from which samples were plated two plates) 1 O.I ml T6r -^ 0.9 ml B; kept 10 min at 37°C; diluted 1 to 10^ 0.05 ml plated 1/10 1/10^ 1318 2 0.1 ml (T(ir 1/10) -^ 0.9 ml B; kept 10 min at 37°C; diluted 1 to 102, 0.05 ml plated 1/10* 1/10^ 474 3 0.1 ml (Tfir 1/10')-^ 0.9 ml B; kept 10 min at 37°C; diluted 1 to 10, 0.05 ml plated 1/10^ 1/10^ 250 4 0.05 ml (T6r 1/10^) plated <1/10* (on plate) I/IO^ 57 The basic observation is illustrated in Table 9-2 and consists in the fact that the same amount of irradiated phage gives a higher activity titer (number of lytic areas or "placiues" on a solid layer of sensitive bacteria) if the bacteria have been allowed to adsorb the phage from a more concen- trated phage .suspension. The effect is not caused by exposure of the infected bacteria to some factor other than phage present in crude concen- trated phage preparations since it occurs equally well with purified phage. RADIATION AND VIRUSES 353 It must be remembered that, in the type of titration employed, only bac- teria which, after receiving phage particles, liberate active phage are measured. Only the.se bacteria are lysed; infected bacteria that fail to liberate active phage die unlysed. If the number of bacteria that liberate active phage is determined and the number of residual active phage particles is subtracted from it, the number of bacteria in which inactive phage was reactivated is obtained. This is never higher than the number of cells that receive two or more inactive particles. For small doses of ultraviolet radiations and for high multiplicities of infection, the two become approximately equal. Thus, reactivation is due to intracellular interaction between phage particles which, if adsorbed on separate bacteria, would have registered as inactive. The interpretation of the mechanism of "multiplicity reactivation" is at this time obscure. The theory originally proposed for its interpreta- tion (Luria, 1947; Luria and Dulbecco, 1949) is undergoing revisions. When the phenomenon was first recognized, it was quickly discovered that reactivation occurs not only among particles of the same phage but also among particles of two related phages. More especially, it occurs among particles of phages T2, T4, and T6. These exhibit the remarkable phenomenon of genetic recombination, in which mixed infection with two different phages results in the production of "hybrid forms," deriving some of their properties from one phage, some from the other (Delbriick and Bailey, 1946; Hershey and Rotman, 1948, 1949). This observation suggested a similarity of mechanism between recom- bination and reactivation, and the hypothesis was formulated that ultra- violet irradiation produced, by discrete hits, a damage localized in discrete gene-like individual "units" in each phage particle and that reactivation resulted from cooperation among the infecting particles. This coopera- tion was supposed to involve the same (unknown) mechanism as that involved in genetic recombination. The requirement for reactivation in a given bacterium was then postu- lated to be the possession by the infecting particles as a group of at least one set of undamaged units. This led to the expression, for the maximum frequency of production of active phage, 00 I x''e J., 11 - (1 - e-"-)']' where x = average number of inactive particles per bacterium, r = average number of hits per particle, k = an integer number, and n = number of the hypothetical units per particle (assumed in first approximation to have equal ultraviolet sensitivity). 354 HADIATIO.N UIOLOGY The denominator in Fa\. (9-2) is the fraction of l)acteria receiving two or more particles; the luimerator is the prol)ahility that the group of k par- ticles infecting a given i)acterium contains one full set of active units. It is possible to determine x and r experimentally. A comparison of ij with the experimental frecjuency w of active phage production gave, at first, results compatible, with some limitations, with E(\. (9-2). The analysis was therefore pushed further along these lines, and estimates were given for the values of n for different phages. The tendency of the experimental ratios w/y toward unity for small values of r (low doses) and high values of .r (high multiplicities) suggested furthermore that any mechanism of recombination, if responsible for reac- tivation, should be an exceedingly efficient one in order to allow an essen- tially full utilization of needed units derived from many different phage particles in the formation of active phage. This led to the "gene-pool" hypothesis, according to which each unit reproduced independently of the others, and the resulting new units reassembled to form the new particles. This hypothesis could explain a number of features of the phage repro- duction process. Additional evidence, however, has forced revision of one basic assump- tion of the theory that multiplicity reactivation is due solely to a highly efficient mechanism of genetic recombination. According to theory, the minimum requirement should be the integrity of at least one full set of units in the infecting particles. If the frequency of reactivation is plotted against the dose of ultraviolet received by the particles, for high doses the curves should tend to an ultimate slope equal to the slope of the inactivation cur\-e for the free phage since both these slopes represent the probability of persistence of one full complement of active units (Dulbecco, 1952). Analytically, it is easily seen that, for very large values of r, Eq. (9-2) tends to the form Dulbecco (1952), having by a special procedure obtained data on the frequency of recombination at very high radiation doses, found that for phage T2 the curves for w versus dose reach their ultimate slope much sooner than expected and that this slope is not the same as that of the inactivation curve of the single particles but only about one-fifth of it (see Fig. 9-1). This result indicates that the simple theory is inadequate. The situation may be summarized as follows: jMultiplicity reactivation represents the result of a cooperation among inactive phage particles in producing active phage. In this cooperation each particle contributes in a more than additive measure. For high ultraviolet doses, for example, bacteria with three particles have a probability of reactivation several UADIATION AND VIRUSES 355 times greater than that of bacteria with two particles. Dulbecco's work on photoreactivation of phage (1951, unpiibhshed results) suggests that some phages (the T2, T4, TG group, in particular) may receive two types of ultraviolet damage, one photoreactivable by a one-quantum process, the other by a multiple-hit process. Multiplicity reactivation can over- come the effects of both types of damage. It may involve some kind of 140 280 420 560 ULTRAVIOLET OOSE.Sec Fig. 9-1. Survival of phage T2r and its multiplicity reactivation after ultraviolet irradiation. Exposure was at 80 cm distance from a 15-watt germicidal lamp (General P^lectric Company). Broken line, free phage survival; solid line, reactiva- tion frequency (fraction of mvdtiple-infected bacteria that liberate active phage). The figures given for each solid-line curve indicate the average multiplicity of infec- tion in the whole population. (Modified frotn Dulbecco, 1952.) very efficient cooperation at the physiological level, together with a mechanism of genetic recombination of a more orthodox nature than the one postulated by the "gene-pool" theory. For phages inactivated by X rays, multiplicity reactivation is very slight, with a frecjuency much lower than with ultraviolet-irradiated phage (Watson, 1950). It has been suggested that it may occur only for that fraction of particles that are inactivated by acts of X-ray adsorption which resemble ultraviolet (juanta in the extent of damage they produce, and possibly in the amount of energy released. Interestingly enough, an 350 UADIATION lUOLOGY ap|)iv('iahk' amount of multiplicity reactivation was found' with phage inactivated by the aftcretTect of X rays (see Sect. 2-1 a). 6. INTRACELLULAR IRRADIATION OF VIRUSES 5-1. IHHADIATION OF CELL8 INFECTED WITH EXOGENOUS VIRUSES Irradiation of cells durinjj; infection with \iruses may be of use in the study of \irus reproduction. This approach has as yet been limited to bacteriophage, but it could be applied to other viruses, particularly in tissue cultures. With bacteriophage the basic experimental procedure (Anderson, 1944; Luria and Latarjet, 1947) consists in infecting a bacterial culture with virus, taking samples at intervals during the period that pre- cedes lysis, and exposing them rapidly to various doses of ratliation. The irradiated infected bacteria are then tested immediately for their ability to liberate phage. This ability can be suppressed by either ultraviolet or X rays; and, if the fraction of bacteria that liberate phage is plotted versus dose of radiation, "suppression curves" are obtained. The sup- pression effect is exerted on the intracellular bacteriophage rather than on the bacterial host. This is shown by the following observations: 1. Active phage can reproduce normally in bacteria exposed to ultra- violet radiation shortly before infection (Anderson, 1948). 2. If infected bacteria are irradiated immediately after infection, the rate of suppression of phage liberation as a function of radiation dose is similar to the rate of inactivation of free virus. 3. In multiple infection the suppression cur\e immediately after infec- tion is of the multiple-hit type and closely resembles the curves obtained for active phage production in multiple infection with irradiated bacterio- phage (see Sect. 4-2a). As the time after infection increases, the suppression curves change in a manner characteristic for the phage. The simplest case is that of phage T7 (Benzer, 1952; see Fig. 9-2). After infection there is no change in ultraviolet sensitivity for 3 or 4 min, but then the suppression curve becomes of the multiple-hit type without any change in the final slope of the curve; this seems to indicate a simple mechanism of multiplication of virus elements having the same sensitivity as the free virus. With phage T2 (Luria and Latarjet, 1947; Benzer, 1952) the first change in bacteria infected with one T2 particle and exposed to ultra- violet (2537 A) is an increase in ultraviolet resistance without appreciable change in the shape of the curve. Several minutes later the inactivation curve changes to a complex type, suggesting an effect on numerous objects within each cell. In the latest stages of infection, radiation sensitivity again increases. The results suggest that phage T2 must perform an early function easily blocked by ultraviolet damage and that the radiation sensitivity increases as this early phase is passed. RADIATION AND VIRUSES 357 Phage T2 has similarly been investigated with X rays (Latarjet, 1948). The picture is simpler than with ultraviolet since the suppression curve remains constant for several minutes, then becomes of a multiple-hit type (with lesser ultimate slope than for free phage), and finally becomes a multiple-hit curve with ultimate slope similar to that of free phage. > o z llJ o UJ a: a. u UJ < q: > > a. (ft 0 05 - 001 0005 100 200 ULTRAVIOLET DOSE, Sec 300 Fig. 9-2. Intracellular irradiation of phage T7. Bacteria were washed in buffer, infected with phage T7 (single infection), and then placed in a nutrient medium. Samples were taken at intervals, chilled, exposed to ultraviolet radiation, and then plated to determine the number of bacteria that still liberate phage. The suppression curves are compared with the inactivation curve for free phage. Exposure conditions were the same as for Fig. 9-1. The time given for each curve is the time between addition of nutrient and chilling previous to irradiation ("growth timeT). {Benzer, 1952.) Thus this type of analysis, although quite incomplete, suggests that a small phage such as T7 reproduces by multiplication of uniform elements, whereas T2 undergoes a complex series of changes, including a process of multiplication, which only in the latest phases leads to the presence of mature fully sensitive phage particles in the infected cells. This con- clusion is in accord with all our information on the reproduction of these viruses. The intracellular irradiation procedure allows the determination of the 358 i{ \i)i AiioN ni(ti.(»r.v stage of iiif(>(t ion from the shape and slope of the suppression curve. This can he utiHzed to identify tiic stages at which a certain Ircatment stops virus reprochu'tion. For example, on exposure of l)acteria infected with phage T2 to a temperature of 45°C, the changes in ultraviolet sensitivity lake place normally for the first 7 min, after which no further change occurs, as though at this time a temp(M-ature-sensitive reaction entered tlie picture (Beii/cer, 19o(), unpublished data). Although no "cure" for phage-infected bacteria has been ol)tained in this work, an extension of these studies to other viruses might produce results of some therapeutic value in \ irus infection. Even if the cells already infected could not be saved by radiation, suppression of their ability to liberate virus might prevent the spreading of infection. The value of such a procedure would depend on the relative sensitivities of the infected cells and of the normal tissues. Experiments on rabbit papil- loma have shown that growth of the papillomas can be suppressed by doses of X rays much smaller than those necessary to inactivate the \^irus in vitro (Syverton, Berry, and Warren, 1941; Syverton, Harvey et al., 1941). Actually, virus can be recovered in undiminished amounts from the irradiated papillomas (Friedewald and Anderson, 1943). Here the radiation probably acts on the host cell, rather than directly on the intra- cellular virus, in the same way as therapeutic doses of X rays affect bacterial infections bj' acting on the tissues of the host. 5-2. RADIATION AND LATENT VIRUSES An interesting possibility is that of affecting, by means of radiation, viruses which may be present in the latent state, i.e., viruses which do not manifest themselves and which behave up to a certain point like normal cell components. The distinction between latent viruses and cell com- ponents is not always easy with the available knowledge and in some cases may actually be academic. Any self-reproducing element of the proto- plasm of a cell might conceivably become a virus if by some evolutionary accident it should ac(iuire the ability to enter other cells and there repro- duce its own kind. Such an origin of viruses has been suggested, but the question will remain academic until more definite knowledge is obtained in regard to the occurrence and properties of self-reproducing units (other than the nuclear genes) in most types of cells. Should such units be more widespread than they appear to be, their origin in the process of cell evolu- tion would still be unknown. It is known that a virus may enter a cell and reproduce while the cell goes through several cell generations, often without causing recognizable cell disturi)ances. Such a virus behaves at least for some time as a cell component. This type of symbiosis often prolongs itself for many cell generations, and in multicellular organisms some viruses are transmitted through the gametes from generation to generation. The recognition of the virus depends then only on indirect RADIATION AND VIRUSES 359 tests, either the inoculation of tissue extracts in a virus-susceptible host or the search for antivirus antibody in the latently infected organisms. The distinction between virus and cell component is thus (luite difficult. The information relevant to this area of biology caiuiot be discussed here. In connection with the possil)ility of affecting latent viruses or self-repro- ducing cell components within cells as a means of analyzing the relation between these entities and the cell as a whole, some radiation results are pertinent. 5-2a. Irradiation of Lysogenic Bacteria. Lysogenic bacteria carry one or more bacteriophages in a latent form without recognizable manifes- tations. In these bacteria the phage is apparently present as immature virus or "prophage." Occasionally a lysogenic cell is lysed and liberates a cluster of mature phage particles, whose presence can be recognized if a susceptible strain of bacteria that responds to phage infection by lysis is available (Lwoff and Gutmann, 1950) . In the course of attempts to define the conditions that lead to the occasional maturation of virus in the lyso- genic bacteria, it has been discovered (Lwoff et al., 1950) that ultraviolet irradiation produces in some lysogenic strains a massive lysis accompanied by liberation of mature phage. This suggests that something in the lyso- genic bacteria prevents the maturation of prophage into bacteriophage, thus preserving the symbiotic relation, and that ultraviolet, by removing the inhibition, releases the maturation process. 5-2b. Irradiation and Cytoplasmic Factors. Another pertinent obser- vation concerns the destruction by radiation of the cytoplasmic factor "kappa" in Paramecium aurelia. Some strains of this organism produce a poison (paramecin) which is lethal for individuals of other strains. The production of the poison is always associated with the presence in the cytoplasm of the killer animals of peculiar Feulgen-positive particles, 0.3- 0.8 M in size, which are the material carriers of a genetically recognizable self -reproducing mutable factor (kappa). The continuous production of kappa depends both on the presence of preexistent kappa and on the proper genetic background. Kappa has been transmitted from one indi- vidual to another by "infection " with cell extracts, thus resembling a virus or a rickettsia (Sonneborn, 1949). X rays (Freer, 1950), as well as ultraviolet (Sonneborn, personal com- munication) and nitrogen mustards (Geckler, 1949), eliminate the killer factor from the protoplasm at a rate that suggests a one-hit inactivation process with an inactivation dose of approximately 4000 r. This dose is comparable to the doses required for sterilization of bacteria which are somewhat larger in size than the kappa particles. This result suggests interesting applications of radiation analysis to the study of cytoplasmic inheritance and encourages speculation on the possible use of selective effects of radiation on cytoplasmic elements in modifying de\-elopment and differentiation (which have been suggested to be controlled by cyto- .3()() IIAIMATIOX MIOLOflY pliismic dotormiiiants of heredity) and in altering the neoplastic properties of tumors. It may he of interest to mention that another self-reproducinj^ mutahle cytoplasmic factor, the virus-like "genoide" for carbon dioxide sensitivity in Drusophila (L'lleritier, 1949), which can he transmitted from fly to fly by cell-free extracts, is inactivated in the extracts by X rays. The inacti- vation dose is around 10'' r, similar to that for medium-sized viruses (L'Heritier and Plus, 1950). REFERENCES Alper, T. (1!)48) Hydrogen peroxide and the indirect effect of ionizing radiations. Nature, 162: 615-616. Anderson, T. F. (1944) Virus reactions insid(> of Ijucterial host cells. J. Bacteriol., 47: 113. (1945) On a bacteriolytic substance associated with a purified bacterial virus. J. Cellular Comp. Physiol., 25: 1-13. (1948) The growth of T2 virus on ultraviolet-killed host cells. J. Bacteriol., 56: 403-410. Hawden, F. C. (1950) Plant viruses and virus diseases. 3d ed., Chronica Hotan., Waltham, Mass. Henzer, S. (1952) Resistance to ultraviolet liKht as an index to the reproduction of l)acteriophage. J. Bacteriol., 63: 59-72. Benzer, S., M. Delbriick, R. Dulbecco, W. Hudson, G. S. Stent, J. D. Watson, W. Weidel, J. J. Weigle, and E. L. Wollman (1950) A .syllabus on procedures, facts, and interpretations in phage. In, Viruses 1950, ed. M. Delbriick. Calif. Inst. Technology Bookstore, Pasadena. Pp. 100-147. Bonet-Maury, P. (1947) The irradiation of viruses. Brit. J. Radiol., Suppl. 1, 21-29. (1948) Irradiation et methodes statistiques de titrage des ultra-virus. In, Les ultraviruses des maladies animales, ed. C. Levaditi, P. Lepine, and J. Verge. Maloine, Paris. Pp. 165-215. Bryan, W. R., and J. W. Beard (1939) Estimation of purified papilloma virus pro- tein by infectivity measurements. J. Infectious Diseases, 65: 306-321. Cohen, S. S. (1949) Growth requirements of bacterial viruses. Bacteriol. Revs., 13: 1-24. Delbriick, M. (1942) Bacterial viruses (bacteriophages). Advances in Enzymol., 2: 1-32. (1950) Viruses 1950, ed. M. Delbriick. Calif. Inst. Technology Bookstore, Pasadena. Pp. 32-34. Delbriick, M., and W. T. Bailey, Jr. (1946) Induced mutations in bacterial viruses. Cold Spring Harbor Symposia Quant. Biol., 11: 33-37. Didbecco, R. (1950) Experiments on photoreactivation of bacteriophages inactivated with ultraviolet radiation. J. Bacteriol., 59: 329-347. Dulbecco, R. (1952) A critical test of the recombination theory of multiplicity reactivation. J. Bacteriol., 63: 199-207. Fluke, D. J., and E. C. Pollard (1949) Ultraviolet action spectrum of Tl bacterio- phage. Science, 110: 274-275. Friedewald, W. F., and R. S. Anderson (1940) Factors influencing the inactivation of rabbit papilloma virus by X-rays. Proc. Soc. Exptl. Biol. Med., 45: 713- 715. RADIATION AND VIRUSES 361 — (1941) Influence of extraneous protein and virus concentration on tlie inac- tivation of the rabbit papilloma virus by X-rays. J. Exptl. Med., 74: 468-487. (1943) The effect of roentgen rays on cell-virus associations. Findings with virus-induced rabbit papillomas and fibromas. J. Exptl. Med., 78: 285-308. Gard, S. (1943) Purification of poliomyelitis viruses. Experiments on murine and human strains. Acta Med. Scand., Suppl. 143. Gates, F. L. (1934) Results of irradiating Stop/iJ/^oroccws awreMS bacteriophage with monochromatic ultraviolet light. J. Exptl. Med., 60: 17'.) 188. Geckler, R. P. (1949) Nitrogen mustard inactivation of the cytoplasmic factor, kappa, in Paramecium. Science, 110: 89-90. Gowen, J. W. (1941) Mutations in Drosophila, bacteria and viruses. Cold Spring Harbor Symposia Quant. Biol., 9: 187-192. Henle, W. (1950) Interference phenomena between animal viruses: a review. J. Immunol., 64: 203-236. Henle, W., and G. Henle (1943) Interference of inactive virus with the propagation of virus of influenza. Science, 98: 87-89. (1945) Interference between inactive and active viruses of influenza. III. Cross-interference between various related and unrelated viruses. Am. J. Med. Sci., 210: 362-369. (1947) The effect of ultraviolet irradiation on various properties of influenza viruses. J. Exptl. Med., 85: 347-364. (1949) Studies on host-virus interactions in the chick embryo-influenza virus system. III. Development of infectivity, hemagghitination, and comple- ment fixation activities during the first infectious cycle. J. Exptl. Med., 90: 23-37. Hershey, A. D., M. D. Kamen, J. W. Kennedy, and H. Gest (1951) The mortality of bacteriophage containing assimilated radioactive phosphorus. J. Gen. Physiol., 34: 305-319. Hershey, A. D., and R. Rotman (1948) Linkage among genes controlling inhibition of lysis in a bacterial virus. Proc. Natl. Acad. Sci. U.S., 34: 89-96. (1949) Genetic recombination between host-range and plaque-type mutants of bacteriophage in single bacterial cells. Genetics, 34: 44-71. Hewitt, H. B., and J. Read (1950) Search for an effect of oxygen on the direct X-ray inactivation of bacteriophage. Brit. J. Radiol., 23: 416-423. Hollaender, A. (1946) Effects of ultraviolet radiation. Ann. Rev. Physiol., 8: 1-16. Hollaender, A., and B. M. Duggar (1936) Irradiation of plant viruses and of micro- organisms with monochromatic light. III. Resistance of the virus of typical tobacco mosaic and Escherichia coli to radiation from X 3000 A to \ 2250 A. Proc. Natl. Acad. Sci. U.S., 22: 19-24. Hollaender, A., and J. W. Oliphant (1944) The inactivating eff"ect of monochromatic ultraviolet radiation on influenza virus. J. BacterioL, 48: 447-454. Hoyle, L. (1948) The growth cycle of influenza virus A. A study of the relations between virus, soluble antigen and host cell in fertile eggs inoculated witli influ- enza virus. Brit. J. Exptl. Path., 29: 390-399. Latarjet, R. (1948) Intracellular growth of bacteriophage studied by roentgen irradiation. J. Gen. Physiol., 31: 529-546. (1949) Mutation induite chez un virus par irradiation ultraviolette de cellules infectees. Compt. rend., 228: 1354-1357. Latarjet, R., and E. Ephrati (1948) Influence protectrice de certaines substances contre I'inactivation d'un bacteriophage par les rayons X. Compt. rend. soc. bioL, 142: 497-499. T-atarjet, R., and R. Wahl (1945) Precisions sur I'inactivation des bacteriophages par les rayons ultraviolets. Ann. Inst. Pasteur, 71: 336-339. :{()2 H \I)I A'IKIN HIOI.OGY LiuilTfT, M. A., and W. C rricc (HMo) Infection by viruses. Arcli. liiocheni., 8: 44S)-tG8. Lea, D. E. (194(5) Actions of radiations on living cells. Cambridge University Press, London (also The Macniillan Company, Xew York, 1947). T/ca, n. 1']., and M. II. Salaman (1942) The inact i\ atiori of wicciiiia virus hy radi- ations. Brit. .1. i;.\|)(l. Patiioi., 2.i: 27-:i7. (194(i) Kxperinients on the inactivation of hacteriophaKc hy radiations and their Ixarin;! on tlie nature of l)a(t(riophaKe. I'roc. Roy. Soc. London, lil33: 4:i4 444. Lea, D. K., K. .M. Smith, H. liulmcs, ami U. .Mari40) KlTcct of radiations on bacteriophage ri6. Nature, 145: 1)35 «>:i(i. Wolliiian, 10., anil A. Lai-assanno (liMO) Evaluation des dimensions des bacterio- phages an moyen des rayons X. Ann. inst. Pasteur, 64: 5-39. Ziegler, J. E., Jr., (!. I. Lavin, and F. L. Horsfall, Jr. (1944) Interforcnce between the inlluciiza viriises. II. The effect of virus rendered non-infective by ultra- vioU't r.uliatioii upon the mult ii)licat ion of iiiHuenza viru.ses in the chick embryo. J. Exptl. Med., 79: 379-400. Manuscript received hii thr ((lilnr Apr. 20, 1951 CHAPTER 10 Effects of Radiation on Bacteria M. R. Zelle Cornell University Ithaca, New York Alexander Hollaender^ Biology Division, Oak Ridge National Laboratory Oak Ridge, Tennessee Bactericidal effects of radiation. High-energy radiations: General results of quatdita- tive investigations. Factors influencing sensitivity to ionizing radiations: Ultravioiet radiation — Extreme ultraviolet radiation — Near- ultraviolet and short-visible radiation. Physiological properties of bacteria following irradiation. Sublethal effects of radiation. Bacterial genetics: Radiation-induced mutations in bacteria — Mechanism of radiation effects. One of the first laboratory observations of the effects of radiations on bac- teria was published by Downes and Blunt in 1877. Since that date a volu- minous literature has accumulated as a result of the wide interest in the effects of radiation on bacteria per se and also because of the relative ease with which quantitative studies of the biological effects of radiation can be made with bacteria. Despite the numerous investigations during the past seventy-five years, understanding of the effects of radiation on bacteria is still only fragmen- tary. Furthermore, the advances since the 1936 predecessors of these volumes, especially during the past decade, have been rapid in comparison to the advances in earlier work. For example, almost all knowledge of radiation-induced mutations in bacteria has been gained since that time. Consequently, in the limited space available, no attempt will be made to review completely the earlier literature. Rather, only those earlier con- tributions considered to be especially significant will be included with corre- spondingly greater emphasis on recent research. The rather numerous reviews concerned with various aspects of the biological effects of radia- tion combine to give a complete coverage of the development of the field ' ^\'o^k performed iiiuier Contract Xo. W-7405-eng-26 for the Atomic Energy Commission. 365 ;^()() UADIATION lilOl.OGY (Dufijrar. \\yM\\ (liese, 1945, H)47. lUoO; K\\\s rl ai. 1041; Latarjet, 1046; Lea, 1017; Loofbourow, 1048; Mitchell, 1051; Rahn, 1045). BACTERICIDAL EFFECTS OF RADIATION When bacteria are exposed to radiation, in either the high-ener{j;y or the ultraviolet range, the most prominent clTcct is the apparent killing of a percentage of the cells, the fraction killed being a function of the absorbed energy. The usual criterion of survival is the ability of the bacteria to form a colony visible to the eye when incubated following plating on ordi- nary culture media. This arbitrarily adopted measure of the bactericidal effects of radiation, although convenient for (|uantitative studies, is influ- enced by a variety of experimental conditions. Ilollaender (1043) showed that prolonged exposure to saline, following long-ultraviolet or short-visible irradiation, reduced the fraction of colony-forming organisms. Roberts and Aldous (1940) made careful studies of various experimental conditions both before and after ultraviolet irradiation which affect the survival of Escherichia coli, strain B. TIkmt results will be considered in more detail later, but for this strain, a hundredfold variation in survival could be produced by changing the postirradiation treatment. Further- more, the shape and slope of the survival curve were markedly influenced by different conditions. Strain B/r, a radiation-resistant mutant deri\'ed from strain B (Witkin, 1946, 1947), did not show similar variation in survival when subjected to the same experimental treatments. Kelner (1949a, b) observed that exposing bacteria to visible light following exposure to ultraviolet significantly increased sur\'ival (photoreacti- vation). Anderson (1949, 1951b) and Stein and Meutzner (1950) have shown that increasing the temperature of incubation increases the sur- vival of ultraviolet-irradiated E. coli B. Survival following X irradiation was shown by Hollaender, Stapleton, and Martin (1951) to be influenced by the oxygen concentration of the medium at the time of irradiation. Stapleton et al. (1953) have discovered that incubation at suboptimal temperatures markedly increases the survival of bacteria exposed to X rays, and Stapleton (1952) has found marked differences in the radio- sensitivity of E. coli cells at different stages of the growth cycle. These findings are mentioned to emphasize the multiplicity of factors which influence the quantitative results obtained in studies of the bac- tericidal effects of radiation. Consequently, in order to obtain reproduc- il)le results, it is necessary that these variables be adequately controlled. Furthermore, it is impossible to determine how much of the variation in the results obtained bj' difl'erent investigators is attributable to differences in their experimental techniques. This is especially true in regard to many of the earlier studies. Bactericidal effect, lethal effect, killing, and inactivation are used EFFECTS OF RADIATION OX BACTKHIA 367 synonymously to indicate the failure of the cells to form a colony visible to the naked eye when plated under the particular conditions of the experiment under discussion. HIGH-ENERGY RADIATIONS GENERAL RESULTS OF QUANTITATIVE INVESTIGATIONS Minch in 1896 was apparently the first to attempt to study the bac- tericidal effects of X rays. His results were essentially negative as were those of man}' other investigators during the next thirty years. Low intensities of X rays and rather insensitive bacteriological techniques seem to be the chief reasons for the conflicting and often negative results of different investigators during this period. Duggar (1936) briefly dis- cusses these early X-ray results, and the bibliographies given by him and by Pugsley et al. (1935) form a helpful guide to the early literature. Diu'ing this period, however, it was convincingly demonstrated by a number of investigators that ionizing radiations do exert a marked bac- tericidal effect. Green (1904)^ employing semiquantitative bacterio- logical techniques, studied the bactericidal effects of radium /3 rays on 23 species of bacteria including five spore formers. In his experiments all species were killed by the /3 rays, and the spore formers were found to be considerably more resistant than the vegetative forms. One of the out- standing contributions during this period was that of Chambers and Russ (1912). These workers studied the effects of radium emanation, pri- marily /3 rays, on distilled water suspensions of Staphylococcus aureus, Escherichia coli, Bacillus pyocyaneus, and Bacillus anthracis. A pro- nounced bactericidal action was observed with all species; anthrax spores were observed to be the most resistant to radiation. Quantitative esti- mates of surviving organisms made by plate counts of an irradiated S. aureus suspension, when plotted semilogarithmically, gave rise to a straight line. This is the first exponential survival curve reported for bacteria subjected to radiation. Furthermore, these workers observed motile cells of B. pyocyaneus in irradiated suspensions in which no colony- forming organisms were present. Similar observations of inactivated but motile cells have been made by Bruynoghe and Mund (1925). Following application of the target theory by Crowther (1924, 1926) to inhibition of mitosis in tissue culture cells observed by Strangeways and Oakley (1923) and to his own data on killing of Colpidium colpoda, a num- ber of investigators applied similar analyses to the bactericidal effects of ionizing radiations. Holweck (1929) and Lacassagne (1929) irradiated "pyocyanique S" with soft X rays of 4 and 8.3 A wave lengths. They observed exponential killing with 4 A X rays, but a multihit or sigmoidal type of survival vxivxe was obtained with 8.3 A. However, Lea, Haines, and Coulson (unpublished, see Lea, 1947) observed exponential sur- 3(1S It \i)i A iio.v iii()L(jr.Y vival curves \vh(Mi icpcMliiifj; this work with the same strain and wave Iciijitlis. Wyckort" (lU:i()a. 1)) and WyckolT and Rivers (llKiO), in a series of care- tul stiuHes, more firmly estal)Msiied the occui'rence of exponential survival curves followiufi; exposure to ionizing radiations. Wyckoff and Rivers ( 1930) studied the bactericidal effects of 15o-kv (8 rays on E. coli, Salmo- nella h/pliimurium, and Staphylococcus aureus cells seeded on the surface of agar plates. Exponential survival curves were obtained for all except S. aureus. By allowing E. coli to divide before irradiation, they showed that clumping of the cells w'as probably the reason that exponential sur- vival curves were not obtained for S. aureus. They conclude that a single electron is sufficient to inactivate a cell of these species. Similar results (Wyckoff, 1930a) wxre obtained in studies with E. coli and *S. tijphimurium using copper-K X rays and the soft general radiation from a tungsten tube operated at 12 kv. Later, Wyckoff (l!)30b) studied the killing of E. coli with X rays of wave lengths varying from 0.5 to 4 A. Exponential killing was observed at all wave lengths. Wyckoff interpreted his results to indicate that a single quantum of X rays was sufficient to kill the bac- teria. His estimated values of the sensitive volume of the organisms decreased with increasing wave lengths as a result of the greater incident energy required for inactivation at the longer w^ave lengths. Similar results have been obtained by other workers with various bac- terial species subjected to various ionizing radiations. Hercik (1933, 1934b) observed exponential sur^•ival curves wuth Serratia marcescens irradiated with a particles emitted by polonium. Pugsley et al. (1935) observed exponential killing for E. coli irradiated wdth 40-kvp X rays but obtained sigmoidal curves for Sarcina lutea. A correction applied for the degree of clumping as determined by microscopic examination of the irradiated suspension resulted in an exponential survival curve. Lorenz and Henshaw (1941) made extensive tests of the bactericidal effects of 200-kvp X rays on Achromobacter fischeri. Statistical analj'sis showed no systematic deviation from an exponential survival curve. During the past decade, in which there has been almost a routine use of radiation for the induction of mutations in microorganisms, numerous investigators have observed exponential survival curves (e.g., Lincoln and Gowen, 1942; Demerec and Latarjet, 1946; Witkin, 1947; Roepke and Mercer, 1947; Anderson, 1951a). Frara et al. (1950) report exponential survival curves for six species following irradiation with 50-kvp X rays. A typical exponential survival curve with 5 per cent confidence limits of the plotted points is shown in Fig. 10-1 (Stapleton, unpublished data). Not all investigators have observed exponential survival curves, how- ever. Observation by Holweck (1929) and Lacassagne (1929) of sig- moidal survival curves with 8.3 A X rays has already been mentioned. Glaus (1933) observed sigmoidal sur\i\al curves for E. coli following EFFECTS OF RADIATION ON BACTERIA 369 100 X irradiation in the presence of heavy metal ions. The sigmoidal curves may have been due to the short wave lengths of the secondary radiations which were considered to be the main cause of inactivation. Luria (1939), employing the same bacteriological techniques in both cases, observed exponential survival curves with polonium a particles and a two-hit sigmoidal curve with 0.7 A X rays. Microscopic examination of the irradiated cells of E. colt revealed that, although some of the cells were killed immediately and did not divide or grow, others continued to grow without dividing and ultimately developed into long filamentous forms. These filamentous forms either divided a few times and then died or else recovered and proceeded to develop normal col- onies. Exposure to both a and X radiation caused filamentous forms, but the proportion was higher with X rays. Luria points out that death by several mecha- nisms is incompatible with the simple mathematical formulation of the target theory. Similar results with Aspergillus terreus spores have been reported by Stapleton, Hollaender, and Martin (1952) and Zirkle et al. (1952) who report sigmoidal sur- \ival curves with hard X rays and exponential curves with densely ionizing protons and a particles. Since in both cases air-dried spores were irradiated, there is no possibility that prodviction of a toxic substance in the irradiated suspending medium could be responsible for the sigmoidal curves. Stapleton (1952) made the important observation that the form of the survival curve obtained after X irradiation (250 kvp) of E. coli B/r cells depends on the stage of the growth cycle of the culture. Cells from fully grown cultures in the stationary phase yield exponential survival curves (Fig. 10-1). However, when cells in the lag phase were exposed to X rays, sigmoidal survival curves were obtained, the deviation from exponential killing increasing to a maximum at the end of the lag phase. Interpreta- tion of the sigmoidal cur\es on the multitarget theory (Atwood and Nor- man, 1949) shows an increase from one to about eight targets during the 0 001 40 DOSE, kr Fig. 10-1. Exponential survival curve with 5 per cent confidence limits for each point; E. coli B/r resting cells irradiated in air- saturated buffer with 250-kvp X rays. (Stapleton, unpuhlished data.) ;^7() RADIATION HIOLOGY lufj; phase. Cytolofiical studies show a hifi;h correspondeiu'e between the iiuniher of ol^servahle nuclear l)odies and the numher of t argots estimated for cells in different f^rowth jihases. The survival curves become expo- nential and the target iniinber ix'comes 1 as growth proceeds through the logarithmic phase and the stationary phase is approached. Stapleton made the further interesting observation that stationary-phase cells sub- jected to 7 rays from a Co^" source exhibit a "two-hit " killing curve as compared to an exponential curve for X rays. Lea et al. (193()), in addition to discussing the principal theories of the mechanism of the bactericidal action of ionizing radiations, present important (luantitative data on survival curves of irradiated bacteria. They studied primarily the survival of Bacillus mesentericus spores and, less extensively, the survival of E. colt and S. aureus. The radiations employed were a particles emitted by polonium and /3 rays produced by radon disintegration. Careful energy measurements were made and the geometrical conditions were controlled. The spores and bacteria were irradiated in dried gelatin films of approximately l-yu thickness. The fraction of organisms surviving was found to be a diminishing exponential function of the time of exposure, or dose, for all organisms with both radiations. The target areas and mean lethal doses (MLD) were com- puted for each organism for both a and 13 rays. The studies with B. mesentericus spores and E. colt were extended to include radium y rays (Lea et al., 1937), neutrons, hard X rays of an effec- tive wave length of 0.15 A, and soft X rays of L5, 4.1, and 8.3 A (Lea et al., 1941). The organisms were irradiated in aqueous suspensions; suspending the cells in previously irradiated distilled water given com- parable exposures was shown to have no effect. Again, exponential survival curves were observed in all cases. Spear (1944), utilizing the same bacterial strains employed by Lea and his associates, presents the most extensive data on the bactericidal effects of neutrons. The neutron source was a beryllium target bombarded by 8-Mev deuterons accelerated in a 37-in. cyclotron. Acjueous suspensions were exposed, and the dose was measured with a Victoreen dosimeter calibrated in roentgens. The sur\i\'al cur\'e did not depart systemati- cally from an exponential curve. The ratio of the T-ray dose in roentgen units to the neutron dose in n units refjuired to produce 50 per cent lethality was 3.2 for E. coli and 5.3 for B. mesentericus spores. Because of the lack of absolute dosimetry with neutrons, Spear presents a curve (Fig. 10-2) showing a systematic decrease in the MLD ratio of B. mesen- tericus spores to E. coli cells as the ionization density increases. This curve includes all the data of Lea and his associates and shows that the neutron data fit into the general relation. No effect of varying temperature between 2.5° and 3()°C at the time of irradiation was observed by Hercik (1934a) in studies employing a par- EFFECTS OF RADIATION ON BACTERIA 371 tides and Serratia marcescens. Lea et at. (1936) found the rate of iuacti- vation of B. mesentericus spores by a and 0 particles to be independent of temperature between -20° and +50°C. No influence of temperature was noted on the rate of inactivation of *S. aureus. These results differ markedly from the high temperature coefficients experienced with chemi- cal disinfectants. Lea e( al. (1937) showed that varying the temperature from 0° 37°C had no influence on the survival of E. colt exposed to y rays. Lea et al. (1936) observed that a sixfold variation in intensity of a-par- ticle flux had no effect on the proportion of organisms surviving a given dose. This was confirmed by experiments in which a given dose was administered in a number of fractions, no effect on survival being observed. 30- _i o o CO o ir uj 20 (ij 5 o I BETA RAYS Oc-GAMMA RAYS SOFT X RAYS I 5 A O' \ O-SOFT X RAYS 83 a O-NEUTRONS (- 10- ALPHARAYS 1^34 IONS PER cm(xlO^) IN BIOLOGICAL MATERIAL Fig. 10-2. Curve showing ratio of MLD mesentericus: MLD coli plotted against ion density for a number of different radiations. {Adapted from Spear, 1944.) Lea et al. (1941) reported further data on the lack of an intensity effect of a particles and 8.3 A X rays. A seventy-fivefold variation in intensity was employed in the latter case. Extremely high intensities of /3 par- ticles have been shown by Huber (1951) to be bactericidally effective. The observation of filamentous forms in bacterial cultures following ultraviolet irradiation was reported by Gates (1933) who concluded that the mechanism of cell division was more sensitive to radiation than the processes of growth. Similar observations have been reported by several investigators (Spencer, 1935; Luria, 1939; Witkin, 1947; Rolierts and Aldous, 1949). Lea et al. (1937) observed such filamentous forms follow- ing continuous y irradiation of growing E. coli cultures. Subsequent experiments indicated that the filamentous cells reacted to y rays in the same manner as the normal cells, i.e., they were equally sensitive to radiation and were inactivated in an exponential manner. Careful studies of the effect of ionization density on the biological effec- 372 u\i)i\ri()N iii()L()(!Y li\ciu>.s.s of poloiiuim (I particles liaxc l)i'cii report (id by Zirkle (11)10) who obtained dilTereiit ioni/atioii densities b}- utilizing dilTerent portions of the path lengtii. An increase in elTectivcness with increasing ionization density was obserxed with Aspenjillns nujer spores whereas, for inactiva- tioii of E. coll, an inverse iclation was observed. Little influence of ionization ciensity on sur\ i\al of yeast cells was noted. In Table 10-1 are assembled the available data on the relation of ioni- zation density and the bactericidal effect of radiations (see also Fano, Chap. 1, and Zirkle, Chap, (i, \'olume I of this series). The estimates made from Spear (1044) and Zirkle (1040) are only approximate but serve to indicate the general relations. It will be observed that the MLD (37 per cent survival dose) for E. coli increases as the iotiization density of the radiation increases. On the contrary, the MLD for B. mesentericus and for .1. nufcr spores decreases with increasing ionization density. This difference is as yet unexplained, and it may indicate that different mechanisms are involved in the lethal effects of irradiation for the two-cell forms. The increased efficiency of more densely ionizing radiations in killing ,4. terrcus spores is confirmed by Stapleton, Hollaender, and Martin (lOo'i) who add the further interesting observation that more densely ionizing a particles are less efficient than hard X rays in inducing mutations. Summarizing, three general conclusions seem warranted: (1) Both exponential and sigmoidal survival curves may be observed, depending on the bacterial strain, the technique of irradiation, the characteristics of the radiation, and the stage of growth of the irradiated cells. (2) The sur\iv- ing fraction for a gi\'en dose of radiation is independent, within limits, of the intensity of the incident radiation or of the fractionation of the dose. (3) For vegetative cells the bactericidal effectiveness of a given dose decreases with increasing ionization density; the opposite seems to be true for spores. The interpretation to be placed on these general results is not clear at this time. Lea et at. (1036) and Lea (1947) have discussed at length the various interpretations proposed for the observed survival curv^es. Interpretation in accordance with the target theory seems the most plausible since the first-order kinetics are a natural consequence of the theory. Similaily, the sigmoidal survival curves are also easily accounted for on the basis of either multiple hits re(iuired in one target or single hits in multiple targets. Atwood and Norman (1940) discuss this latter interpretation. Lea (1947) especially has been a strong proponent of the target theory. Il(> interprets the results to indicate that a single ioniza- tion is sufficient to inactivate a bacterial cell. He has developed the hypothesis, first suggested by Rahn (1020, 1030), that the bactericidal effects are due to lethal mutations induced by the radiation. Following elaborate analysis of the results obtained with E. coli exposed to radiations EFFECTS OF RADIATION ON BACTERIA 373 Table 10-1. Mean Lethal Doses" of Various Rai)l\tions on Various Bacteria Reference Radiation MLD E. coll (doses in 10' r or 10' n) Wyckoff (H)30b) X rays, A: 0.56 4.2 0.71 4.6 1.5 4.3 2.3 6.7 4.0 8.4 Zirkle (1940) X rays 0.3 A 3.9 a Particles, Mev: ~5 5.7 ~2 6.4 heaetal. (1941) ^ rays 4 7 rays 5.2 X rays, A: 0.15 6.0 1.5 6.5 8.3 7.5 Neutrons 7.1 (4.1)'> OL Partic iles 24 B. mesentericus spores (doses in 10^ r^ LeaeiaL (1941) ^ rays 7 rays X rays, A: 1.5 4.1 8.3 Neutrons a Particles 1.1 1.3 1.3 1.1 1.5 0.61'- 0.26 A. niger spores" (doses in 10^ r) Zirkle (1940) a Particles, Mev: ~4 11.0 ~3 3.6 ~2 4.7 ~1.5 3.9 ~1 3.2 " MLD, 37 per cent survival dose. * Estimated from Spear (1944). « Spore germination, not colony growth, wasmeasured: estimated from Zirkle (1940). 874 UADIATION ItlOI.OOY of (lilTcrcMit ionization (ioiisitics, Leu (11)47) coMcludos ih.il the hiictcricidal cITccts can be accounted for on the hypothesis of lethal niiitalions induced aiuoiifi; 2')() ^enes liavinj; an avera}j;e diameter of 12 ni^u. An iucreasinii; amount of evidence has l)een accumuhiting to indicate; that a major porti(in of tlie effects of ionizing radiations may he iiuhrect. Conse(iuently there is consi(hMal)le doubt that the target theory, without modification, may Ix' Nalidly ajjplied to interpretation of those bac- tericidal phenomena in which indirect actions aic known to be involved. Further discussion is gix en in a later section. FACTORS INFLUENCING SENSITIVITY TO IONIZING RADIATIONS Few comparisons have been made of the X-ray sensitivity of spores and the parent vegetative forms. In general, spores have been found to l^e more resistant (Green, 1904; Chambers and Russ, 1912; Baker, 1935; Lea et al., 193G, 1941). The greater resistance to radiation of spores may be partially due to the lower water content (Stapleton and Hollaender, 1952). Of equal interest is the generality of the inverse relation of sensi- tivity of spores and vegetative cells to radiations of different ionization densities (Table 10-1). The relative sensitivity of E. coli B/r cells to 250-kvp X rays at different stages of the growth cycle has been studied by Stapleton (1952), as dis- cussed earlier. There is a sharp decrease in sensitivity during the lag phase, followed by a marked increase in sensitivity during the logarithmic phase. The maximum sensitivity is reached at the end of the logarithmic phase, and as the stationary phase progresses, the sensitivity gradually declines to the initial level. Table 10-2. Mean Lethal Doses" of Escherichia coli Strains Irradiated DURixr, the Stationary Phase in Air (Incubatkd at 37°C and Irradiated with 250-kvp X Rays) (PVoin Stapleton, pensonal (.'ommunication.) Strain .MLD, (r) X 10' B 3.5 B/r 6.2 Tennessee 6.0 86G 6.2 Gratia 6.5 n-52 6.5 • Crook 8.0 Texas 10.0 "• MLD, 37 per cent survivaL The relation of genetic constitution to radiation resistance wall be dis- cussed later. That strains within a species maj^ vary widely in sensitivity to radiation is showai in Table 10-2, in which the MLD \alues for eight E. coli strains exposed to 250-kvp X rays are showai (Stapleton, personal EFFECTS OF RADIATION ON BACTERIA 375 communication). Although strain B/r shows about the median resist- ance in this group, strain B is significantly more sensitive than any of the others. Relation of Oxygen Concentration to X-ray Effects. The current interest in the relation of oxygen tension to the effects of X rays on living cells stems largely from the work of Thoday and Read (1947j, although earlier investigators had made similar ol)servations. The relation of oxygen concentration to cytogenetic effects is discussed by Giles (Chap. 10, volume I of this series) along with possible interpretations to be placed upon the observations. A similar relation between oxygen concentration and bactericidal effec- tiveness of X rays has been observed for E. coli B/r by Hollaender and coworkers (Hollaender, Stapleton, and Martin, 1951; Hollaender, Baker, and Anderson, 1951: Hollaender, Stapleton, and Burnett, 1951; Hol- laender and Stapleton, 1953). This same group has obtained interesting results on the closely allied problem of chemical protection against X rays. Except when studying temperature effects, all irradiations were per- formed at 2°C with washed cells suspended in M/lb phosphate buffer. Reduction of oxygen tension was accomplished by partial evacuation fol- lowed by saturation with nitrogen, helium, hydrogen, or carbon dioxide. The particular gas used to replace oxygen was of no importance. Figures 10-3 and 4 from Hollaender, Stapleton, and Martin (1951) show that lowering the oxygen tension changes not only the slope of the sur- vival curve but also the shape, although in a later publication (Burnett et al., 1951) exponential survival curves are shown for both oxygen-satu- rated and nitrogen-saturated suspensions as well as for suspensions con- taining 0.04 M concentrations of sodium hydrosulfite (Na2S204), British anti-Lewisite (BAL), and ethanol. Equally apparent in Figs. 10-3 and 4 is the lower sensitivity of cells grown in glucose broth and of cells grown anaerobically, in contrast to the sensitivity of aerobically grown cells. Although the ultimate slope of the nitrogen-saturation survival curves is less steep than the slope of those for oxygen saturation, cells grown in glucose broth or nutrient broth, either aerobically or anaerobically, exhibit parallel survival curves once the threshold dosage is exceeded. The threshold dose is a function of the method of culturing. Hollaender, Stapleton, and Martin (1951) observed that cells irradiated in oxygen-saturated suspensions were more sensitive at 2°C, whereas cells irradiated in the absence of oxygen were more sensitive at 37°C. As dis- cussed earlier, Hercik (1934a) and Lea et al. (1936) failed to detect an effect of temperature. The apparent disagreement is probably attribut- able to the solubility of oxygen at different temperatures and to differ- ences in the techni(iue of irradiation, since the latter workers exposed the cells either on the surface of dried agar plates or in dried films of gelatin. Hollaender, Stapleton, and Martin (1951 ) further observed that the cells 376 UAIMATIO.N lilOLOOY were more seiisiti\'e as their coiiceiitration in the irradiated suspeiision.s was rechiced and that nutrient broth and certain amino acids afforded some protection. All these observations indicate that an indirect action of X rays is involved in bacterial inactivation, which may be similar to the indirect action of X rays on dilute suspensions of enzymes and viruses (for discussion, see liea, 1947). 40 60 80 X-RAY DOSE , kr 120 Fig. 10-3. Comparative sensitivity of aerobic and anaerobic cells irradiated in high and low oxygen tensions. I, aerobic broth cells irradiated in oxygen-saturated buffer; II, aerobic broth cells irradiated in nitrogen-saturated buffer; III, anaerobic glucose cells irradiated in oxygen-saturated buffer; IV, anaerobic glucose cells irradiated in nitrogen-saturated buffer. {HoUaender, Stapleton, and Martin, 1951.) Current experiments (Stapleton, personal communication) indicate that since the cell suspensions in the earher studies were prepared at room temperature and were not chilled until just before irradiation, the appar- ent decrease in sensitivity with increased bacterial concentration may be attributed to the greater decrease in oxygen concentration resulting from endogenous oxygen utilization by the cells in the more concentrated suspensions. The existence of a critical oi- threshold concentration of oxygen l)elow which survival is greatly increased is indicated by Fig. 10-5 (from Morse, Burke, and Burnett — see HoUaender and Stai)leton, EFFECTS OF RADIATION ON HACTKRI A 37' 1953) which nicely shows the relation between dissolved oxygen at the time of irradiation and survival of X-irradiated E. coli B/r cells. Chemical Protection against X rays. Some of the more salient findings in the studies on chemical j)rotecti()n against X rays are illustrated in Table 10-3, adapted from HoUaender and Stapleton (1953), and in the 20 30 40 X-RAY DOSE, kr Fig. 10-4. Effect of cultural conditions on X-ray sensitivity of cells irradiated in oxygen-saturated buffer. I, aerobic broth cells; II, anaerobic broth cells; III, aerobic glucose cells; IV, anaerobic glucose cells. (HoUaender, Stapleton, and Martin, 1951.) following crude classification of compounds so far determined to possess a protective effect: 1. Sulfhydryl compounds: viz., cysteine, mercaptosuccinate, 2,3-di- mercaptopropanol (BAL), and 2-(2-mercaptoethoxy)-ethanol. 2. Sodium hydrosulfite (Na2S204). 3. Alcohols and glycols: viz., methanol, ethanol, isopropanol, pro- panediol, glycerol, triethylene glycol, and propylene glycol. 4. Metabolic intermediates and products: viz., formate, succinate, pyruvate, fumarate, lactate, and malate. In general, a preincubation at 37°C is required for maximum protective effect for the metabolic intermediates and alcohols in low concentration 378 hadiation hiolocjy l)Ut not tor tlic suiriiydiyl compoimds and sodium li_\-di()sid(il('. Sui\i\al ratios lip to .')()() times tliosc of the control aic ohscrxcd witli the optimal concentrations of the various compounds. liAL is rou}i,ldy twice as effi- cient as eysteini', wliicli niny l)e tlic result of its possessing two sulfhydi-yl groups or of the hj^droxyl group also present. Increasing the concentra- io--t o < a: o > 10— > q: lo-i I 10 Mg OF OXYGEN /LITER OF SUSPENSION BUFFER 100 Fig. 10-5. l"]ffec't of oxygen concentruliou on the survival of bacteria exposed to 80,000 V of 250-kvp X rays. (Burnett and Burke, unpublished; see Hollaender and Stapleton, li)53.) Table 10-3. Comparison of the Protective Action of Various Groups OF Compounds on Escherichia coli B/r (From Hollaender and Stapleton, 1952.) Lower Upper Protec- Incuba- Class limiting concen- limiting concen- tion fac- tor with tion condi- tration" tration'' agenf tion Sulfhydryl compounds (cysteine and BAL) ().<)()25 0.04 3.2-4.0 Without Inorganic sulfur compounds (sodium hydro- sulfit*;) 0. 00001 0.02 4.0 Without Alcohols and glycols (ethanol) 0 . 005 0.8 2.5-3 0 With 0.05 3.5 Without Carl)()X3'li(' acids (sodium formate) 0. 00005 0.001 3.0 With " Ttie concentration of the compound giving a significant increase in survival. * The lowest concentration required to give the highest survival achieved under indicated conditions. "^ The protection factor is the ratio of X-ray dose required to inactivate a given fraction of cells in the presence of a protective agent to that required in its absence. EFFECTS OF RADIATION ON BACTERIA 379 tions of the various compounds results in an increasing protective effect up to a certain level, after which the curves reach a plateau. Tests for additivity show that compounds within a group are com- pletely additive as long as the total concentration is below the plateau level. There is no increase in protection when increasing amounts of cysteine, for example, are added to optimal or plateau concentrations of BAL. In contrast to this, combinations between groups of compounds do give additive protection when combined at plateau level. Thus all combinations of alcohol, BAL, and sodium hydrosulfite by pairs give additive protection, but combinations of all three give no further increase in protection. This may indicate that the residual killing is the direct effect of the X rays on the cells. Stapleton, Billen, and Hollaender (1952) have studied the mechanism of the protective action of those compounds which reciuire preincubation for maximum protection. The necessity of preincubation suggests an enzymatic mechanism, and the evidence indicates that only those com- pounds utilized as oxidative substrates are effective. This was further indicated by tests in which inhibition of respiration was shown to cause a corresponding decrease in protection. In general, respiratory inhibitors had no influence on the degree of protection afforded by sulfhydryl com- pounds and sodium hydrosulfite although the protective efficacy of cys- teine was reduced. Varying degrees of respiratory inhibition with cyanide were accompanied by a corresponding decrease in protection b}^ succinate, suggesting a critical intracellular oxygen concentration. This is also suggested by the sharp increase in protection afforded by an increase in sodium hydrosulfite concentration from about 2 X 10~^ M to 8 X 10~^ M, there being no protection at concentrations lower than 2 X 10~^ M. Studies of cells possessing an active hydrogenase system indicated that neither hydrogen donation nor production of a highly reduced state within the cell is a major mechanism in chemical protection. The respiration studies are compatible with the hypothesis that either enzymatic or nonenzymatic removal of oxygen from around or within the cells is a major mode of protection. Various hypotheses to explain the mode of action of the different pro- tective substances have been advanced by these workers. In general, these hypotheses involve either a reduction in the yield of toxic radio- decomposition products due to the removal of oxygen from the cellular environment or a competition for highly reactive radiodecomposition products by the protective compound. Some similar observations have been reported by Thompson ei al. (1951). They found that the presence of 0.5 per cent pyruvate during irradiation partially protected the bacteria from the lethal and mutagenic effects of X rays and extreme ultraviolet. Addition of pyru\ate after irradiation had no effect. Wyss (1951) reported that X or extreme ultra- 380 RADIATION BIOLOGY xiolet inucliation of hydroj^cii-saturated sii.spoii.sions of Azolohaclrr colls, contaiiiiiig an actixe hydrojicnase, results in less killing and fewer muta- tions than irradiation of nitrof^en- or methane-sat uraled suspensiru\ate may exert its elTeet by reacting with hydrogen peroxide and tiiat the hydrogenase may permit the organisms to destroy oxidizing radicals and peroxide formed by the radiation. Although speculation al)out the mechanism of the protective elfect of X rays on bacteria may be unprofitable since so little is known concerning this phenomenon, there are a few considerations which may be worth pointing out. First, it seems clear, from the influence of concentration of bacterial cells in li(}uid suspensions on the inactivation rate and the pro- tective effect exerted bj' the constituents of nutrient broth and other compounds, that at least part of the inactivation of bacteria by X rays is by an indirect mechanism. Such indirect action of X rays has been shown for bacteriophages by Luria and Exner (1941), for tobacco mosaic virus by Lea et al. (1944), and for rabbit papilloma virus by Friedenwald and Anderson (1941). Latarjet and Ephrati (1948) studied the protective effect for bacteriophages of certain amino acids and physiological reducing compounds. In discussing the indirect effects on virus inactivation, Lea (1947) showed that the inactivation dose (37 per cent survival) increases as the nonvirus protein content of the irradiated suspension increases until, at sufficiently high concentrations of nonvirus protein, the inactivation dose essentially ecjuals that for the presumed direct effect as determined by irradiation of dried purified virus protein. He attributed the influence of nonvirus protein to the competition for the active decomposition products formed in water by the X rays. Lea showed that exponential survival curves are obtained by either the direct or indirect action of X rays. It therefore seems logical to assume that decrease of the oxygen ten- sion or addition of a protective compound is influencing the indirect bac- tericidal effects of X rays and that the residual killing in the absence of oxygen may result from a direct effect on vital elements of the bacterial cell. Certain complications in the kinetics observed arise on this assump- tion. Thus, if the presumed direct effect is trul}' sigmoidal as shown in Fig. 10-3, then the indirect bactericidal effects, when plotted semilogarith- mically, must show a very steep negative slope initially which increases as the direct-effect slope decreases and becomes constant w^hen the direct- effect curve becomes semilogarithmically linear. It is difficult to explain such a curve for the indirect effects. If, however, the residual or direct survival curve is exponential as shown l)y Burnett ct al. (1951), no diffi- culty arises. In this connection, it should be pointed out that Lea rial. EFFECTS OF RADIATION ON BACTERIA 381 (1936) observed exponential s\ii\iAal curves when E. coli cells were exposed to /3 and a particles in dried gelatin films, in which any indi- rect effects involving radiodecomposition products of water would be minimized. To attempt any explanation of the observed results, one can only turn to the knowledge of the radiodecomposition of pure water. This extrapo- lation is questionable since it is known that slight impurities in the water have a profound influence on the results obtained. Hence, greatly dif- ferent phenomena may occur in the complex chemical milieu within the bacterial cell. However, to attempt any explanation encompassing these considerations seems hopeless in the present state of knowledge (for a brief informative discussion, see Burton, 1951). The significant observation would seem to be the large effect of oxygen concentration. Since, under irradiation, oxygen is known to be reduced to peroxide in two steps — as shown in the following equation (Allen, 1948) O2 + H = HO2 HO2 + H = H0O2, the chief effect of reducing the oxygen concentration would be to reduce the concentration of peroxide and HO2 radicals formed. It would seem therefore that the indirect bactericidal effects of X rays are mediated through either peroxide, HO2 radicals, or other unknown radiation prod- ucts, possibly organic peroxides, depending on oxygen for their formation. It seems equally probable that H atoms and OH radicals are not effective in the indirect action since they are formed independently of oxygen, and, indeed, the presence of oxygen would reduce the number of such radicals free to react with the protoplasmic components involved in the indirect mechanism. It has been shown that varying the concentration of oxygen has little effect on the frequency of chromosome aberrations produced in Trades- cantia by a particles (Giles, Chap. 10, volume I of this series). No similar studies have yet been made with bacteria. This observation, however, is not incompatible with the hypothesis that HO 2 and/or peroxide are the biologically active w^ater decomposition products, since it is knowai that detectable amounts of peroxide are formed by irradiation of oxygen-free water wdth a particles and negligible quantities are produced by X irradi- ation. Allen (1948) has shown a general relation between the steady- state hydrogen pressure and hydrogen peroxide concentrations produced in pure water by radiations of different ionization densities, higher concen- trations being observed as the ionization density increases. In summary, the indications are that the indirect effects of X rays are mediated in part by either HO2 radicals and/or hydrogen peroxide and that the protective compounds exert their effect either by reducing the oxygen concentration within or immediately surrounding the cells or by 382 radiation: hiolooy coinpct iii^ t'oi- llic a('ti\"c products produced l)y radiation. In addition, llollacnder and coworkcns liiivc suggested that certain comi^ounds may give protection l)y supplying a metabolic intermediate which has been blocked temporarily by the irradition effect. This possibility would seem to l)e subject to direct test sinrc, in this case, supplying the particular compound after the irradiation should bi' effective. 10- I0--2- i io--iJ o 4 K b. O Z > > ^ io--± I0=i4 10- 18 -I— 30 ~~1 42 TEMPERATURE, 'C Fig. 10-6. Survival of E. coU B/r at several X-ray doses as a function of ineubation temperature. Solid circles, 40,000 r; open circles, 60,000 r; triangles, 80,000 r. {Stapleton, Billen, and Hollaender, 1953.) Incubation at Suboptimal Temperatures. Latarjet (1943) has reported a partial recovery of yeast cells subjected to X rays of wave length 1.54 A when the irradiated cells were held for varying numbers of days at 5°C. No such recovery was observed with bacterial cells similarly tested. Ultraviolet irradiation with 2537 A wave length was followed by a similar recovery of the yeast cells. Stapleton et al. (1953) have observed more than a hundredfold recovery of E. coli cells subjected to various doses of X rays when incubated after irradiation at sul^optimal temperatures for 24 hours before incubation at 37°C. Figure 10-6 shows typical results. EFFECTS OF RADIATION OX BACTERIA 383 The optimum temperature for E. coli B/r was 18°C, and for the Texas and Crook strains, the optimum temperatures were 26° and r2°C, respectively. The rate of the recovery has been found to be exponential when plotted against time with the length of the exponential phase varying greatly at the different temperatures. An exponential survival curve is found when the surviving fraction following maximal recovery is plotted against X-ray dose, the slope being less steep than that of the curve for the control cells. Studies of the lag phase before cell division and of the rate of recovery sug- gest that the recovery process is terminated by cell division. Appreciable recovery does not occur in buffered inorganic salt solutions or in a glucose synthetic medium which supports growth of the cells but does occur in nutrient broth or yeast extract solutions. This eliminates the theory of simple decay of a toxic product resulting from irradiation and indicates that a metabolic process is involved in the recovery. This process could involve either the enzymatic destruction of a toxic product or the synthesis at low temperature of compounds necessary to overcome the potential damage produced by the radiation. The correspondence of the division time with the time required for twofold recovery plus the failure to observe significant recovery in a synthetic medium with a utiliz- able energy source favor the latter hypothesis. Since it has been shown that irradiation initiates a series of reactions which ultimately are lethal to the cells, Stapleton and coworkers speculate that the optimal temperature of 18°C may be the optimum equilibrium between opposing processes, the recovery process and the unknown processes leading ultimately to inacti- vation, each with a high temperature coefficient. These significant obser- vations form the first well-substantiated case of recovery of X-irradiated bacterial cells. The important cjuestion of the effect of similar recovery on induced mutations is now under investigation. ULTRAVIOLET RADL\TION Duggar (1936), Ellis et al. (1941), and Loofbourow (1948) have so ade- quately review^ed the early development of the knowledge of ultraviolet effects on bacteria that it is unnecessary to do so here. Few of the experi- ments in the fifty years following the original observations of Downes and Blunt (1877) yielded information of a quantitative nature. In addition to the rather qualitative bacteriological methods of demonstrating the bactericidal properties of ultraviolet radiation, the use of nonmonochro- matic light sources, the failure to measure intensities, the failure to correct adequately or to control absorption of the incident energy in the sus- pending medium, and the lack of information concerning the absorption of ultraviolet radiation by bacterial protoplasm were the main factors responsible for the lack of accurate ciuantitative data. However, research during this period had adequately shown that all bacterial species subjected to ultraN'iolet radiation of appi'opriate wave lengths were inacti- 384 UADIATION IJIOLUUV vatetl and that wave k'lijjjtlis i)el()\v alxjut 31)50 A were hactericidally effec- tive. Numerous workers had shown that the bactericidal eifectiveness increased greatly for those wave lengths helow about 29()7 A and extended to the siiortest wave lengtlis convenienlly studied. Furthermore, several early investigators had suggested that absorption of ultraviolet radiation by specific structures within I he ceil was responsible for the bactericidal effects. For example, Henri (1914) emphasized absorption within the nucleus as being primarily responsible for the inactivation and pointed out the possibility, later realized in experiment, that sublethal doses might induce hci'ilable modifications. I- 8- V 1^ E ACTION SPECTRA In 1928 Gates made the preliminary aiuiouiiccmcnt of the action spec- trum or relative bactericidal effectiveness of diffei-cnt wave lengths of monochromatic ultraviolet radiation and pointed out the probable rela- tion to the absorption of ultraviolet by deoxyribonucleic acid (DNA) derivatives. His detailed results were presented in a later series of ./• 'X publications (Gates, 1929a, b, 1930). Cells of S. aureus were irradiated on the surface of agar plates with beams of monochromatic ultraviolet radia- tion isolated by means of a large monochromator with quartz prisms, the incident energy being measured by means of a calibrated thermopile. The studies were later extended to E. coll (Gates, 1930), and measure- ments were made of the absorption coefficients of a thin layer of bac- terial cells pressed between quartz cover slips. For both species the curves of the reciprocal of the inci- dent energy required for 50 per cent killing plotted against wave length were similar to the absorption spec- tra with a maximum of bactericidal I 8 6- O 4 0 23 0 25 0 27 WAVE LENGTHS,/! — I — 029 0 31 Fui. 10-7. Curve of the reciprocals of the incident energies required for inacti- vation of 50 per cent of E. coli. (Adapted from Galen, 19:i0.) effectiveness and absorption of about 2000 A and a minimum at about 2380 A with indications of another maximum at wave lengths shorter than 2300 A. The action spectrum for E. coli is reproduced in Fig. 10-7. Similar bactericidal action spectra have been observed by other workers (Ehrismann and Noethling, 1932; Wyckoff, 1932; Duggar and HoUaender. 1934a, b; Ilollaender and Glaus, 1936; HoUaender and Duggar, 193(); EFFECTS OF RADIATION ON BACTERIA 38.) Luckiesh, 1946). Ehrismann and Noethling (1932) report a maximum of sensitivity at 2650 A for B. pijocyaneus, Micrococrus candicans, S. aureus, a vibrio, and one species of yeast. For E. coli they report a maximum sensitivity at 2510 A and for Serratia marcescens at 2804 A. Duggar and Hollaender (1934a, h) found a maximum of sensitivity at 2650 A for S. marcescens, and Wyckoff (1932), Hollaender and Claus (1936), and Hol- laender and Duggar (1936) found the maximum sensitivity of E. coh to be at 2650 A. An improvement in technique was developed by Hollaender and Claus (1936) who studied the inactivation of E. coli cells suspended in a non- absorbing physiological salt solution. This was a modification of the technique used by Duggar and Hollaender (1934a, b). The concentration of bacteria in the suspensions was so great that, except for the small por- tion of light scattered back into the beam, all the energy incident on the suspension was absorbed. This method reduces the amount of nonspe- cific absorption which may occur when cells, seeded on the surface of agar plates, are irradiated with ultraviolet. Furthermore, the energy absorbed per bacterium can be calculated directly without the additional source of error involved in estimating absorption coefficients. The bacteria were grown on agar slants and were either suspended in saUne solution and irradiated or else washed one or more times before irradiation. Washed suspensions gave a lower MLD than unwashed suspensions, indicating either that washing made the cells more sensitive to radiation or that there was a considerable degree of nonspecific absorption by metabohc products and nutrients in the unwashed suspensions, or both. The action spectra obtained by Hollaender and Claus (1936) showed a sharp maximum at 2650 A. The evidence is clear that there is a maximum of bactericidal effective- ness at 2650 A suggesting that absorption of ultraviolet by nucleic acids or by nucleic acid components is the first step in the reactions resulting ultimately in death of the cell. Loofbourow (1948) has given the theoretical basis underlying the action-spectrum technique. His analysis showed that the following assumptions are inherent in the action-spectrum method : 1. The biological effect observed is, on the average, attributable to photochemical change in a given number of molecules of an essential substance. 2. The quantum efficiency of the photochemical process is independent of wave length within the region studied. 3. The attenuation of the intensity of radiation before reaching the sensitive substance is either independent of wave lengths or so small in magnitude as to be ignored. 4. The relative absorption of suspected sensitive substances hi the cell as a function of wave length either can be estimated with sufficient accu- 38() ltAi>IAT10.\ l5lOL(){JV racy or can l)«' asisunicd to he o(juivalent to the relative extinction eocffi- eients of tlie su.sp(>('t(>cl substances. 5. Th(^ reciprocity law is xalid i'oi' t lie I inics iMuploycd in t lu'cxpcriincnt . Since in many cases the \ali(lity ol all lliese assumptions is difficult or impossible to demonstrate, the action-spectrum techni(|ue can be sug- gestive, l)ut the results must be interpreted with caution. Nevertheless, much useful information has been gained by this technicjue. Giese (1945) has summarized the data on action spectra of various photobiological effects and has listed seven general types. The observation of the maxi- mum efficiency of bactericidal effects at ^2600 A early focused attention on the purine and pyrimidine constituents of nucleic acids. SHAPE AND SIGNIFICANCE OF SURVIVAL CURVES Less uniformity in the form of survival curves has been observed with ultraviolet than with ionizing radiations. Coblentz and Fulton (1924), working with thickly seeded plates of E. coll, observed distinctly sigmoidal survival curves with a long threshold exposure before any bactericidal effects were noticed. In some cases the threshold exposure was nearly half the exposure recjuired to kill 90 per cent of the organisms. It is doubtful if the bacteriological techniques employed by these investigators were sensitive enough to detect small amounts of inactivation. Similarly, Gates (1929a, b, 1930) observed sig- moidal survival curves which he interpreted to indicate differences in the sensitivity of individual cells. He computed the extremely skewed distri- bution necessary to account for the observed curves on this hypothesis. Baker and Xanavutty (1929) observed survival curves with increas- ingly steeper slopes when plotted semilogarithmically. They postulated a cumulative action of a toxic substance produced by the ultraviolet radiation as the mechanism. Wyckoff" (1932) obtained exponential survival curves when E. coli cells were irradiated on the surface of agar plates with monochromatic ultra- violet. Extending the analysis applied to his results Avith ionizing radia- tion, Wyckoff suggested that the exponential curve indicated that the absorption of a single quantum in a vital structure was sufficient to kill the cell, but calculations using absorption data of Gates (1930) showed only one in about 4 million quanta absorbed by the cell was effective. In general, the quantal efficiency of ultraviolet killing of bacteria is very low. HoUaender and Glaus (1936) observed some deviations from exponen- tial killing but, if their data were corrected for the known proportion of double cells determined by microscopic examination, the observed sur- vival could be fitted best by an exponential curve, and their theoretical analysis was based on such a curve. Hercik (1937) observed exponential killing with monochromatic ultraviolet irradiation of Bacillus megatherium spores and vegetative cells. EFFECTS OF RADIATION ON BACTERIA 387 Sharp (1939) presents survival curves for ten species irradiated on agar surfaces with 2537 A ultraviolet. Although much scatter is evident in the points plotted, in seven of the species there is no systematic deviation from an exponential. The three exceptions were S. aureus, S. albus, and B. anthracis, and clumps or chains of cells could be at least partially responsible. Lea and Haines (1940), in very careful studies, observed exponential survival curves for S. marcescens, E. coli, and B. mesentcricus spores when aqueous suspensions were exposed to 2537 A ultraviolet. Witkin (1946, 1947) observed exponential survival curves with E. coli B when exposed to 2537 A ultraviolet but sigmoidal curves with strain B/r. Numerous other workers have made similar observations with these strains. Hence in this case, a bona fide sigmoidal survival curve has been observed since the same bacteriological and physical techniques yield an exponential curve for strain B. Both strains B and B/r are inactivated exponentially by X rays. The explanation for these results is as yet luiknown. In conclusion, it appears that, as with high-energy radiation, exponen- tial or sigmoidal survival curves may be obtained following ultraviolet irradiation, depending on the strain of bacteria and the technique of irra- diation including precautions against clumping in the preparation of the bacteria to be irradiated. It should be remembered that it is difficult to determine the precise shape of the killing curve since the low dose range where killing is slight is the most important (see Fano, Chap. 1, volume I of this series). A number of workers, e.g.. Gates (1929a) and Rentschler et al. (1941), have attempted to account for the observed survival curves on the basis of variable resistance among the cells making up the population. Others, notably Wyckoff (1932), HoUaender and Claus (1936), and Lea and Haines (1940), interpret the exponential survival curves as indicating that a single quantum is sufficient to kill, sigmoidal survival curves from this point of view being accounted for by the multihit or multitarget theory. Most investigators favor the Cfuantum-hit interpretation since the expo- nential distribution of resistance necessary to explain cases of exponential survival curves is highly improbable. EFFECT OF INTENSITY The Bunsen-Roscoe reciprocity law states that the effect of exposure to radiation is a function of the total energy and is independent of intensity and time. Loofbourow (1948) points out that the reciprocity law is meaningful for photobiological phenomena only when it is restricted to periods of time so short that other kinetic and metabolic activities do not influence the reaction. Numerous workers have tested the applical)ility of this law with some- 388 RADIATION BIOLOGY what conHictinf^ results. Cohlentz and Fulton (1924) concluded that the law did not apply, since they observed that a reduction in intensity to one- liftieth required an increase in exposure of seventy-five- to eightyfold to obtain the same killing elYect. These same workers, however, observed no (lirtcrcncc in inactivation when the same total energy was given in one contiinu)Us dose or in as many as sixteen intermittent doses with varying int(M-vals between. (Jates (19291)) studied the elTect on an approximately fourfold variation in intensity and observed small differences in survival at the two intensities. The difference in survival at the two intensities diminished as the survival ratio approached zero. Lea and Haines (1940) observed no intensity effect on the inactivation of B. mesentericus spores and A', coll when the intensity was varied a hundredfold. Roller (1939), in his studies of th(> lethal effects on air-borne bacteria, observed reci- procity when the intensity of 2537 A ultraviolet radiation was varied about fifteen hundredfold. The most extreme variations in intensity were those employed by Rentschler ct al. (1941 ) w^ho gave the same dose of 2537 A ultraviolet in periods of time varjdng from a few microseconds to several minutes. No effect in intensity was observed as long as the total length of exposure w^as small relative to the generation time of the bac- teria. Therefore it appears that the reciprocity law holds approximately for the inactivation of bacteria by ultraviolet radiation if all other factors are held constant. For very low intensities given over long periods of time, the deviations from reciprocity are greater. Since the energy emitted in the various lines of the mercury-arc spectrum varies widely, it is difficult to avoid differences in intensity of the various wave lengths in action-spectrum studies. However, it would appear that the variation in intensity could have only a very minor influence on the results obtained at the different wave lengths. RELATION OF TEMPERATURE The early somewhat conflicting results of studies concerned with the relation of temperature to the bactericidal efficiency of ultraviolet have been discussed by Duggar (193()). Bayne-Jones and Van der Lingen (1923) observed temperature coefficients {Q^n) of 1.06 and 1.04 for the temperature ranges 2°-12°C and 30°-40°C, respectively. Gates (1929b) similarly observed a small temperature coefficient of about 1.1. Exposure at 5° and 37°C had no effect in the studies of Rentschler et al. (1941). Heinmets and Taylor (1951) have studied the effect of temperatures as low as — 50°C. No pronounced influence of temperature is apparent until -35°C when survi\al begins to dei^rease rapidly for a given dose. The low temperature coefficients ol)served agree well with those expected on the hypothesis that the bactericidal effects of ultraviolet result from a primary, simple photochemical reaction. EFFECTS OF RADIATION ON BACTERIA 389 RELATIVE SENSITIVITY OF VARIOUS SPECIES Comparison of the sensitivity of different species in absolute energy- units must be made with caution since there are many factors which can influence the results obtained by different investigators in different labo- ratories. In Table 10-4 are shown the incident energies of 2537 A ultra- violet in ergs per square centimeter necessary to inhibit colony formation in 90 per cent of the organisms. This table is taken from Hollaender (1942) and includes some estimates of the sensitivity of E. coli B and B/r made from the data of Demerec and Latarjet (1946) and Witkin (1947). These estimates show a striking difference between the two strains of bacteria, one of which is a radiation-resistant mutant of the other. It is of interest that E. coli B is the most sensitive strain for which data are available. FACTORS INFLUENCING SENSITIVITY pH. Bay ne- Jones and Van der Lingen (1923) and Gates (1929b) \'aried the pH of the medium on which the organisms were irradiated between 4.5 and 9. No appreciable influence of pH on the bactericidal effect was observed although the former workers observed more rapid inactivation below pH 4.6. Stage of Growth. Relatively few investigations have been concerned with the comparative sensitivity of bacteria to ultraviolet at different stages in the growth cycle. Morse and Carter (1949) and Morse (1950) have shown about threefold variation in the DNA content of cells of E. coli B and B/r at different stages of the growth cycle. In view of the maximum at 2600 A in the bactericidal action spectra, it would be sur- prising if the sensiti\'ity of bacteria remained constant during the growth cycle. Hollaender and Claus (1936) found 7-hr agar slant cultures of E. coli to be more resistant to ultraviolet than their standard 15-hr cul- tures, whereas 10-day-old cultures were more sensitive. Microscopic examination of the 7-hr cultures revealed that 70 per cent of the cells were double, which may account for a large part of the difference in resistance observed. Demerec and Latarjet (1946) observed that growing cells of E. coli B/r were more sensitive to 2537 A ultraviolet than were resting cells. Witkin (1951) reported greatest sensitivity to ultraviolet in the loga- rithmic phase, greater resistance in the resting stage, and the greatest resistance during the lag phase. These differences in resistance did not parallel differences observed in the mean number of nuclei per cell. Relative Sensitivity of Vegetative Cells and Spores. Few investigators have compared the sensitivity of spores with that of vegetative cells of the same strain. Duggar and Hollaender (1934b) observed B. subtilis spores to be about twice as resistant as the vegetative cells. This was confirmed by Hercik (1937) with B. megatherium and l)y Rentschler et al. (1941) with B. .mbtilis (Table 10-4). 31 K) RADIATION BIOLOGY Tablk lO-l. Incident Energies at 2537 A Necessary to Inhibit Colony Formation in 90 per cent of the Organisms Organisin lliicrgy (ergs/cni2) X 10* Reference Bacillus (inlhrdcis 452 Sharp (1939) Bacillus mcijathviiuin sp.: Vegetative cells IV.i Hercik (1937) Spores 273 Bacillus subtilis: MLxcd 710 Rentschler <-< cil. (1941) 600 Keller (1939) Spores 1200 Rentschler et al. (1941) Corynebacterium diphtherine 337 Sharp (1939) Eberthclla tijphosa 214 Micrococcus candidus (505 Ehrismann and Noethling (1932) Aficrococcus piltoriensis 810 1000 Rentschler ct al. (1941) Micrococcus sphaeroides Neisseria catarrhalis 440 440 Pht/tomonas tumefaciens Proteus inilgaris 264 Pseudonionas aeruainosa 550 Ehrismann and Xoethliug (1932) Pseudomonas fluorescens 350 Sarcina lutea 1970 242 Rentschler et al. (1941) Serratia marcescens 220 Sharp (1939) 83 Ehrismann and Noetliling (1932) Shigella paradysenteriae 168 Sharp (1939) Spirillum rubruni 440 Rentschler ct al. (1941) Staphylococcus albus 184 Sharp (1939) 330 Rentschler et al. (1941) 184 Staphylococcus aureus 218 Gates (1929a, 1930) . 200 Sharp (1939) 495 Ehrismann and Noethling (1932) Streptococcus heniolyticus 216 Sharp (1939) Streptococcus lactis 615 Rentschler et al. (1941) Streptococcus viridans 200 Sharp (1939) Escherichia coli 240« 550" Gates (1929a, 1930) Ehrismann and Noethling (1932) 640" Wyckoff (1932) 211'' Hollaender and Glaus (1936) 500" (est.) Roller (1939) 245" Sharp (1939) 250" Rentschler et al. (1941) Escherichia coli: Strain B 160,''« Strain B/r 1200/''= 4 4 " Agar surface. '' Liquid suspension. " Estimated from \\itkin (1947) and Dcmerec and Latarjet (1946). EFFPX'TS OF RADIATION ON BACTEUIA 391 Moisture Content. Somewhat conflicting results have been obtained by different investigators who studied the relation between sensitivity to ultraviolet and moisture content of the colls. Thus Roller (1939) and Wells (1940) reported that air-borne K. coli are more sensitive than the same organisms floating in liciuid suspension. Rentschler and Nagy (1940) reported the same sensitivity for air-borne bacteria and for bacteria exposed on the surface of agar plates. Wells and Wells (1936) and KoUer (1939) observed that air-borne bacteria were more resistant to ultraviolet irradiation at high than at low relative humidities. On the other hand, Rentschler and Nagy (1940) found no difference in sensitivity of air-borne bacteria at different relative humidities. A direct relation between X-ray sensitivity of spores of A. terreus and their relative water content was observed by Stapleton and Hollaender (1952). Spores containing approx- imate!}'^ 25, 42, and 80 per cent moisture had relative sensitivities of 1 : 1.7:2.4, respectively. Enzymatic Constitution. The enzymatic constitution of bacterial cells is known to depend on the conditions of growth. Thus the enzyme sys- tems of resting bacteria differ from those of growing cells. Similarly, resting cells grown in broth, glucose broth, or synthetic medium differ in enzymatic constitution. Roberts and Aldous (1949) have shown corre- sponding variation in the ultraviolet sensitivity of cells of E. coli B. Resting cells grown in broth (final pH 8) were much less sensitive than resting cells grown in glucose broth (final pH 5.5) or bacteria grown in broth with a final pH of 7.0. Two-hour cultures of E. coli B grown in broth and in synthetic medium were more sensitive than cultures of resting cells grown in broth. Not only did the sensitivity change, but the form of the survival curve changed with different growth conditions. In all cases, greater survival was observed when the irradiated cells v.-ere assayed on synthetic agar plates than when assayed on nutrient agar plates. Strain B/r, the radiation-resistant mutant of strain B, did not yield similar results. Genetic Constitution. Several references have already been made to E. coli B/r isolated by Witkin (1946, 1947). This radiation-resistant mutant was first isolated as one of four surviving colonies from a sample of strain B, which had survived a dose of 1000 ergs/mm- of 2537 A ultraviolet radiation. The mutant strain, which shows greatly increased resistance to ultraviolet and ionizing radiations, has remained stable throughout numerous transfers and has been widely used by many investigators in radiobiological investigations. Witkin found that cells of the parent strain, when subjected to low doses of ultraviolet and plated on agar, formed almost 100 per cent of long filaments. Strain B/r, on the other hand, after a normal lag period of about 1 hr, divided normally. Witkin utilized this observation in a clever double-irradiation technique which permits quantitative estimation of the numbers of resistant cells in 392 RADIATION HIOLOGY samples of strain B. Tlic mctluxl consists in j)lutiM{i; samples of the bac- teria on agar, irradiating with oO ergs/mm-, and incuhating at 37°C' for 3 hr. At the end of this time, all the surviving sensitive cells will have formed long filaments. The resistant cells, however, will have divided normally and formed a microcolony of 50 1 00 cells. The incubated plates are then given a of 700 ergs/mm'-. This exposure will leave fiom 10 to 20 resistant cells in each microcolony of strain U/v, whereas, if the fila- ments of sensitive cells behave as single bacteria rather than as chains of bacteria, all the sensitive bacteria will be killed by the second heavy irra- diation. This was found to We the case and corresponds to the observa- tion of Lea et al. (1937) that the filamentous forms were, like individual cells, killed by a single hit and w^ere equally sensitive to radiation. Uti- 300 600 900 1200 1500 1800 2 Ergs per mm^ Fig. 10-8. Sensitivity of E. coli B and B/r to ultraviolet radiation. Witkin, 1947.) (Adapted from lizing this quantitative double-irradiation technique, Witkin (1947) demonstrated the mutational origin of the resistant cells and estimated the mutation rate from radiation sensitivity to resistance to be about 10^^. By concurrently testing the resistance of the radiation-resistant mutants to penicillin and sodium sulfathiazole, at least four different types of radiation-resistant mutations were demonstrated. Bryson (1947, 1948) has extended Witkin's observations to include mustard and nitrogen mustard. Survival curves obtained with E. coli B and B/r exposed to 2537 A ultraviolet radiation are shown in Fig. 10-8. Although strain B is killed exponentially with a change in the slope of the curve at about 1 per cent survival, strain B/r follows a sigmoidal survival curve which, interpreted within the framework of the target theory, would indicate that the muta- tion to resistance causes a change from a single hit to multiple hits to be necessary for lethality. Both strains are killed exponentially by X rays. EFFECTS OF RADIATION ON BACTERIA 393 The physiological nature of these genetically determined differences resulting in radiation resistance are as yet unknown. Po stir radiation Treatment. Hollaender and Claus (1937) found that holding ultraviolet-irradiated cells in distilled water or physiological salt solution resulted in significant increase in survival. Reference has already been made to the work of Roberts and Aldous (1949) in which they discovered that \'arious postirradiation treatments produced as high as one-hundredfold increases in survival of E. coli B which had been exposed to 2537 A ultraviolet. For example, significantly greater survival was obtained when the irradiated cells were plated on syn- thetic agar plates than when plated on nutrient agar plates. Furthermore, holding the irradiated cells in fluid media resulted in striking increases in survival. This recovery did not depend to any great extent on the presence of specific factors in the hold- ing fluid; distilled water, saline, syn- thetic medium, synthetic medium without an energy source, and nutrient broth gave approximately the same results. Varying the pH from 5 to 9 or the concentration of bacteria from 10^ to 10^ per ml likewise had no effect on the extent of recovery. Factors which did influence the recovery in fluid media were the radiation dose and the temperature. As the radiation dose was increased, the rate and final level of recovery attained decreased. The rate of recovery was found to be directly correlated with temperature. When the logarithm of the survival ratio was plotted against dose of ultra- violet for varj^ing periods of recovery, significant changes in the slope and the shape of the survival curves were observed. In Fig. 10-9 are shown the various survival curves obtained. It will be observed that the survival curves change progres- sively from a concave through a straight-line condition to a convex form as the recovery approaches the maximum possible. Obviously, caution must be exercised in the interpretations placed on the shape of survi\-al curves. In discussing their results, Roberts and Aldous (1949) point out that very careful control of technitiues must be exercised in order to obtain 40 80 120 160 DOSE, relative units Fig. 10-9. Survival curves of E. coli B after 0-4.5 hours of recovery from 2537 A ultraviolet. {Adapted from Roberts and Aldous, 1949.) 394 RADIATION BIOLOGY reproducible results. They postulate a poison produced intracellularly by photochemical action which selectively inhibits the mechanism of cell division, one molecule bein^ elective. The recovery is explained by simple exponential decay of the poison. However, they emphasize that since no recovery is observed with strain H/r, a (lualitatively different mechanism must be assumed for this strain. Furthermore, since no recovery was observed in cither strain with X rays, they point out that the mechanism of X-ray action is qualitatively different from that of ultraviolet. Heat Eeactivation. Anderson (1949, 1951a) and Stein and Meutzner (1950) independently observed that incubating ultraviolet-irradiated cells of E. coli B at temperatures higher than 37°C results in greater survival. The magnitude of the increased surxival is of the same order as that observed when irradiated cells are exposed to visible light (Kelner, 1949b, see section on photoreactivation). The dose-reduction ratios (the ratio of the ultraviolet exposure of cells incubated at 40°C to that for cells incu- bated at 30°C' which produces the same level of survival) for strain B were found by Anderson (1951a) to be a decreasing function of the total dose of radiation, the decrease for heat reactivation being greater than the corre- sponding decrease for photoreactivation. E. coli B/r exhibited only a small heat reactivation, and neither strain show'ed appreciable heat reacti- vation following X irradiation. Among ten E. coli and seven yeast strains tested by Anderson only two E. coli strains were found capable of heat reactivation. It would appear therefore that heat reactivation is not a general phenomenon. Harm and Stein (1952) have shown that the large difference between strain B and its mutant strain B/r in ultraviolet resist- ance, when the irradiation cells are incubated at 37°C\ disappears when the cells are incubated at 44.5°C, equal survival being observed in both strains. Ultraviolet-irradiated cells of strain B remain fully heat reac- tivable for 1 hr at 37°C (Stein and Harm, 1952). If a longer period elapses before incubation at the reactivating temperature, the amount of reactivation decreases rapidly with no reactivation occurring after 3 hr. Photoreactivation. Among the more significant recent developments is the discovery of the phenomenon of photoreactivation. No exhaustive discussion will be attempted here since Dulbecco reviews the available data in Chap. 12 of this volume. Although Whitaker (1942) presented conclusive data showing that visible light partially counteracted the effects of ultraviolet radiation in Fucus eggs, the present interest in photoreactivation stems largely from ■work by Kelner (1949a, b). Dulbecco (1949) independently observed photoreactivation of ultraviolet-irradiated bacteriophage adsorbed on sensitive host cells. Kelner (1949a) showed that exposure to visil)le light subseriuent to exposure to uiti-aviolet radiation would result in as high as 300,000-fold recovery of Strcptomijces griseus conidia. He later (1949b) EFFECTS OF RADIATION ON BACTERIA 395 extended his observations to E. coli B/r, Pcnicillium notatum, and Saccha- romyces cerevisiae. By employing a standard photoreactivation treat- ment which gave maximum recovery, Kehier showed that the effect of photoreactivation was, essentially, to decrease the inactivation rate per unit dose of ultraviolet. Defining the dose-reduction ratio as the ratio of the ultraviolet dose followed by maximum photoreactivation to the ultra- violet dose with no photoreactivating light for the same inactivation, Kelner observed a constant dose-reduction ratio for E. coli of about 2.5, which was independent of the survival ratio of the ultraviolet-irradiated suspension. Similar conclusions were reached by Novick and Szilard (1949) who demonstrated that a simple linear relation existed between the survival curves of photoreactivated and nonphotoreactivated ultraviolet- irradiated bacteria. Johnson et al. (1950) found that photoreactivation of E. coli B following exposure to ultraviolet radiation was independent of the presence of oxygen and that vegetative cells of Bacillus cereus showed but little photoreactivation. The action spectra for photoreactivation of E. coli cells and S. griseus conidia were investigated by Kelner (1951). For reactivation of S. griseus conidia, the effective spectral region extended from 3650 to about 5000 A with the most effective wave lengths lying near 4360 A. For E. coli, however, the effective wave lengths extended from 3650 to 4700 A with the most active wave length lying near 3750 A. Kelner suggests the sharp peak for *S'. griseus conidia at 4360 A may indicate that porphyrins are involved in photoreactivation. Heinmets and Taylor (1951) have shown that cells inactivated by ultra- violet at temperatures as low as — 70°C can be photoreactivated when in the liquid state but not in the frozen state. They further showed that cells which have been inactivated by wave lengths of 3000-4000 A while in the frozen state do not exhibit photoreactivation when in the liquid state, suggesting a qualitatively different mechanism for inactivation by this spectral region. Photoreactivation seems to be a rather general phenomenon, having been reported for several species of bacteria and bacteriophage (Dulbecco, 1949, 1950), Paramecium aurelia (Kimball and Gaither, 1950), Amhlij- stoma larvae (Blum and Mathews, 1950), and gametes of the sea urchins, Arbacia punctulata (Marshak, 1949; Blum et al., 1950), and Strong ylocen- trotus purpuratus (Wells and Giese, 1950). Bawden and Kleczkowski (1952) observed similar visible-light-induced recovery of ultraviolet- irradiated tobacco necrosis virus inoculated onto French bean leaves and of tomato bushy stvmt \'irus inoculated onto Nicofiana glutinosa. No recovery was noted for tobacco mosaic virus inoculated onto A'', glutinosa. The same workers observed that visible light prevented the ultraviolet- induced necrosis of epidermal cells of Phaseoleus vulgaris leaves. Beckhorn (1952) and Kelner (1952) have shown that the extension of SOfi RADIATION HIOLOGY the liig pluuso follow iiif^ ultraviolet irradiation is photoreversible by visible light. Latarjet (1951) has shown that the induction of active bacterio- phage in lysogenic cultures l)y ultraviolet and also by X rays is similarly photoreversible. Except for Latarjet's observation (19ol), no similar visible-light- induced recovery from the effect of ionizing radiations has been reported, any recovery which may occur being so small in magnitude as to be diffi- cult to demonstrate. This would indicate again that quite different mechanisms are involved in damage by ultraviolet and by ionizing radia- tions. Furthermore, not all the ultraviolet effects can be reversed by visible light since, in every instance thus far studied, recovery has not been complete. This seems to indicate that more than one mechanism exists by which ultraviolet radiation produces lethal or other effects in cells. Other Types of Reactivation. Working with K. coli K12, Monod et at. (1949) observed that treatment of the irradiated cells with catalase increased the survival ratio following exposure to 2537 A ultraviolet. Latarjet and Caldas (1952) have studied catalase restoration in greater detail. Catalase restoration is enhanced by small doses of visible light. The greatest degree of catalase restoration has been observed with E. coli K12 and with B. megatherium 899, both of which are lysogenic. A non- lysogenic B. megatherium strain and E. coli B/r show only a slight degree of catalase restoration, whereas E. coli B shows none. Neither strains B nor B/r are known to be lysogenic. Catalase restoration requires only small amounts of catalase in contact with the cells for a short period; 5 min is sufficient, and maximum catalase restoration is observed only with rather heavy doses of ultraviolet. The catalase restorability of cells persists for about 2 hr after ultraviolet irradiation and then drops rapidly. No catalase restoration is observed following exposure to X rays. Lembke ei al. (1951), in a preliminary note, reported partial reversal of the effects of ultraviolet by treatment of the irradiated cells with certain chemicals. Phenol, glycine, and hydrogen sulfide were effective, whereas chloroform resulted in no reversal. EXTREME I'LTRA VIOLET RADIATION Very few data are available concerning the bactericidal or other effects of the extreme ultraviolet (Schumann region) on bacteria. The tech- nical difficulties involved in bactericidal studies in this region are consider- able, owing to the absorption of air below 1850 A. However, Bovie (1910) and Blank and Arnold (1935) have demonstrated the lethal effects of radiation between 1850 and 1100 A. Curran and Evans (1938) found that ultraviolet radiations of 2537 A wave length and in the Schumann region were bactericidal and also that EFEFCTS OF RADIATION ON BACTERIA 397 this wave length sensitized bacterial spores to subseciuent exposure to heat. Schumann rays of 1250-1 GOO A were several times as effective as 2537 A in producing this heat sensitization. NEAR-ULTRAVIOLET AND SHORT-VISIBLE RADIATION Somewhat conflicting results have been obtained by different investi- gators in studies of the bactericidal action of the near ultraviolet. The upper limit of the bactericidally effective wave lengths as reported by dif- ferent investigators has ranged from 2967 to as high as 3650 A. However, there seems little doubt that, although the efficiency of the w^ave lengths above about 2967 A decreases greatly corresponding to a similar low absorption of these wave lengths by bacterial cells, exposure to large amounts of radiation in the near-ultraviolet and short-visible regions will produce bactericidal effects as measured by viable count. Duggar (1936) reviews many of the earlier data. The most extensive data are those of Hollaender (1943) who showed a significant reduction in viable count following large exposures to radiation in the region from 3500 to 4900 A but greatest near 3650 A. Typical survival curves for the same culture of E. coli exposed to 2650 A and 3500-4900 A radiation are shown in Fig. 10-10. The survival curve obtained with the latter range of radiation is definitely of a threshold type, no bactericidal effects being noticed until large amounts of energy have been absorbed. Several differences were noticed in the studies of near-ultraviolet and short-visible radiation as compared to the bactericidally effective wave lengths. A temperature coefficient of about 1.7-2.2 for the near ultraviolet contrasts with the value of 1.1 for ultraviolet (Gates, 1929b). The incident energy for a given lethal effect is much greater for the near ultraviolet, and the exten- sion of the lag phase is more pronounced. Reference has already been made to the observation that cells exposed to near ultraviolet are more sensitive to the toxic effect of suspension in saline at 37°C. In Table 10-5 the differences between near-ultraviolet and the bactericidally effective wave lengths are summarized. Hollaender postulates the destruction of an essential cell component, or components, probably generally distrib- uted throughout the cell, which cannot be repaired or replaced from the other cell constituents but can, in many of the cells, be replaced from factors which are present in nutrient broth as the mechanism for the lethal action of near ultraviolet. The effect of treatment with near-ultraviolet and short-visible radiation in causing photoreversal of ultraviolet effects has already been discussed briefly. In this connection, Heinmets and Taylor (1951) have studied effects of ultraviolet and near-ultraviolet radiation and visible light on frozen bacteria. Photoreactivation was observed to occur only in the li(iuid phase. Bacteria in the frozen state, whether or not previousl^y exposed to ultraviolet, were inactivated by radiation in the 3000-4000 A 398 RADIATION BIOLOGY •40 X 10 Ergs 1 00 "TcAy'*"'^"''''^.— 10- 5 > (£ (ft UJ o < t- Z y I- (£ liJ a 01- 001' \ ^ ^x \ \ 2650 A '\ 3500-4900 6 °- \ X \ X \ X \ \ qo \ \ X —I — 100 — I — 300 400 200 X 10'^ Ergs Fig. 10-10. Survival ratio plotted against energy per orgaiii.sni for E. roll in liquid suspension. Lower scale, 2050 A; upper scale, 3500-4900 A. {After Hullaendcr. 1943.) Table 10-5. Comparison of Effects of the 3500 to 4900 A and the 2180 to 2950 A Region.s [From Hollaender (1943)] 3500-4900 A 2180-2950 A Shape of killing curve (log survi\al ratio Threshold type Approaching versus energy) straight line Incident energj^ for 50 per cent survival ratio (ergs/cm-) 2 X 10* 5 X 10^-103 Temperature coefficient 1.7-2.2 1.1 Time that sublethal effects ajjpear Before any organisms After 60-90 per cent are killed (in thresh- of organisms are old part of killing killed curve) Extension of retarded growth phase for 10 per cent survival ratio (per cent). . Up to 1000 50 Time that toxicitv of certain salt solu- Inunediately after ir- In (iOO mill at 32°C' tions can be recognized radiation Mutation production None Mutations pr()duc<'d in fungi and Dro- soph ila KFFIOCT.S OF UAOIATIOX ON HACTKKIA 399 range. Such frozen cells inactivatetl by 3000-4000 A radiation showed no l)ii()toreacti\'ation in the liciuid phase. Thus, Heinmets and Taylor eon- firm that radiation in this range can cause loss of viability of Ji. roll B cells and that the mechanism of this inactivation is probably (luite different from that of ultraviolet radiation of wave length shorter than 3000 A. No studies of mutation induction in l)acteria by irradiation at wave lengths greater than 3800 A have been published. HoUaender and Emmons (1946) observed morphological mutations following irradiation of A. terreus conidia at wave lengths 2967 and 3130 A. The energy required was much higher and the maximum proportion of mutants was lower than for 2650 A ultraviolet. The expected mutagenicity of sun- light, which contains appreciable amounts of energy in the 2900-3150 A range, was realized experimentally (HoUaender and Emmons, 1946; HoUaender et al, 1946). McAulay and his associates (INIcAulay and Ford, 1947; McAulay et al. 1949; Ford and Kirwan, 1949) report muta- tions in the fungus, Chaetomium glohosum, following irradiation with wave lengths of 3354, 3654, and 4047 A. This work is discussed in more detail later. In conclusion, it appears that, although irradiation with wave lengths greater than 3000 A may produce mutations, the mutagenic efficiency is markedly lower than for wave lengths near 2600 A. An interesting effect of visible light is photodynamic action, a complex function of visible light and photodynamic dye which recjuires the pres- ence of oxygen in order to be functional. Duggar (1936) briefly re\dewed the earlier work in this field. That permanent hereditary changes in microorganisms can be produced by photodynamic action has been demonstrated by Kaplan. Thus microcolonies of a permanent nature were observed in S. marcesccns following exposure to visible light in the presence of erythrosin (Kaplan, 1950b). Similarly, reverse mutations in a histidineless strain of E. coli (Kaplan, 1950c) and mutations to resist- ance to coliphage T7 (Kaplan, 1950a) were produced by photodynamic action. Perhaps the most striking effects were obtained with P. notatum (Kaplan, 1950d) in which significant increases in colony morphology mutations w^ere observed. Further studies of photodynamic action seem indicated since, as yet, the mechanism and significance of this phe- nomenon are not very well understood. PHYSIOLOGICAL PROPERTIES OF BACTERIA FOLLOWING IRRADIATION Surprisingly few investigations have been concerned with the physi- ology of irradiated bacteria, and much more work is needed in this field. The observation of filamentous cells following irradiation has been dis- cussed. Chambers and Russ (1912) and Bruynoghe and Mund (1925) •100 RADIATION HIOLOGY have observed motile cell.s in irratliated suspensions eontaining no cells capable of forming visible colonies, indicating that the energy-conversion enzyme systems were still timet ional. Anderson (1948) has shown that bacteriophage synthesis can occur in ultra xiolet-irradiated cells. The phosphorus uj)take and distribution were found to remain normal in irradiated cells (Abelson and Roberts, 1948). Morse and Charter (1949) have studied riboiuu-leic acid (RXA), DXA, and protein synthesis in normal and irradiated cells of A', coli B. Normally, the UNA content per cell increases five- to tenfold, and DXA and nitrogen two- to threefold dining the lag phase. After multiplication commences, nucleic acid per cell, especially RX^A, decreases. Doses of ultraviolet sufficient to reduce survival to 3-5 per cent produce no delay in the synthesis of nucleic acid. Synthesis of RXA is of normal magnitude, but syntheses of DX"A and nitrogen are reduced. Doses sufficient to reduce the viable count to 1 per cent interfere immediately with all syntheses. Relatively little material is synthesized during the normal lag period, after which RXA synthesis appears normal, whereas DX"A and nitrogen syntheses are reduced. Giese (1941) studied the respiration and luminescence of Achromohactcr fischeri cells after exposure to ultraviolet. Doses, which just inhibited division of most of the cells, permitted a normal respiration rate for 5 hr, followed by a decline in rate. The length of the period of normal respira- tion was an inverse function of dose, whereas the amount of the decline was proportional to the dose. Very large exposures resulted in an almost immediate large decline in respiration. Luminescence was intermediate in radiation sensitivity between division inhibition and respiration inhibition. A similar period of apparently normal respiration of X-irradiated K. coli B/r cells followed by a decline was observed by Billen et al. (1953), who further observed that the duration of the normal period was substrate dependent, being longer on pyruvate and succinate than on glucose. With the Texas strain of E. coli, however, an immediate inhibition of respiration was observed on pyruvate. The inhibition of respiration was more pronounced at 37° than at 2C°C. Brandt H al. (1951) found that ultraviolet radiation inhibited the adajjtive formation of galactozymase in Saccharomyccs cerevisiae. Doses which completely inhibited synthesis of the enzyme had little effect on preformed enzyme. Exposure to 4850 r of X rays did not inhibit forma- tion of galactozymase, even though division was inhibited in 90 per cent of the irradiated cells. In similar studies, Billen and Lichstein (1952) observed that increasing doses of X rays caused an increasing inhibition of the adaptive formation of formic hydrogenlyase in E. coli. Doses of 60,000-90,000 r had no measurable effect on preformed enzyme but completely inhibited the adajitive synthesis of formic hydrogenlyase. Although the inhibition of EFFECTS OF RADIATION ON BACTERIA 401 (Mizyme formation was proportional to dose for exposures less than 00,000 r, the amount of enzyme synthesized in such irradiated cell suspensions was much greater than could be attributed to the cells still capal)le of forming colonies. These studies suggest that inhil)ition of enzyme syn- thesis may be more important in the bactericidal effect of radiation than inactivation of enzyme molecules already present in the cells, as suggested by Dale (1940, 1942). It appears that irradiated cells rendered incapable of forming visible colonies are still able to perform many if not most of the normal metabolic functions. Complex processes such as growth and phage synthesis would seem to indicate that no gross disturbances of metal)olism result from irradiation and that the cell membrane remains essentially intact. In this latter connection, however, Loofbourow and associates (for review^ see Loofbourow, 1948) have shown the accumulation in the suspending medium of growth-promoting factors from irradiated cells, and Billen (personal communication) has shown a leakage of adenosinetriphosphate from E. coli cells following exposure to X radiation. The bactericidal effects of radiation have long been recognized more as a specific inhibition of cell division than as a general inhibition of metabo- lism, and the results discussed indicate greater radiosensitivity of cell division as compared to the radiosensitivity of respiration and of certain synthetic processes. Morse and Carter's investigations of nucleic acid synthesis are an example of further studies of specific metabolic functions intimately related to cell division which are needed to elucidate the par- ticular metabolic processes involved in the inactivation of bacterial cells by radiations. SUBLETHAL EFFECTS OF RADIATION The genetic changes are the most widely studied sublethal effects of radiations. Before considering the genetic phenomena, however, some other sublethal effects will be considered. The extension of the lag phase observed by numerous workers has been studied in some detail by Hollaender and Duggar (1938). Working with E. coll, they had observed a delay in the appearance of colonies following irradiation with ultraviolet of 2650 A wave length. During their study of the delay in growth of irradiated organisms in liquid cultures, they observed a second effect, an apparent initial increase in the number of cells. Thus, although the control suspensions showed no increase in numbers of cells for the first 2 hr of incubation, the irradiated suspensions showed a significant increase. The apparent early increase in rmmbers of cells did not eliminate the extended lag phase, the duration of which was estimated by extrapolating the logarithmic portion of the growth curve back to zero growth. The extension of the lag phase is evident with survivals as high as 70 per cent, whereas the apparent initial increase does 402 RADIATION UIOLOOY not appear until doses resulting in JO per cent survival have been given and is more pronounced with doses resulting in less than 8 per cent sur- vival. Keillor (U)o2) and Beckhorn {\9rt2) have shown that the exten- sion ot the lai-'; phase is photorevcrsihle by visible light to about the same degree as the bactericidal effects. BACTERIAL GENETICS Although very little was known concerning bacterial genetics prior to about 1940, the development ol knowledge in this field has been rapid since that time. Inasmuch as excellent reviews are available (Lederberg, 1948, 1951; Braun, 1947; Luria, 1947), no extended discussion is neces- sary here. The methods of classical Mendclian genetics are not applicable to genetic analysis since, in general, bacteria reproduce asexually. Instead, the chief tool of the bacterial geneticist must be an analysis of mutation. The case for the genie nature of heredity in bacteria, therefore, must rest largely on a number of analogies to the behavior of genes in higher, sexu- ally reproducing organisms. That the fundamental unit of inheritance in bacteria is the gene is strongly indicated by the repeated observations of the permanence of mutated characteristics, the spontaneous occurrence of these characteristics independent of specific environmental stimuli at defi- nite rates, the indepeiulence of different mutations in the same organism, mutation inducibility by mutagenic agents known to be effective in higher organisms, the reversibility of mutations, and the occurrence of mutations with physiological effects similar to known gene mutations in sexually reproducing organisms. Luria and Delbriick (1943) showed that the numbers of mutant cells in parallel cultures of bacteria should follow a clonal rather than a random sampling distribution if they resulted from preadaptive mutation. Such was found to be the case for bacteriophage-resistant mutants, and their so-called "fluctuation" test has since been applied successfully to other types of bacterial mutations. More direct demonstrations of the muta- tional origin of bacterial variants have been given by Xewcombe (1949) and Lederberg and Lederberg (1952). In addition, Lederberg's thorough analysis (Lederberg, 1947, 1949; Lederberg et al., 1951) of the phenomenon of recombination in E. coli \\\2 (Tatum and Lederberg, 1947) shows the physical basis of inheritance in bacteria to be essentially similar to that in higher organisms. Haas et al. (1948) have shown that sublethal doses of ultraviolet increase the fre- fiuency of sexual recombination in strain K12. Although the actual fusion of cells of two mutant strains to form hetero/ygotes has not been demonstrated, Zelle and Lederberg (1951), by means of single-cell studies EFFECTS OF KADI A TlOX ON' BACTElllA 403 Oil heterozygous strains, haxc shown fon\ inringl}' that individual cells contain the genetic potentiahties of the two parent strains. C'ytologically, it is known that bacteria possess Feulgen-positi\'e bodies which Robinow (1945) considers as chromosomes. Furthermore, the number of such bodies is known to vary from one to four or more per cell depending on the species, the stage of the growth cycle, and the stage in the division cycle of the individual bacterium observed. Observations indicating a genetic role for the so-called "chromatinic bodies" are just beginning to appear. Lederberg et al. (1951) have made comparative cytological studies of E. coli cells known to be haploid or diploid from genetic evidence. Perhaps the strongest evidence indicating an actual genetic function for these structures is that of Witkin (1951) who found a correspondence between the average number of chromatinic bodies per cell and the size of nonlactose-fermenting mutant sectors in colonies of E. coli. Zelle and Lederberg (1951, and unpublished observations) have genetic evidence that individual cells can be multinucleate, since a single cell has been observed to divide and form a diploid and haploid cell, the differentiation being based on subseciuent genetic behavior. The fact that bacterial cells do not necessarily have a constant nuclear constitution has an important bearing on the mechanism of bactericidal effects of radi- ation as well as an obvious relation to genetic phenomena in bacteria. If it is granted that the basic unit of inheritance in bacteria is the gene and that gene mutations are responsible for the observed heritable varia- tions, bacteria become a very valuable experimental tool because of the relative ease of making quantitative studies of both spontaneous and induced mutations. Systems of bacterial mutations which are particu- larly valuable in such quantitative studies include mutations to resist- ance to certain antibacterial agents such as bacteriophages, antibiotics, radiation, and metabolic inhibitors; reverse mutations of biochemical mutations in which the ability to carrj^ out certain syntheses has been lost; and mutations affecting the fermentation reactions. It has generally been assumed that the kinds of viable mutations observed following irradiation do not differ from those which occur spon- taneously, and that irradiation merely increases the rate of occurrence. Bryson and Davidson (1951) have studied a series of independently occur- ring, spontaneous, and ultraviolet-induced mutations to resistance to Tl bacteriophage in E. coli B/r. The mutants were analyzed for resistance to the other phages of the T series, for the tryptophane requirement which Anderson (1946) has shown generally accompanies spontaneous mutation to resistance to phage Tl, and for other biochemical require- ments. Significant differences were observed in the proportions of Tl- resistant and Tl- and T5-resistant mutations in the spontaneous and irradiated series. Similarly, the frequency of tryptophane requirement 101 l>\l)I\rio\ lUOLOGY was sif^iiificantly lowci- in I he inadiuted series. No complex phaj^c- resistant imitaiits were observed in the it radiated series, and, alth()up;h no other biochemical re(jiiirements were obsciNCMl in the spontaneous mutants, ti\'e biochemical r(M|uirements other than tryptophane were noted in th(^ 1 14 radiation-induced mutants studied. These are the most critical data bearing on this important (piestion and indicate that radia- tion-induced mutations may be qualitatively different from those that occur spontaneously. RADIATIOX-IXDUCED MUTATIONS I\ BACTERIA Stable heritable variation in colony morphology, cellular morphology, and pathogenicity were observed by Henri (1914) among the surviving cells of B. anthracis cultures exposed to ultraviolet radiation. In addi- tion, unstable variants occurred which reverted to the normal type. Stable variants were produced in numerous experiments and appear to have been bona fide mutations although, of course, the underlying cause was not proved to be genie. Shortly after Muller's announcement of the mutagenic effect of X rays in Drosophila (1927), Rice and Reed (1931) studied I'ough-to-smooth variation in a bovine strain of Mycobacterium tuberculosis irradiated with 88-kvp X rays. Their results are hard to evaluate in that the rough-to- smooth change occurs spontaneously and the smooth forms are quite unstable. Haberman and Ellsworth (1940) qualitatively demonstrated that X rays increased the so-called "dissociative" changes in .S. aureus and 8. marcescens. The first quantitative study of X-ray-induced mutations in bacteria was that by Lincoln and Gowen (1942) in which various colonial charac- teristics of Phytomonas stewartii were utilized. The data show clearly an increase in the frequency of mutations among the cells surviving X irradi- ation, and the rate of mutation for the three major characteristics studied was about 3.7 X 10"** per character per roentgen. Croland (1943) observed a hinidredfold increase in mutants per 10^ cells in studies of the succinate-miiuis to succinate-positive mutation in Moraxclla hvoffi with X rays. He observed an increase in the absolute number of mutants as well as in the proportion of mutants among the survivors. Biochemical mutants that affect specific syntheses were reported by Roepke ct al. (1944) and Gray and Tatum (1944) following X-ray treat- ment. Since that time, numerous in\(^stigators have succeeded in iso- lating biochemical mutations in various species of bacteria. The work of Demerec and Latarjet (1946) and Anderson (1951b), who have published the most precise ciuantitativo studies of induced mutations in bacteria, will he discussed later. Burkholder and Giles (1947) induced biochemical mutations with ultraviolet and X-ray treatment of B. subtilis spores, and Devi ct al. EFFECTS OF RADIATION ON BACTERIA 'iOo (1951) isolated mutations affecting the nutritional requirements of Aero- hacter aerogenes when the cells were exposed to X rays in the dry state. DELAYED EXPRESSION OF INDUCED MUTATIONS Demerec (1946) observed a delay in the phenotypic expression of ultraviolet-induced mutations to resistance to Tl bacteriophage. Only about 1 per cent of the induced phage-resistant mutations were observed as mutants when irradiated cells were plated upon the surface of agar plates containing an excess of phage. The peak expression of induced mutations occurred after one or two generations of growth, and mduced mutations were observed to continue to appear until 12 or 13 generations of growth had occurred. The technique which made these observations possible consisted in infecting the bacteria, after varying periods of growth on agar plates, by exposing the plates to an aerosol of the bacterio- phage, a method which does not disturb the distribution of cells on the plates'. Later, Newcombe (1948) observed a similar delay in the expres- sion of spontaneous mutations to phage resistance although the appear- ance of mutations did not continue through as many generations of growth. Newcombe and Scott (1949), in studies of the factors responsible for the delayed appearance of radiation-induced mutants, concluded that a phe- notypic lag in expression of mutations, similar to that occurring m spon- taneous mutations, plus a variable delay in the onset of division of the irradiated cells are responsible. Demerec, Dollinger, and FUnt (1951) found quite different patterns m the phenotypic expression of induced phage-resistant and streptomycin- resistant mutations. Witkin (1951) employed a double-screemng tech- nique to obtain evidence that induced mutations in E. coli were sometimes expressed in only part of a clone derived from a surviving irradiated cell. These results indicate that nuclear segregation, a physiological lag, and a variable onset of division are not sufficient to explain the delay in expres- sion of radiation-induced mutations. A complete discussion of delayed expression of mutations is beyond the scope of this chapter. It is obvious, however, that this delay m expression is an important factor which must be considered in quanti- tative studies of radiation-induced mutations in bacteria. QUANTITATIVE STUDIES OF RADIATION-INDUCED MUTATIONS In an important paper, Demerec and Latarjet (1946) have reported studies on ultraviolet- and X-ray-induced mutations to resistance to Tl bacteriophage in E. coli B and B/r. Both zero-point mutations (i.e. mutations expressed immediately) and end-point mutations (i.e., total mutations induced including those expressed immediately and after a delay) were assayed. In Fig. 10-1 1 are shown the results for ultraviolet irradiation of strain B/r. lUC UADlATiO.N lUol.dC'* For /cro-poinl nuitatioiis tho mto of inciviiHc in tlif iiumhcr of iniitji- tioiis with incrcasiiijj; dose excoods an exponential liinction of dose. The frcMiiienc}' of mutations increases until doses eausinj; ah<»ut 10 ' sur\i\al are exeeeded, at wliich tim(> a plateau followed l)y a slight decline is oliserved. A similar (leclinc in mutations at high doses has been observed by IloUaender and Emmons (l<)89a. 1941 ; Kmmonsand Ilollaender, 1939) with the fungus Trichophyton mcntagrophi/tes, Ilollaender, Raper, and Coghill (1945) with ,1. terreus, and Hollaeiider . . . Demerec (1945) for Ncitrospom. Xo good explanation exists for the occurrence of this maximum in the mutation-dose relation. 1 000 ?00Q 3000 4000 DOSAGE. Ergs « mm'' 50O0 6000 Fig. 10-1 1. Zero-point and end-point mutants induced by ultraviolet 2537 A in E. roli B/r, and ultraviolet svirvival curve of B/r (seniiiojrarithmic plot). Solid circles, zero- point mutants; open circles, end-point mutants. (Adapted from Demerec and Latarjet, 1946.) For end-point mutations, Demerec and Latarjet (1946) observed a rapid increase in mutations at low doses, but as the dose continued to increase the rate of increase in mutations decreased and contirmed more or less exponentially to the highest doses tested, no maximum being observed for the end-point mutations. The frequency of end-point mutations was higher at all doses than that of zero-point mutations. With X rays, essentially similar results were obtained for both zero- point and end-point mutations, the latter being approximately 200 times as freciuent at all doses. The mutation-dose curves were approximately linear, which is compatible with a one-hit mechanism, whereas the much- greater-than-linear increase in mutations with increasing dose observed for ultraviolet irradiation indicates a multiple-hit relation. In comparative experiments with strains B and B/r, nearly identical phage-resistance mutation rates were observed at a given dose of either ultraviolet or X ravs even though the inactivation of strain B was much EFFECTS OF RADIATION ON BACTERIA 407 greater. This observation has important implications with respect to the mechanism of the lethal action of radiation. On the basis of energy absorbed per bacterium, X rays were found to be approximately 200 times as efficient in producing inactivation and about ten times as efficient in the production of mutations. In later studies, Demerec (1949, 1951) observed a linear increase in reverse mutations of the SD-4 streptomycin-dependent mutant of strain B/r (Bertani, 1951) with both X and ultraviolet irradiation. With this mutation system, no zero-point mutations occur since no mutations are expressed until at least one division has occurred. In this connection, Crigg (1952) has shown that, in quantitative studies of reverse mutations of biochemical mutations in Neurospora conidia, precautions must be taken to ensure that the observed reversions are actually mutations and are not already present in the irradiated culture. He found that large numbers of mutant conidia which refjuire a particular growth factor inhibit the growth of a known smaller number of wild-type cells when plated on minimal medium. He suggested that, in induced- reverse-mutation experiments, the radiation merely kills enough of the growth-factor-requiring cells to remove the inhibition, thus permitting spontaneous reverse mutations already present in the culture to develop. This was demonstrated in tests in which the apparent proportion of wild- type nuclei increased tenfold when the culture was plated at low dilutions. Somewhat similar observations have been reported by Bryson (1950) with streptomycin-resistant mutants of E. colt. Still another type of mutation-dose relation has been observed by Newcombe and Whitehead (1951) in studies of color-response mutants of E. coll B/r on mannitol-tetrazolium agar. With this system the frequency of mutation rises very rapidly with ultraviolet doses below 500 ergs/mm- and is nearly constant for doses of 1000 5000 ergs/mm^. No decline in the percentage of mutations is observed at the higher doses. Extensive quantitative studies of reverse mutations of the same SD-4 mutant of strain B/r used by Demerec (1949, 1951) and of a purineless mutant of the same strain were made by Anderson (1951b). The chief results of his experiments, w^hich were designed to test whether the oxygen concentration at the time of X irradiation had a parallel influence on mutagenesis and inactivation, are shown in Figs. 10-12 and 13. Survival curves for the two mutant strains were nearly identical. However, greatly different response curves were obtained for the two reverse muta- tions. Thus Fig. 10-12 shows that, for the streptomycin-dependent strain, a linear relation exists between induced mutations and dose and that only a slightly higher rate of induced mutation was observed when the organisms were irradiated in the presence of oxygen. For the purine- less strain, an approximately exponcMitial increase in mutation rate was observed with increasing dose, the frequency of mutations observed for a 408 HADIATION Mlol.OGY f^ivoM close ol radiation Ix'iiiK much fi;ri'al(.'r in the picscncc of oxygen tluiii in its iihsence. I'lie same mutation data are plotted against survival ratio in Fig. 10-18 to illustrate more elearly that, for the streptomycin- dependent strain, the freiiuency of back mutation is greater for a given survival lexcl when the cells were irradiated in the absence of oxygen and 20 40 60 80 100 120 140 1600- 1400- 1200- 1000. 800- 600- 400- 200- nr OXYGEN 1 1 — 40 50 EXPOSURE.!" Ix 10* 9x10' SxlO-* -7x10* •6x10' -5x10^ -4x10' -3xlO' -2x10' IxlO^ 90 Fig. 10-12. X-ray induction of back mutations as a function of kiloroentgens of exposure. Left and bottom scales, comparison of back-mutation rates to strepto- mycin nondependence of the streptom.ycin-dependent strain when irradiated in oxygen (curve I) or in nitrogen (curve II). Right and top scales, comparison of the back-mutation rates of the purineless strain to purine nondependence when irradiated in oxygen (curve III) or in nitrogen (curve IV). (Anderson, 1951b.) hence had received approximately two and one-half times as much inci- dent energy as the cells irradiated in oxygen. For the purineless back mutation, the mutation-survival curves are identical for cells irradiated in the presence and in the absence of oxygen. These results are significant in indicating that greatly different quantitative relations may exist between the induced-mutation rates and radiation dosage for different mutation systems. Furthermore, although the oxygen-sensitive indirect mechanism which influences inactivation of both strains appears to have EFFECTS OF RADIATION OX IJACTERIA 409 a quantitatively equal effect on the purineless reverse mutation, it does not seem to be a major factor in the induction of streptomycm-dependent reverse mutation. Demerec et al. (1952) have studied the reverse-mutation response ot H 2400 2200 2000- 1800 -II X 10 o 3 o Q 1600- 1400 1200 1000 800 600- 400 200- SURVIVAL RATIO, N/N^ Fig 10-13 Rate of X-rav induction of back mutations as a function of survival ratio Left scale, comparison of the back-mutation rates to streptomycin nondependence of the streptomycin-dependent strain irradiated in oxygen (curve I) or m nitrogen (curve II). Right scale, comparison of the back-mutation rates to purme nonde- pendence of the purineless strain irradiated in oxygen (curve III) or m nitrogen (curve IV). {Anderson, 1951b.) mutant strains of E. coli to a variety of mutagenic treatments. The most striking observation was that in four of the strains the spontaneous rate of back mutation could not be increased by any of the mutagenic treatments which included X and ultraviolet radiation, manganous chloride, and ^-propiolactone. In addition, the quantitative response of the other strains to ultraviolet and manganous chloride varied differentially. It seems clear that all bacterial mutations do not exhibit the same 410 RADIATION UlULOGY • luaiititativc relations to radiation or other mutagens and that some may not respond to radiation or other mutajj;ens at ail. That such diverti;enl results should he obtained with the first few specilic mutations aderiuatcly studied indicates that caution should he exercised in generaliziu};, as regards the mutation-dose relation, and that (Miual caution should he used in interpreting radiation-induced mutation rates which are the sum of the mutation rates of an unknown number of loci. PHOTOREACTn VTION OF ULTKAVIOLKT MUTAGENIC EFFECTS Although this subject is covered more comi)letely in Chap. 12 by Dulbecco, a brief discussion seems appropriate heie. Since photoreacti- vation indicates an indirect mechanism for a major proportion of the ultraviolet bactericidal efl'ects, it is of interest to know if the same indirect mechanisms are involved in the production of mutations following ultra- violet irradiation. Kelner (1949b) and Novick and Szilard (1949) were the first to study this question and, although Kelner's results were some- what inconclusive, Novick and Szilard observed a reduction in mutagenic effects to a degi-ee corresponding roughly with the reduction in the lethal effects. Similar observations have been reported for Paramecium aurelia (Kimball and (iaither, 1950; Kimball, 1950), Neurospora (Goodgal, 1950: Brown, 1951), PeniciUium chrysoyenum (Roegner, 1951), and for the polar cap cells of Drosophila (Meyer, 1951). Roegner's results are of interest in that the mutation-dosage relation exhibited a maximum, as discussed earlier. When the photoreactivation treatment followed high doses of ultraviolet, i.e., on the descending portion of the curve beyond the maxi- mum, Roegner observed an increase in the fre(iuency of mutation, but at lower ultraviolet doses in the ascending portion of the curve, photoreacti- vation resulted in a decreased proportion of mutations. The most extensive studies are those of Xewcombe and Whitehead (1951) with the color-response mutations of E. coli Br on mannitol-tetra- zolium agar. A relatively constant proportion of mutations was observed for ultraviolet doses greater than 1000 ergs/mm-'. Photoreactivation of low doses of radiation resulted in a dose-reduction factor of 5 which is greater than that observed for the bactericidal effects. For very large doses of ultraviolet, no effect of photoreactivation in lowering the muta- tion frofiuency was observed. To explain their results, Newcombe and Whitehead postulate that both a phot()stal)le and photosensitive mutagen poison are produced by the ultraviolet and that the plateau in the muta- tion-dose curve is due to a common limiting step in the reactions by which both of these mutagen poisons cause mutations. Thus, at high doses, a sufficient amount of the photostable mutagen poison is produced to cause a maximum mutation effect, and no photoreactivation is possible. In studies of ultraviolet-induced mutations from streptomycin depend- ence to independence in the Sn-4 strain of E. coH, Beckhorn (1951) EFFECTS OF RADIATION ON BACTERIA "ill observed a decrease in induced mutation rates following photoreactivation at all ultraviolet doses. In this case, however, there is no maximum or plateau in the mutation-dose curve. ULTRAVIOLET ACTION SPECTRUM OF MUTATION INDUCTION \ction spectra of mutation induction with maxima in the neighborhood of 2G00-2650 A have been reported by Knapp et al. (^^^Q) for .Sp/.aero- carvus donnelli sperm; Hollaender and Emmons (1939a, b, 1941 and Emmons and Hollaender (1939) for Trichophyton mentagrophytes ;hiad[ev and Uber (1942) for maize pollen grains; Hollaender . . . Demerec (1945) for Neurospora; Hollaender and Zimmer (1945) for P. notatum; and Hollaender, Raper, and Coghill (1945) for A. terreus. The similarity between the action spectrum for mutation production and the absorption spectrum for nucleic acid is commonly interpreted to indicate that the nucleic acid portion of the gene acts as the chromophore for mutation production. Changes in viscosity, stream birefringence, and colloid osmotic pressure of deoxyribonucleate following ultraviolet irradiation have been studied by Hollaender ./ al (1941). The doses required to produce a detectable effect in vitro are large compared to those required for the bactericidal or mutagenic effects. Noethling and Stubbe (1934; Stubbe and Noethling, 1937) found 2967 \ to be the most efficient wave length of four tested in producing muta- tions when pollen of Antirrhinum majus was irradiated. Differential absorption of the irradiated pollen grains and the small number of tests cast doubt on the significance of this observation. McAulay and his associates (McAulay and Ford, 1947; McAulay et al, 1949- Ford and Kirwan, 1949) have published some unusual results obtained in studies of mutation induction with the fungus, Chaeto7mum^ globosuni. The most efficient ultraviolet wave length is 2804 A and mutations are reported following irradiation with wave lengths of 33d4, 3654 and 4047 A. They report that the shorter wave lengths, i.e., 2654, 9804' and 2967 A, selectively induce the so-called "K mutant," whereas very'few K mutants are found following the near-ultraviolet and short- visible treatment. The statistical validity of this conclusion seems questionable since the sizes of samples observed are quite small. No Iv mutants were observed following X irradiation, whereas, with large doses of 2804 A ultraviolet, as high as 62 per cent of K mutants were observed. The mutation-dose curves for "lethal mutations" (mutations resulting m cessation of growth while the colony is still very small) indicate that, although two quantum hits of 2804 A ultraviolet will induce the mutation, five quantum hits of X rays are necessary for lethality (Ford and Kirwan, 1949) These results are difficult to evaluate since, except lor the lethal- mutation studies, sample sizes are very small owing to the characteristics of the fungus which produces huge colonies. That the lethal mutations 412 RADIATION HIOIiOGY are actu;ill>' jfeiie niutatioii.s (scems dou^ttul since up to lOU per eeiit ot such mutations can l)e induced at liifi;li doses. Ivaplan (1952) was the first to puhHsii an action spectrum tor mutation induction in bacteria. He studied the muta}i;enic efficiency of (i\(' wave lengtlis ranging from 2480 to 'HYM) A. A maximum at 2()50 A was observed with eacli of tlie three types of mutations in Serratia marcescens studied. Simihir results have been obtained by Zelle and Ilollaender (unpublished data), who used six wave lengths from 2378 to 29()7 A. Maximum efficiency of mutation induction at 2V)i)0 A was observed for both the streptomycin dependence to independence and the purineless reverse mutations in E. coli B/r. These are the same mutation systems that were employed by Anderson (19511)) in his study of the relation of oxygen concentration to mutation production by X rays. Absolute increases in the number of mutant cells were observed by Kaplan and by Zelle and Ilollaender, thus eliminating differential survival of spontaneous mutants as the reason for the increased mutation rate. The observation of a maximum efficiency at 2(350 A in bacteria, as in sexually reproducing forms, is additional supporting evidence of the genie nature of bacterial variation. INDUCTION OF MUTATIONS BY IRRADIATED MEDIA In many of the earlier studies of bactericidal effects, the bacteria were irradiated on the surface of agar plates. To test the possibility of an indirect action through the photochemical production of toxic substances in the medium, several workers irradiated media before inoculation. In general, it was possible to demonstrate toxic properties of the irradiated media, but the doses required to produce an appreciable degree of toxicity were far beyond those utilized in the bactericidal tests by direct irradia- tion of the organism (Loofbourow, 1948). The discovery of photoreactivation and of the influence of oxygen concentration on the bactericidal and mutagenic effects of ultra- violet and X rays, respectively, indicate clearly that at least part of the bactericidal and mutagenic effects of radiation are indirect and involve largely unknown reactions which, initiated by the radiation, ultimately alter a vital structure of the cell and cause either mutation or inactivation. Interest in the indirect effects of the irradiation of substrates has been revived by a series of publications by a group at the University of Texas. Mutations to resistance to antibiotics in S. aureus were usually studied. Thus Stone et al. (1947) demonstrated that irradiation of the substrate with the entire spectrum of a (luartz mercury arc lamp, prior to inocu- lation with bacteria, caused an increase in the mutation rate. Chemical treatment of the substrate or of amino acids added to the substrate with hydrogen peroxide caused a similar increase in mutations (Wyss et al., EFFECTS OF RADIATION ON BACTERIA 413 1947). Evidence was presented (Stone et al, 1948) that selective growth of spontaneous mutations dready present in the inoculum could not account for the observed results. More critical evidence that irradiated substrate or peroxide-treated substrates could induce mutations was presented by Wagner et al. (1950) who observed increases in mutations in Neurospora conidia treated with irradiated broth. An increase in mutation rate when inhibitors of cata- lase and the cytochrome system were incorporated into the medium, thus permitting an accumulation of hydrogen peroxide within the cell, was reported by Wyss et al. (1948) and Wagner et al. (1950). Haas et al. (1950) summarize these results and, in addition, report that the most effective wave lengths in producing mutagenic substrates are those below 2000 A. Increasing the temperature of the substrate during the preinoculation irradiation from 0° to 60°C results in a significant increase in the mutagenic properties. They report that the bactericidal and mutagenic effects of irradiated medium were partially reversed by subsequent exposure to visible light. Wyss et al. (1948) failed to demonstrate an increase in mutation rate by direct treatment of bacteria with hydrogen peroxide, although positive results were obtained by Wagner et al. (1950) with Neurospora. They conclude that formation of organic peroxides is responsible for the muta- genic effects of peroxide treatment of the broth and probably also of ultra- violet irradiation. This conclusion is strengthened by the work of Dickey et al. (1949), who showed that various organic peroxides are effective in inducing reverse mutation in an adenine-reciuiring strain of Neurospora. In their experiments the efficiency of the mutagenic peroxides was con- siderably less than that of direct ultraviolet irradiation. Summarizing, it seems established that the mutation rate is increased when the cells are grown in ultraviolet-irradiated or hydrogen peroxide- treated substrates. It is difficult, however, to assess properly the signifi- cance of these results as they relate to the induction of mutations by direct irradiation of the cells with ultraviolet. In general, the mutagenic effi- ciency of the various substrate treatments is low; where comparisons were made, it is lower than that of direct ultraviolet radiation. Furthermore, there is a complete lack of quantitative data as to the amounts of radia- tion absorbed by the substrates. It is certain, however, that doses far greater than those employed in direct irradiation of cells are employed hi the substrate treatments. Furthermore, the wave lengths effective in producing mutagenic compounds in irradiated substrates lie mainly below 2000 A and are not present in the spectrum of the widely used germicidal lamps with glass envelopes or in monochromatic beams of ultraviolet radiation of the wave lengths most efficient in producing mutations by direct irradiation. Loofbourow (1948) points out that, although the formation of hydrogen peroxide by ultraviolet radiation is essentially con- U4 KADI \I'I()N HI()L(»(;V lined lo wave lenfi;ths shortci- ilcm iMIOO A, the pliotodccoiupo.sition of peroxide occurs throujilioiit the ultruviolet spcictrum. All these considerations plus the selective absorption of ultraviolet Wy certain protoplasmic constituents indi<'ate that only a very minor portion of the effects of direct ultra\iolet irradiation of bacterial cells by the wave lengths usually employed can be ascribed to an indirect mechanism which has as its first step the formation of hydrogen peroxide from water. A more conservative estimate of the significance of these results is that treatment of nutrient broth or solutions of certain amino acids with hydrogen peroxide or with ultraviolet of wave lengths shorter than 2000 A can produce chemical mutagens. Such irradiation of so complex a sub- strate as nutrient broth would be followed by a variety of chemical changes including formation of hydrogen peroxide and organic peroxides. Since both hydrogen peroxide and certain organic peroxides have been shown to be mutagenic (Dickey et al., 1949; Wagner et al., 19o0; Demerec, Bertani, and Flint, 1951), it is not surprising that broth irradiated under such conditions is also mutagenic. MECHANISM OF RADIATION EFFECTS Various hypotheses have been proposed to explain the bactericidal effects of radiations. In view of the recent evidence that a large propor- tion of the effects of both ionizing and ultraviolet radiation are produced by indirect mechanisms, many of the discussions are no longer appro- priate, and no attempt will be made to reconsider them completely. Such discussions have generally been concerned with two central prob- lems: (1) how best to account for the kinetics observed, and (2) the nature of the damage and the mechanism by which it is produced. In regard to the former problem, several waiters (e.g.. Gates, 1929a) have explained the occurrence of the survival curves observed on the basis of variation in resistance of the individual cells comprising the bacterial population. Although the occurrence of the sigmoidal survival curves can l)e explained on this basis with not too violently skewed distributions, the distribution recjuired to explain an exponential survival curve, namely, an exponential distribution of resistances, is implausible. By contrast, the first-order kinetics indicated by exponential survival curves are a natural consequence of the target theory in its simplest form. Sigmoidal survival curves are easily accounted foi' on the basis of the multihit theory. Consequently, very few investigators still hold to the distri- bution theory. In regard to the latter prol)lem, a number of explanations have been proposed which are discussed by Lea et al. (1936). These may be broadly grouped into two categories: (1) secondary poisoning of the cell by chemical substances produced by the radiation and (2) decomposition of molecules vital to the organism. Lea and cow^orkers reject the poison EFFECTS OF RADIATION' ON BACTEIUA 415 hypothesis on several grounds. It is difficult to account for the kinetics observed without making assumptions which are somewhat contradictory to the behavior of chemical disinfectants in bacteria. Furthermore, the independence of bactericidal effect and intensity and the lack of a large temperature coefficient characteristic of chemical disinfectants are like- wise difficult to explain unless it is assumed that a cell poison produced internallj^ by radiation behaves quite differently from a chemical disin- fectant applied externally. Postulating that a cell poison is produced by radiation really does little to explain the mechanism of radiation damage except to infer that an indirect mechanism exists between the initial chemical change produced by the radiation and the lethal end result. To be meaningful, the hypothesis must be elaborated to indicate the nature of the lethal effect produced and to take into account the kinetics observed. This has been done by Roberts and Aldous (1949) to explain their results on recovery of E. coll B from the effects of ultraviolet irradiation. They postulate that a cell poison is produced by the radiation by photochemical reaction and that this cell poison selectively affects the division mechanism. To explain the kinetics involved in the recovery, they further postulate that one molecule of this poison is effective in inhibiting division and, further- more, that the poison exponentially disappears independently of ultra- violet irradiation. Similarly, although a target-theory interpretation can adequately account for the kinetics observed, it has little meaning unless it is expanded to indicate the nature of the target and the change induced in the target by the radiation. Lea and his associates (1941; Lea, 1947) have done this and postulate the nucleus as the target and the induction of lethal mutations as the damage causing inactivation. On this hypoth- esis, Lea has estimated that E. colt possesses 250 genes capable of lethal mutation, each 12 m^t in diameter. In view of the developments since 1947. however, a reexamination of the lethal-mutation interpretation seems indicated. The growing body of evidence indicates that a major proportion of the effects of both ultraviolet and ionizing radiation on bacteria are indirect and involve a largely unknown chain of reactions occurring betw^een the initial ionization, or quantum absorption, and the final lethal or muta- genic change. The assumption that the residual effect of radiations, i.e., the nonphotoreactivable portion of ultraviolet effects or the residual bactericidal effects of ionizing radiations in the absence of oxygen, is due to direct effects of the radiation on vital cell structures is by no means proved; the "residual effect" may actually involve still other indirect mechanisms. The existence of an indirect mechanism, however, does not necessitate abandoning the assumption that the final decisive end product of the 416 RADIATION TUOLOGY chain of reactions is the induction of lethal or viable mutation.s. Indeed, the evidence indicates that with ultraviolet radiation the same photo- reactivahle process affects both the bactericidal and mutagenic elTects of radiation. Similarly, Anderson (19olb) has shown that in the case of one mutation system the lethal and mutagenic effects of X rays may result from the same indirect mechanism. Thus, although the existence of indirect mechanisms necessitates a modification of the concept of lethality t)y chemical change in one molecule, presumably the gene, by direct ionization, it does not require abandonment of the hypothesis that the final product of the indirect process may be lethal mutations. The observation of exponential survival curves when E. coli are X-irradiated in oxygen-saturated media indicates that first-order kinetics result, even though it is known that a large proportion of the effects are indirect. Thus the problem of determination of the best means to account for the observed first-order kinetics still remains, and the plausibility of the lethal-mutation theory is in no way impaired. There are a number of observations, however, which bear directly on the lethal-mutation hypothesis. Lea et al. (1937) and Witkin (1947) have observed that the long fila- mentous forms W'hich result from bacteria subjected to sublethal doses of radiation react to subsequent exposure to radiation in the same manner as normal cells and appear to be equally sensitive to radiation. Delaporte (1949) has studied the filamentous forms of E. coli B cytologically and reports them to be multinucleate. It would thus appear that the mechanism of killing of such an elongated, multinucleate cell would not be due to recessive lethal mutations, and, if the lethal-mutation hypothesis is to be retained, it must be postulated that the induced lethal mutations are dominant and can be expressed equally well in uninucleate and multinucleate cells. Demerec and Latarjet (1946) in comparative studies of E. coli B and B/r have shown that, with a given dose of ultraviolet, the rate of mutation induction is the same for the two strains, whereas the bactericidal effects are greatly different. A similar lack of correlation between the induction of mutations and the lethal effects was observed when comparisons were made with ultraviolet and X rays on strain B/r where, for a given survival ratio, the number of mutations induced by X rays was significantly less than the number induced by ultraviolet. Beckhorn (1950) and Lederberg et al. (1951) have approached this question more directly by comparing the sensitivity of E. coli K12 cells, known genetically to be diploid, with that of haploid cells of the same strain. No systematic differences in sensitivity to ultraviolet radiation were observed, and the most prominent effect of irradiation appeared to be the conversion of diploid cells to the haploid condition. This haploidi- zation occurs at doses smaller than those required for appreciable killing of EFFECTS OF RADIATION ON BACTERIA 417 the cells. Beckhorn demonstrated that this unusuul effect of ultraviolet was photoreactivable to about the same degree as are the bactericidal and mutagenic effects. Lederberg et al. have shown that X rays produce a similar effect and have found that, on continued incubation, most of the apparent haploid colonies develop spots of growth which are diploid. Thus, although diploid cells surviving the irradiation are temporarily- altered so as to produce many haploid segregates, they retain the ability to transmit the diploid condition. These interesting results indicate that recessive lethal-mutation induction is not an important mechanism in killing by irradiation in these strains. If such were the case, marked differences in sensitivity should be observed between haploid and diploid cells and segregation of diploids to haploids should be decreased rather than stimulated since recessive lethals, although masked in the diploid, are expressed in the haploid cells. That the nuclear constitution of microbial cells does influence the sensi- tivity to the lethal effects of radiation is indicated by the results of Latarjet and Ephrussi (1949) in which haploid and diploid cells of the same yeast strain were irradiated with X rays. They obtained exponen- tial survival curves for the haploid cells and sigmoidal survival curves for the diploid cells. The sigmoidal curve corresponded approximately to a two-hit curve, as had been observed by other workers in studies of diploid yeast. Similar results have been obtained by Tobias (1952) who used haploid, diploid, and presumed tetraploid yeasts in tests of the diffusion model of the biological effects of high-energy radiations. Atwood (1952) has devised a technique employing heterokaryotic conidia of Neurospora which permits, in addition to determination of the surviving fraction of cells, the determination of the surviving fraction of nuclei, the fraction of nuclei containing at least one recessive lethal muta- tion, the frequency with which separately induced recessive lethal muta- tions are homologous, and the degree to which all these effects are independent of one another. Although these studies are still in the preliminary stage, it seems warranted to conclude that killing of Neuro- spora conidia by X rays is primarily, if not entirely, a nuclear phenomenon and that recessive lethal mutations, although more important in uni- nucleate conidia, have but a minor effect in the inactivation of hetero- karyotic nuclei since homologous recessive lethal mutations must be induced for their expression. Utilizing similar techniques, Norman (1951) has published extensive experiments on the ultraviolet inactivation of Neurospora conidia. He concludes that inactivation of conidia is a consequence of the inactivation of nuclei and that nuclei exhibit first- order kinetics. Two kinds of inactivation processes are postulated: recessive lethal mutations and a nongenetic effect on the nucleus. The two mechanisms are intimately related, however, since the same action spectrum is obtained with monochromatic ultraviolet radiation and each 118 RADIMIo.N MIOLOGY is photorcaclivatxHl to al)()iit the .same cxtciil. Xo spcculal ions con- cerning the nature of the uongenetic nuclear daraage are given, hut it would seem that it may be of a gross nature, and hence may involve a number of genes. The observation of fiist-order kinetics indicates that the ultraviolet quantum is capable of producing this effect. These observations with yeast, and especially with Ncuronpora, indicate that the damage causing lethality is damage to the nucleus, i.e., to the genetic apparatus. Furthermore, the Ncuroapora results indicate that recessive lethal mutation is of relatively minor importance in the inacti- vation of multinucleate cells by irradiation. Since there is an increasing amount of evidence that bacterial cells of certain species may be multi- nucleate, at least at certain stages of development of the culture, a similar situation may obtain in bacteria. This has been indicated by the results of Stapleton (1952), who found differences in X-ray resistance and in the kinetics of inactivation which were correlated with differences in the mean number of nuclei of E. coli B/r cells at different stages of the growth cycle. Witkin (1951), however, found resistance of E. coli B/r to ultraviolet to be lowest in growing cells, greater in resting cells, and highest in cells in the lag phase. These differences in resistance did not parallel differ- ences in the average number of nuclei — 4,2, and 4, respectively — for the three stages of the growth cycle and seem incompatible with the recessive lethal-mutation hypothesis of radiation killing. Furthermore, the cor- relation between nuclear number and the size of lactose negative sectors in colonies derived from cells which survive irradiation strongly suggests that nuclear segregation is partially responsil^le for the sectored colonies. If this is actually the case, it is questionable if ultraviolet killing is nuclear at all since, as Witkin points out, wdth survivals as low as 10"'' as in her experiments, the probability of a surviving cell having more than one viable nucleus would be very low. Dale (1940, 1942) has suggested that the bactericidal effects of irradia- tion may not involve lethal mutations but rather the inactivation of enzymes. Although objections to this hypothesis have been made on the basis of the relative insensitivity of enzymes irradiated in vitro, Dale has shown that dilute solutions of purified enzymes are quite sensitive to radiation, and, indeed, this sensitivity is the l)asis for his suggestion that enzyme inactivation may be a factor in the bactericidal effects of irradia- tion. Barron et al. (1949) have showni that the sulfhydryl-containing enzymes are particularly sensitive to ionizing radiations. It is known that dilute solutions of enzymes are inactivated primarily by an indirect effect of ionizing radiations and that a great variety of compounds protect the enzyme against radiation by competing for the highly reactive prod- ucts formed from water. It would appear that a similar competitive pro- tection would occur within the cell, since a great variety of prolc'iiis are EFFECTS OF RADIATION ON BACTERIA 419 present A further difficulty arises in accounting for the first-order kinet- ics frequently observed. If there are many molecules of a given enzyme nresent in the cell, it is difficult to visualize that destruction of a single such molecule would be lethal to the cell. Mcllwain (194G) has suggested that only one or a very few molecules of certain enzymes may be present in the cell If this is the case, the first-order kinetics can be explained. If the extreme possibility, suggested by Mcllwain (1947), that the one or few units of enzyme present in the cell are actually the units of inheri- tance is correct, the lethal-mutation and enzyme-destruction hypotheses of the ultimate damage responsible for the bactericidal effects merge. There is increasing evidence to indicate that attempts to explain the bactericidal effects of irradiation on the basis of one mechanism are not realistic Luria (1939), on the basis of microscopic examination ot incubated irradiated cells, suggested that more than one mechanism of killing existed and that an attempt to explain the bactericidal effects of irradiation on the basis of a single mechanism did not seem warranted. For both X rays and ultraviolet, an indirect mechanism is known to pro- duce a portion of bactericidal effects, whereas apparently quite different mechanisms account for the residual effects, although the ultimate dam- age may be the same in both cases. It is interesting to compare some of the known facts concerning E. coU B and B/r from the standpoint of multiple mechanisms. Apparently, these two strains differ by mutation of a single gene. Even though closelv related, they show many differences m behavior fol- lomng ultraviolet irradiation. Strain B is much more sensitive than the usual E coli strain (Tables 10-2 and 4), and Witkin (1947) and Roberts and Aldous (1949) have shown that a large part of the killing seems to involve damage to the cell-division mechanism. E. coli B exhibits expo- nential killing with ultraviolet, is one of the few bacterial strains found to exhibit heat reactivation (Anderson, 1949, 1951a; Stein and Meutzner, 1950), recovers partially when held in hquid media (Roberts and Aldous, 1949)' and does not exhibit catalase reactivation (Latarjet and Caldas, 1952)' Strain B/r is much more resistant both to ultraviolet and X rays than strain B, exhibits a sigmoidal survival curve with ultraviolet, shows only a small degree of heat reactivation, does not recover from ultraviolet irradiation in liquid suspension (Roberts and Aldous, 1949), and exhibits a small but definite amount of catalase restoration. Both strains exhibit photoreactivation, and Demerec and Latarjet (1946) have shown that, for equal doses of ultraviolet, the rate of phage resistance-mutation induction is the same in each strain. At least two different mechanisms must be postulated to explain these differences in behavior of the two strains following ultraviolet irradiation and the catalase restoration may necessitate a third. Since both B and B/r exhibit photoreactivation, it seems likely that the mitial photo- 420 RADIATION BIOLOGY chemical events may be similar in the two orf^anisms, and involve the pro- duction of a product sensitive to \isihle lijj;h1 w Inch subsecjuently initiates ill llic two orjianisms two different series of reactions leadinji; t(j different, ultimately lethal effects. It is possible that both mechanisms are oper- ative in both strains witli the diricrence in sensitivity due to the single gene mutation being a shift in the relati\(' jiroportion of effects of radi- ation produced by the two mechanisms. With both B and B/r a non- photoreactivable residue exists, making it seem necessary to postulate in each strain an additional mechanism which is at least partially distinct from the photoreactivable mechanism. To these mechanisms involving ultraviolet must be added still different mechanisms for X rays since no photoreactivation occurs and, in strain B, no filamentous forms are produced (Roberts and Aldous, 1949). The X-ray effects, in turn, are subdivided into an oxygen-influoncod and an oxygen-independent mechanism. As with the photoreactivable and non- photoreactivable mechanisms of ultraviolet, it is not clear if the oxygen- dependent and oxygen-independent effects of X rays produce the same or different kinds of ultimately lethal damage to the cells. Thus the obser- vations on these two closely related strains indicate that a variety of mechanisms exist by which radiations produce bactericidal effects. In view of the ionization-density studies, it seems likely that different mechanisms are involved in the inactivation by high-energy radiations of spores and vegetative cells. A quite different mechanism by which radiation can cause the killing of bacterial cells has been demonstrated by Lwoff et al. (1950) in studies of lysogenic strains of bacteria. It was found that small doses of ultra- violet will induce, in nearly all the cells, the formation of active bac- teriophage resulting in lysis. Latarjet (1951) has shown that small doses of X rays have a similar effect and, furthermore, that visible light reduces the effect following both ultraviolet and X irradiation. Similar ultra- violet treatment will cause phage production and lysis of E. coli K12 (Weigle and Delbriick, 1951) which E. Lederberg (personal communica- tion) discovered to be lysogenic. A similar variety of mechanisms may be involved in mutation produc- tion since it is known that mutations are induced by the photoreactivable and nonphotoreactivable ultraviolet mechanisms and by X rays both in the presence and in the absence of oxygen. In addition, it is becoming apparent that different loci may respond quite differently to a particular radiation, as evidenced by the different types of mutation-dose curves and the differential response of two different mutations to X rays in the presence and absence of oxygen. Many problems have been uncovered which, when more thoroughly analyzed, promise to add much to the knowledge of the effects of irradia- tion in bacteria. In the more detailed analysis of the problems, parallel I EFFECTS OF RADIATION ON BACTERIA 421 studies of the genetic and lethal effects seem desirable, whenever possible, in order to determine the extent to which the mechanisms involved in producing these effects are common or distinct. Some of the problems which seem worthy of more detailed study are photoreactivation, the indirect and presumed direct effects of X rays, the various types of recovery which have been observed, the differences in response of cells grown under different conditions, and chemical protec- tion. Additional investigations of the physiological properties of irra- diated cells may help to determine the nature of the ultimate lethal dam- age produced by irradiation. 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R., anl potassium cyanide . . . fJaffeine Neurospora crassa Ophiostoma multiannu Ophiostoma multianm latum (latum Wagner ct al., 19.50 Fries, 1950 Methvl xanthines Fries and Kihlman, 1948 F)iazomethane Neurospora crassa So cch a ram i/crs cerev isiae .Jensen rt al., 1949 Acrifla\'ine Ephrussi and Hottinguer, 1950 It should be noted, particularly for chemical mutagens, that the muta- tions observed may not always be caused directly by the agents used or in the manner anticipated. Ulie unmasking of genetic variability, which is concealed in polyploid or particularly in heterokaryotic complexes, is a basis for induced variation in fungi which, if unrecognized, may lead to erroneous conclusions about the actions of inducing agents. It is also important to examine the experimental conditions to evaluate the possi- bility of selection of preexisting spontaneous mutants as opposed to direct causation (Lederberg, 1948), an evaluation which has not always been critically attempted. Spontaneous genetic changes are well known both RADIATION STUDIES ON FUNGI 433 in natural and laboratory cultures and many of the induced changes resemble those already seen in nature, a fact appreciated very early by investigators of fungi (e.g., Nadson and Philippov, 1932). This reflects the nonspecific character of the action of radiation and radiomimetic chemicals which typically increase the incidence of many different kinds of mutations simultaneously. It is reasonable to suppose that there must be many separate effects of radiation which pass unnoticed because the means for detecting them are lacking. Some of these may be biologically unimportant, w^hereas others may provide the underlying basis for some of the major observed results. For example, the number and character of the lethal effects are not known, although there is reason to suspect that many alternatives are involved. It will often be difficult to find adequate criteria to distinguish between primary effects of radiation and the secondary consequences of cell morbidity, a ubiquitous problem in radiation biology. ULTRAVIOLET RADIATION Several recent articles are available on general biophysical aspects of ultraviolet radiation, an agent which has been wddely used in experiments with fungi (Loofbourow, 1948; Giese, 1945, 1950; McLaren, 1949). Effects of ultraviolet on growth and respiration have been noted in yeasts and molds, as shown in Table 11-2. Table 11-2. Some Nongenetic Effects of Ultraviolet Irradiation OF Fungi Type of effect Organism Remarks Reference Stimulation Various yeasts, and Low doses only Nadson and Philip- mucors pov, 1928a Delayed budding or Various yeasts: Low doses Lacassagne, 1930: germination, retarded Rhizopus suinus Dimond and Duggar, growth rate Ustilago zeae 1940a, b; Landen, Aspergillus niger 1939; Zahl d al., 1939 Changes in respiration Saccharotnyces cerevisiae Giese, 1942; Giese and Swanson, 1947 Stimulation by slight doses has been reported with various yeasts {Saccharomyces cerevisiae, Saccharomyces ellipsoideus, Nadsonia fulvescens, Zygosaccharomyces priorianus) and molds (Mucor genevensis, Mucor guilliermondi) , based on increased growth on plates exposed to a graded amount of radiation (Nadson and Philippov, 1928a). The interpretation of such experiments is made uncertain by the possibility that stimulation is brought about by substances released from the few killed colls. 43-t RADIATION BIOLOGY Yeajst cells irradiated with ultraviolet of wave leufitlus 2800-3800 A (at somewhat higher do.ses than used l)y Xadson and Philippov, 1928a) did not begin to hud as early as the controls — in some cases they never budtled, whereas in others they finally grew like the controls (Lacassagne, 1930). Other investigators, working with somewhat shorter wave lengths, also ft)und delayed budding phenomena and killing in various species of Saccharomyces (Wyckoff and Luyet, 1931; Oster, 1934a, b, c). The spores of Rhizopiis suiniis show a similar behavior in that, after irradi- ation at 2050 A, they exhibit delayed germination or complete lack thereof, as well as a somewhat retarded growth rate of the germinated spores (I)imond and Duggar, 1940a, b). Usti- lago zeae has- been reported to behave in this way (Landen, 1939) , as has Aspcryillus niger (Zahl et at., 1939). Effects on respiration have been ob- served by various w-orkers (Oster, 1934a; Giese, 1942; Giese and Swanson, 1947). There appears to be an increase in en- dogenous respiration with a decrease in exogenous respiration of various sub- strates by S. cerevisiae (experimental conditions used by Giese, 1942, and Giese and Swanson, 1947: 2 X 10^^ cells/ml in a quartz Warburg vessel, Sterilamp as source of ultraviolet radia- tion, 10- to lo-min exposures, approxi- mate dose of 70 ergs/mm^/sec). A de- creased fermentation rate was also ob- served (Giese and Swanson, 1947). Wave-length Dependence in the Ultra- violet. For yeast, maximum killing effici- ency was found to lie between 2600 and 2700 A, and Fig. 11-1 (Oster, 1934c) is representative of the action spectra. The suggestion was made that "the effects of ultraviolet irradiation may result from the absorption of energy by . . . nucleoproteins" (Oster, 1934c), because of the corre- spondence between the killing action spectiiun and the absorption spec- trum of nucleoproteins (see Loofbourow, 1948; Giese, 1950, for discussions of the significance of action spectra). Similar results for killing and mutation have been obtained in various fungi as shown in Table 1 1-3. In a series of papers dealing with Chaetominm glohosum a sectorial colonial change, designated as "K/' has been found to be selectively produced at 0.22 0,30 0 24 0 26 0 28 WAVE LENGTH,^ Fig. 11-1. Comparison of the de- structive efficioncy of ultraviolet energy on yeast at different wave lengths. Ordinate, reciprocals of the energies required to "iciH" 50 per cent of the yeast cells. {Oster, 1934c.) RADIATION STUDIES ON FUNGI 435 wave lengths around 2800 A but not at longer wave lengths (around 8300 A), while "saltations involving modifications of growth rate and form, mycelium and perithecia are produced" in constant proportion to lethal effect at all wave lengths tested (2300-4047 A) (McAulay et al., 1945; Ford, 1947). However, experiments of 7-10 days duration (as were the irradiation exposures at wave length 4047 A) are open to question on the grounds of contamination by shorter wave lengths, as well as of changes in the organism. The percentage of colonies showing the K-type sector has reached as high in some experiments as 30-50 per cent (Ford, 1946), with the peak activity at 2804 A (McAulay and Ford, 1947). Table 11-3. Organisms Showing Wave-length Dependence for Killing AND Mutation with Ultraviolet Radiation Organism Remarks Reference Saccharomyces cerevisiae 2600-2700 A max. kill Oster, 1934c { stilago zeae 2400 A max. activity; 1.5 X 10'' ergs /mm 2 at 3130 A produced no kill Landen, 1939 Aspergillus niger 2537 A max. activity; 2 X 10^ ergs /mm 2 gave 30% kill at 3129 A; no effect noted at 3650 A Zahl et al., 1939 Trichophyton mentagrophytes 2650 A max. activity; kill Hollaender and Emmons, but no mutation between 1939, 1941, 1946; Em- 3400 and 4400 A mons and Hollaender, 1939 Aspergillus terreus 2537-2650 A max. activ- Hollaender and Emmons, ity; 2967 and 3150 A pro- 1946; Hollaender, San- duce mutation but less some, et al., 1945 efficiently Neurospora crassa 2650 A max. activity ; 2280 Hollaender, Sansome, et A gave kill but little mu- al, 1945 tation: at 2967 A both were low Penicillium notatum 2650 A max. activity- Hollaender and Zimmer, 1945 Chaetomium glohosum Exception to general ob- McAulay et al., 1945; Ford, servation ; see text for dis- 1946, 1947; McAulay and cussion Ford, 1947 The wave-length dependence of the production of K type and other saltants led to two conclusions: (1) a protein excitation near 2800 A gives rise to K type, and is responsible for most efficient production of other mutants, and (2) a second, less efficient process, can occur at all wave lengths tested to produce mutants other than K type (McAulay and Ford, 1947). Figures ll-2a,6 graphically summarize these points. The major emphasis was that protein absorption, not nucleic acid absorption, gave 130 RADIATION BIOLOGY ii«o to K mutations in this material. X rays wen^ found not to produce the K saltation (Ford and Kirwan, 1949; Ford, 1918; McAulay et ai, 1949), hut other morpholofjjical changes were produced. So-called "lethal mutations" were found with all wave lengths of ultraviolet examined and with X rays (Ford and Kirwan, 1949; Ford, 1948; McAulay et ai, 1949). These were, in fact, spores which germinated, produced a small amount of mycelium, and then stopped growing; it is impossible to say whether the cessation of growth is attributable to genetic mutation in the usual sense. Since C. (jlobomni is apparently unsuitable for ordinary genetic experi- ments, the basis for the K saltation remains (Mnially uncertain. In view 2500 3000 3500 WAVE LENGTH, A (a) 4000 2500 3000 3500 WAVE LENGTH, A (b) Fig. 11-2. Curve a sho\v.s the ratio of percentage K saltants to the percentage "oilier" saltants at different wave lengths. Curve h shows the optimum doses in joules per square centimeter for saltant production. Both curves are plotted against wave length: curve a, natural scale; curve b, logarithmic scale. Doses read downward to bring out the similarity between the curves. {McAulay and Ford, 1947.) of these uncertainties and of the general finding in other material of action spectra suggesting nucleic acid rather than protein excitation it seems reasonable to suppose that the interesting findings in Chadomium require an ad hoc explanation, and are not to be regarded as serious obstacles to the hypothesis that nucleic acid excitation is the most effective primary action of ultraviolet. It should be noted that the finding of a marked effect (in terms of killing and mutation) of ultraviolet-irradiated medium (Wagner et al., 1950) raises some question as to the complete propriety of any interpre- tation based solely on a consideration of certain absorptive characteristics of the organism (Bacq, 1951). The experiments show that toxic and mutagenic substances are formed by the action of ultraviolet on nutrient broth. Similarly, treatment of nutrient broth with hydrogen peroxide produces mutagenic activity. There are important differences between RADIATION STUDIES ON FUNGI 437 direct irradiation of the cells and irradiation of the medium, however. The former is so much more effective, dose for dose, that it seems hardly possible to interpret the difference solely in terms of greater absorption by the cells. Since activation of the medium is produced only by lamps with a quartz envelope, it appears that very short wave lengths, producing high yields of peroxide, may play an important role. Action spectra for mutagenesis by direct cell irradiation are clearly unrelated to the yield of peroxide. It is not yet possible to state to what extent the results pro- duced by irradiating the cells directly are brought about by the same mechanisms which operate when the medium is irradiated in the absence of cells, but these mechanisms are most likely of secondary importance. Table 11-4. Types of Survival Curves Obtained after Ultraviolet Irradiation Type of Morphological Organism element Reference curve irradiated Rhizopus suinus"\ Mucor dispersus] Sigmoidal Conidiospores Dimond and Duggar, 1941 Aspergillus melleus. . . Exponential Conidiospores Dimond and Duggar, 1941 Trichophyton nwnta- Sigmoidal Conidiospores Hollaender and Emmons, grophi/tes 1939, 1941, 1946; Emmons and Hollaender, 1939 Aspergillus terreus . . . Sigmoidal Conidiospores Hollaender, Sansome, ei al., 1945 Snccharomyces cerevisiae (haploid) Exponential Resting cells DeLong and Lindegren, 1951 Saccharomyces Sigmoidal Resting cells Sarachek and Lucke, 1953; cerevisiae (haploid) Pomper, unpublished Saccharo?7iyces Sigmoidal Resting cells Wyckoff and Luyet, 1931; cerevisiae (diploid) DeLong and Lindegren, 1951 ; Pomper, unpublished Ustilago zeae (haploid Sigmoidal Sporidia and Landen, 1939 and diploid) chlamydospores Streptomyces flaveolus Exponential Conidiospores Kelner, 1948 Streptomyces griseus. . Sigmoidal Conidiospores Savage, 1949 Aspergillus niger Sigmoidal Conidiospores Zahl et al, 1939 Rhizopus nigricans. . . Sigmoidal Conidiospores Luyet, 1932 Neurospora crassa. . . . Exponential Uninucleate microconidia Norman, 1951 Neurospora crassa . . . . Sigmoidal Multinucleate conidia Norman, 1951 Survival and Mutation Kinetics. There appears to be no clear sepa- ration of lethal and mutagenic effects on the basis of wave-length depend- ence, although nonmutagenic wave lengths can be lethal at very high doses. On the other hand, the dose-response curves for the two effects show marked differences. Table 11-4 lists the types of ultraviolet sur- i;i8 RADIATION H1()I-0(;V vival curx cs thai Ikiac been rcpoiicd with \aiii)iis liiii;;!. ()ii I lie liasi.s of tlio analysis carriod out with Nnirosjjora (Xorinaii, lUol ). the occurrence: of hotli sigmoidal and exponential sur\i\al curves amonji; tlie funsi may reflect dilTerenees in the number of inu-lei per spore. Exponential sur- vival was obtained with uninucleate conidia of Neurospora, whereas sigmoidal survival was correlated with a nnilt inucjeate condition (Atwood 001 0 1 2 3 4 5 6 7 DOSE, quanta X cnn"2 x 10''^ Fig. 11-3. Inactivation of Neurospora conidia by ultraA'iolet radiation. Uninucleate and multinucleate survival curves. Average number of nuclei per coiiidiuni: curve I, 1.0; curve II, 2.3; curve III, 4.2; curve IV, 5.9. {Norman, 1951.) and Norman, 1949; Norman, 1951). Figure 11-3 shows the survival curves obtained by Norman with uni- and multinucleate conidia of Neurospora. Similarly, the ploidy has been implicated as an important factor in j^east. The ultraviolet survival curves in yeast are generally sigmoidal, but those of haploids are of much lower order than those of polyploids (Sarachek and Lucke, 1953; Caldas and Constantin, 1951; Warshaw, 1952; Pomper, unpublished). Mutation frequency curves have been obtained with various fungi. Trichophi/ton mcntagrophytcs (Hollaender and Emmons, 1941), Neuro- spora crassa (Hollaender, Sansome, et al., 1945), Aspergillus terreus RADIATION STUDIES ON FUNGI 439 - 100' 50 iO (Hollaeiider, Raper, and Coghill, 1945), and Penicillium notatum (Hol- laender and Zimmer, 1945) exhibit more or less similar behavior, in that mutation increases to a maximum and then decreases rather erratically (Fig. 11-4). Decrease in the mutation f requeue}^ at higher doses was not observed with Streptotnyccs flaveolns (Kelner, 1948), although rather erratic behavior was noted. A series of inositolless mutants of .V. crassa have been used (Giles and Lederberg, 1948; Giles, 1948, 1951) to study the frequency of induced reversions to inositol independence. Similar ex- periments have been carried out with mutants of S. cerevisiae requir- ing adenine and uracil (Pomper, unpublished). Figure 11-5 shows a mutation frequency curve ob- tained with inositolless Nciirospora (Giles, 1951). The mutation fre- quency in measurements of rever- sions of this sort is usually fairlj^ low, so that accurate determina- tions cannot be made when the sur- vivors fall below a certain level. On the other hand, the experiments described at the beginning of this paragraph measured occurrence of morphological mutations, which in some instances reached as high as 30-80 per cent. Hence, the curves could be followed over greater dose ranges using small cell populations. Also in these studies, many different "mutations" of uncertain genetic status were obser^'ed as compared to the more restricted assay when using reversions under conditions of controlled genetics. These differences may be part of the explanation for the difference in shape of the mutation curves. An anomalous relation between the ultraviolet dose-effect curves for survival and mutation pro- duction is particularly apparent in Neurospora. Although uninucleate microconidia of Neurospora give exponential survival curves, suggesting a single-hit or single-target event, they do not give linear mutation curves. It would seem logical to expect that the frequency of the mutational event should be simply proportional to dose, since deviations from such proportionality would, in the case of lethal mutation, produce corre- sponding de\'iations from the exponential survival curve. It has been 50 «^ 10 ) \ 5 " \ 1 05 - •\ 01 - \ v» 0 05 - V 001 - o y /^ ""sV^^ o o - / ° o n 1 1 1 1 1 1 1 1 - 5 3 5 0 123456789 X 10"' ERGS/SPORE Fig. 11-4. Curve showing mutation pro- duction and killing. Top curve: change of survival ratio of fungus spores with in- creasing energy using 2650 A radiation. Lower curve: variation of percentage mutations of surviving spores. Both curves were obtained from the same material. [Adapted from Hollaender and Emmons, 1941.) 140 HADIATION mOLOGY shown l)y tlu* hctciokaryoii inctliod (Atwood, l!)5()) tliat sufliciciit reces- sive lethal mutations are induced in the nuclei of macroconidia to distort noticeably the exponential survival curve if these mutations were induced in micronidia according to the same dose-effect relation as in macro- conidia. It is possible that an inteiaction of unknown nature between the nuclei of the multiiuideate cells may be the explanation t'oi' this discrepancy. Data on ('. (ilohosKm have been interpreted as signifying a re(|uirement of two quantum hits at 2804 A to cause lethal mutation (Ford and Kirwan, 120 37401 37102 64001 0 100 200 300 400 ULTRAVIOLET OOSE, seC Fig. 1 1-5. Relation between ultraviolet dosage and frequency of reverse mutations in three inositolless mutants (all in c m f stocks). The dotted line for nmtant 89(501 is added to indicate the exceedingly low reverse-mutation rate for this mutant. {Giles, 1951.) 1949; McAulay et al., 1949), and earlier work with yeast (Oster and Arnold, 1934) suggested that different numbers of hits are required to bring about different degrees of ultraviolet action, as judged by the cri- terion of the amount of budding prior to cessation of growth. Such observations may be indicative of a manifold inactivation unit allowing budding to continue until mutually complementary surviving parts have been segregated from one another. Detailed studies of cell lineage in irradiated yeast might be very instructive. Modifying Effects on Ultraviolet Action. Various factors have been studied which modify the effects of ultraviolet radiation on fungi; some of these are listed in Table 11-5. Perhaps the most important is the marked RADIATION STUDIES ON FUNGI 441 ameliorating effect of visible light applied following irradiation with ultraviolet, a phenomenon which is fully discussed by Dulbecco (Chap. 12 of this volume). This has been shown for several kinds of ultraviolet effects on numerous organisms including the killing of Strcptomyces griseus, P. rtotatum, and S. cerevisiae (Kelner, 1949a, b), killing and muta- tion of .V. crassa (Goodgal, 1949), reverse mutation of inositolless Neuro- spora (Brown, 1951), killing and mutation of haploid and diploid Saccha- romyces (Pomper, unpublished), and suppression of enzymatic adaptation in Saccharomyces (Swenson, 1950). Photoreactivation provides a striking demonstration of the existence of intermediate states between the absorp- Table 1 l-fi. Factors Which May Alter the Effectiveness of Ultraviolet Factor Organism Remarks Reference Visible light Various fungi Reverses killing and mutation Kelner, 1949a, b Pretreatment with Aspergillus terreus Increases mutation Swanson and Good- nitrogen mustard rate gal, 1948 Pretreatment with Neurospora crassa Increases mutation Swanson et al., 1949 nitrogen mustard rate Pretreatment with Aspergillus terreus Increases mutation Swanson and Good- dinitrophenol rate gal, 1950 Methylcholanthrene Saccharomyces Sensitization to Hollaenderefa/., 1939 cerevisiae longer ultraviolet Pretreatment with Aspergillus terreus Slight increase in Swanson et al., 1948 infrared radiation mutation rate Postheating Yeast Increased killing Anderson and Dug- gar, 1941 Age of cells Various fungi Varying results Dimond and Duggar, 1941; Oster, 1934b; Dickson, 1932 Temperature at time Yeast Irradiation less effi- Oster, 1934b of exposure cient in killing in cold than at higher temperatures tion of ultraviolet cjuanta and their final biological effects, and it is economical to suppose that killing, mutation, and impaired enzymatic adaptation are mediated through similar mechanisms at the stage where photoreactivation occurs. It should be emphasized that all work reported with ultraviolet radiation must be scrutinized carefully to see whether or not the results have been influenced in an unrecognized manner by the effects of visible light. This is especially true of material published before 1949, when the photoreactivation phenomenon was so clearly demonstrated (Kelner, 1949a). Other factors or agents have been considered which might influence the sensitivity of microorganisms to ultraviolet radiation. Nitrogen mustard, 442 H\I)I\|-1()\ KIOLOGY ill siiliinuta^cnic concent r.it ion when adniinisterod with ultraviolet radi- ation (2r)87 A), has Ween toiind to jiixc an iiici'casc in imitation rate of A. Iirrciis of liOO 100 per cent oxci- that caused hy the iiiadiat ion aionc^ (Swaiisoii and (ioodgal, 1948). This work was iiniitcd to oIiscia at ion of morph()l()j>;ical \ariants. A microeoiiidial strain of M earuapora has been employed, both morphological and nutritionally exacting mutants being scored, in similar experiments leading to the same results, i.e., an increase in mutagenicity of ulti-aviolet radiation after a 30-min pretreatment with 0.1 per cent aciueous solution of nitrogen mustard (Swanson d al., 1949j. Pretreatment with dinitrophenol also increased the morphological muta- tion rate of A. terreus, whereas potassium cyanide had no effect (Swanson and Goodgal, 1950). Posttreatment with dinitrophenol and submuta- genic levels of nitrogen mustard also increased the mutagenic effectiveness of the radiation, suggesting to these authors an indirect action of the radiation. An interpretation based on direct action is, of course, e(iually tenable, since in our present state of ignorance it is as reasonable to assume semistable intermediate states of the genie material as extragenic chemical intermediates. A photodynamic sensitization by methylcholanthrene of S. cerevisiae to hght in the 3450-4500 A range has been noted (Hollaender d al., 1939). Pretreatment with infrared radiation has been found to raise slightly the mutation frequency at high doses of ultraviolet with .4 . terreus (Swanson et al., 1948). Prior exposure to heat did n(jt sensitize yeast cells to the lethal action of subsequent ultraviolet radiation, although reversal of the sequence did have a sensitizing effect (Anderson and Duggar, 1941). Older yeast cells have been found to be more resistant to killing than younger cells (Oster, 1934b) and a similar result has been reported for the spores of Rhizopus suinus (Dimond and Duggar, 1941). There appears to be a slight temperature effect with yeast, since for the same killing more energy is required at 8°C than at higher temperatures (Oster, 1934b). No effect of humidity was noted with .4. niger spores (Zahl ct al., 1939). The significance of such isolated observations remains obscure, but it is likely that satisfactory interpretations and comparisons of experimental data have been greatly hindered by lack of awareness of the influence of seemingly irrelevant factors on the final outcome. 44iere is a very con- siderable need to explore more thoroughly and under controlled genetic conditions the effects of age, nutritional state, and other eiu'ironmental factors upon the sensitivity of cells exposed to ultraviolet radiation. IONIZING RADIATIONS The ionizing radiations are free from the dependence on specific absorp- tion which is an outstanding characteristic of ultraviolet. The elemen- tary physical processes which they bring about within the cells involve RADIATION STUDIES ON FUNGI 443 much greater energies than in the case of ultraviolet. The much milder processes brought about by ultraviolet or by photodynamic action of even longer wave lengths are entirelj^ adequate, however, to produce the same biological effects which are apparently caused by ionizing radiation. It is to be expected, then, that comparison of the biological effects of different radiations solely on the basis of their energies is singularly uninformative. The major differences are found in comparisons of kinetic experiments with the different radiations, and of environmental modifying factors. Thus the survival of Neurospora microconidia is exponential with ultra- violet (Norman, 1951), but is complex with a higher order component with X rays (Giles, 1951). In yeast the order of the survival curves is similar to the ploid}^ with X rays, but much higher with ultraviolet (Pomper, unpublished; Sarachek and Lucke, 1953). Photoreactivation, the strongest modifying factor with ultraviolet, is not observed with X rays, whereas oxygen tension during irradiation, the strongest modifying factor with X rays, is not influential with ultraviolet. A^arious general effects of ionizing radiations on fungi have been reported. With yeast, no stimulation of growth was obser\'ed at low doses with X or a rays (Lacassagne and Holweck, 1930). Cell division of yeasts was retarded (Holweck and Lacassagne, 1930a). After higher dosages of X rays, cells often underwent two divisions before swelling up and dying (Holweck, 1930; Wyckoff and Luyet, 1931; Holweck and Lacassagne, 1930a; Brace, 1950; Henshaw and Turkowitz, 1940). The phenomenon of delayed death has been emphasized (Holweck, 1930; Wyckoff and Luyet, 1931) as a major difference between ultraviolet and X radiation but Oster (1934b) has observed the delay following ultra- violet. It has been reported that some enlarged cells obtained after X irradiation or exposure to radiimi have continued in culture as enlarged cells (Brace, 1950; Bauch, 1943). A suggestion of induced polyploidy has been made but cannot be accepted without genetic proof. Experiments with a rays (Lacassagne, 1930) and cathode rays (Wyckoff and Luyet, 1931) suggest that death occurs after a cell division. X-irradiated spores of Chaetomium cochtiodes all germinated but only a fraction survived to maturity (Dickson, 1932). Changes in the sexual pattern of certain yeasts and molds have also been reported. In a culture of normally isogamic Mucor genevcnsis, fre- quent heterogamic conjugations were observed after X irradiation (Nadson and Philippov, 1925). Two strains of this mold were isolated, one showing an increased amount of zygote formation with a decrease in sporangia, the second the reverse. Zijgorhynchus moUeri also exhibited the latter behavior (Nadson and Philippov, 1928b). The yeast Nadsonia fulrcsccns, normally heterogamic, responded to X rays by developing a series of abnormal sexual reactions, leading finally to complete loss of sexuality with greater doses (Nadson and Philippov. 1926). 444 RADIATION BIOLOGY Comparison of Different Ionizing Radiations. The dis.sipatioii of energy along the paths traversed by ionizing particles, rather than randomly throughout the material, is undoubtedly the major reason tor dilTerences in biological etTectiveness of physically equivalent doses of ionizing radi- ations of different quality (Lea, 1947). Some studies on the comparative etTectiveness of different ionizing radiations are summarized in Table I l-li. With A. terreus as test organism, the interesting observation has been made that densely ionizing radiations (neutrons and a rays) were more effective in killing but less effective in mutating than 7 and X rays (Stapleton and Martin, 1949). A further difference has been ol)served in that survival followed a sigmoidal curve whereas mutation production was linear with dose (Stapleton and Martin, 1949). Aspergillus niger spores behave similarly to .1. terreus, as regards comparative lethal effec- tiveness of densely ionizing and more disperse radiation, while .S. cere- Table ll-C. Relative Effectiveness of Ionizing Radiations in Causing Killing and Mutation in Fungi Organism Effect Order of decreasing effectiveness R(>ferencc Aspergillus terreus . . Killing Neutrons and a rays > X rays and Stapleton and 7 rays Martin, 1!)4<) Asperf/illus terreus. . Mutations 7 rays > X rays > neutrons and Stapleton and a particles Martin, li)49 Aspergillus niger. . . . Killing Dense ionization track > disperse Zirkle, 1(140 Yeast Killing a rays > X rays Zirkle, 1940; Tobias, 1952 Yeast Killing X rays > a rays Holweck, 1930 visiae showed only a slight effect, and Escherichia coli a complete reversal of effectiveness (Zirkle, 1940). There appears to be some contradiction in the data on yeast, since a rays have been reported to be more effective (Tobias, 1952) and less effective (Holweck, 1930) than X rays; and longer X rays (8 A) to be less efficient in killing than shorter radiation (2 A) (Holweck and Lacassagne, 1930b). At the point of 50 per cent lethality, twice the cathode-ray dose, three times the X-ray dose, and six times the ultraviolet exposure are required for Rhizopus nigricans spores, as compared to yeast cells (Luyet, 1932). In general, such differences between the effectiveness of radiations of different quality are indicative of a rather close localization of the effects to points along the paths of ion- izing particles. If the effects were not localized, it would be impossible to explain the results in terms of the spatial relations of the biological targets and the nonrandom distribution of elementary processes. Present knowledge provides no reasonable alternative. Dose-effect Curres. Survival curves have been obtained with various fungi, a few of which are listed in Table 11-7. The survival curves RADIATION STUDIES ON FUNGI 445 Tablk 11-7. Types ok Survival Curves Obtained with X Rays for a Few Fuxfii Organism Type of curve Remarks Reference Sarrharomyces cerevisiae (diploid) Saccharomyces cerevisiae (haploid) ToruJopsis rremoris . . . . Chaetomiuni (/lobonuin. . Rhizopus nigricans Streptouuices flaveolus . . . Sigmoidal Exponential Exponential Sigmoidal Sigmoidal Exponential Exponential Sigmoidal Exponential curve has been reported (Hen- shaw and Turkowitz, 1940) No genetic analysis Five hits to kill Wyckoff and Luyet, 1931; Latarjet and Ephrussi, 1949 Latarjet and Ephrussi, 1949 Anderson and Turkowitz, 1941 Ford and Kirwan, 1949 Luyet, 1932 Kelner, 1948 Streptoinijces griseus. . . . Savage, 1949 Stapleton and Mar- tin, 1949 Aspergillus terreus 2 0 reported for yeast exposed to X rays have been sigmoidal with diploid S. cerevisiae (Wyckoff and Luyet, 1931 ; Latarjet and Ephrussi, 1949) but exponential with a haploid strain (Fig. 11-6). Independent confirmatory re- sults have also been obtained, showing that, with ionizing radiations, a hap- loid yeast is killed logarithmically, a diploid sigmoidally, and a tetraploid also sigmoidally (Pomper, unpub- lished; Tobias, 1952; Lucke and Sarachek, 1953). An exponential survival curve has been reported for presumably diploid S. cerevisiae ex- posed to X rays (Henshaw and Turko- witz, 1940), and here a genetic analy- sis of the presumed diploid would be a helpful datum in appraisal. Tobias (1952) has reported that certain stocks obtained from originally diploid cells surviving X irradiation were found to have survival curves differ- ing from those of their diploid pro- genitors in being more nearly expo- nential. One is tempted to interpret this finding as indicative of the loss of a large portion of the genetic appa 8 16 24 DOSE, rx 10^ Fig. 11-6. Survival of haploid and diploid S. cerevisiae exposed to X rays. The inactivation of yeast cells by X rays. Curve I, haploid cells; curve II, diploid cells. (The data, from Latarjet and Ephrussi, 1949, were adapted by Norman.) 1 ir> RADIATION niol.oOY rntiis, l(';i\iii^ tlic initially diploid cell in a ('ondition more nearly' appioxi- inating the liaploid. Kxpcrimonts with genet ically marked dij)l()i(ls would he especially informative on tliis point. There has been consideruhle confusion in interpreting sigmoidal sur- vival cur\-es, particularly with regard to dctcriniiiing tlic number of events necessary to kill. The dithculty arises largely from failure to recognize that one must distinguish between two completely difTerent meanings of the luimber of events: (1) number of hits that are necessary, and (2) number of units that must be inactivated. The difference between these interpretations and the reasons for preferring the latter in most cases have been discussed by Atwood and Norman (1949). A study of the survival kinetics in material in which the mitotic stage, the ploidy, or the number of nuclei per cell can be systematically varied will be most profitable. The lack of such criteria precludes, in our present rudimentary state of knowledge, any satisfactory interpretation of sur- vival data, and for this reason most of the experimental observations in Tables 11-4 and 7 must be simply noted without comment. Curves relating mutation production to radiation dosage have been obtained for X rays with A. terreus (Stapleton and ]\Iartin, 1949), N. crassa (Sansome et al., 1945), and P. notatum (Hollaender and Zimmer, 1945), with morphological mutations as the criterion, mutation being essentiall}^ proportional to dose in all cases. Streptomyces flaveolus differed in that the curve broke consistently downward between 100,000 and 200,000 r (Kelner, 1948). A critical study of mutation frequency is that recently carried out with Neurospora (Giles, 1951) using reversions to inosital independence as assay procedure. A linear relation between dose and frequency of mutations was obtained with X rays, as shown in Fig. 11-7. Modifying Factors. Certain environmental or cultural conditions have been found to exercise a considerable effect in modifying results obtained with ionizing radiations (Table 11-8). Rather striking effects have been obtained, both in augmenting and in decreasing the magnitude of the results expected from a given dosage. Pretreatment with infrared radi- ation has been found to increase significantly the morphological mutation rate with A. terreus and Trichophyton mentagrophytcs (Swanson et al., 1948; Hollaender and Swanson, 1947). The range reported as active was 7000-18,000 A, with a maximum at 10,000 A. Heat had no saltating effect on Chactomium cochliodes (Dickson, 1932). That temperature may play an important role is suggested by the obser- vation with Saccharomyces ellipsoideus of a reduction in killing from 37 to 19 per cent at 13,000 r by holding the cells on ice for 10 days after irradi- ation (Latarjet, 1943). This observation led to the suggestion that the radiation damage may actually be caused in large part by the secondary chemical reactions occurring after irradiation, rather than the primary, RADIATION STUDIES ON FUNGI 447 Table 11-8. P" actors Which May Alter X-ray Effects Factor Organism Effect Reference Pretreatment with Aspergillus terreus and Increase muta- Swanson et al., infrared radiation Trirhophi/ton iiii-iitn- tion rate 1948; Hollaender grophyteN and Swanson, 1947 Postincubation at Saccharomijces Decrease killing Latarjet, 1943 reduced temper- elUpsoideus (in some experi- atures ments) Age of culture Saccharoiiu/ces Older cells more Lacassagne and elUpsoideus easily killed Holweck, 19:30 Age of culture Chaetoniium rorhliodes Older mycelium higher mutation rate Dickson, 1932 Moisture content. . . Sarrharonn/rcs Dried cells higher Dunn et at., 1948 cerevisiae survival Moisture content. . . Aspergillus hircus Dried spore.s Stapleton and higher survival Hollaender, 1952 Absence of oxygen . . Aspergillus terreus Reduction of Stapleton and killing Hollaender, 1952 Absence of oxygen . . Torulopsis cremoris Reduction of Anderson and killing Turkowitz, 1941 50 < 40 - O o V3 z ? 30 2 0 z o I- < 3 I 0 - > UJ r 37401 ,-89601 10 20 30 X- RAY DOSAGE, r X lO' 40 Fig. 11-7. Relation between X-ray dosage and frequency of reverse mutations in two inositolless mutants (in c //i/ stocks). {Giles, 1951.) 448 KADIATION HIOLOGY tompcraturo-insenhiitivo roMction, and tliat the crt'cct of the cokl treatment was to slow clown these injurious reactions so that the cell might recover (Latarjet, 1913). Another paper notes no effect on this same yeast of holding cells on ice for several days after X or a irradiation (Lacassagne and Hoi week, 1930). Old resting cells of S. ellipsoideus have been found to be more sensitive than young dividing cells (Lacassagne and Holweck, 1930; cf. Oster, 19341)). Time of irradiation apparently has little effect (Lacassagne and Holweck, 1930), although one might expect that too prolonged an expo- sure might lead to experimental complications. Toxic substances formed in the suspending medium might play an important role if experiments are prolonged, although under ordinary circumstances this has not been found to be a serious factor. Experiments in which two other variables, oxygen and humidity, have been shown to be important in modifying the lethal effects of X rays have aroused considerable interest. Dried S. cerevisiae was found to be more resistant to X rays than normal (wet) cells (Dunn et ai, 1948). Wet spores of A. terreus were more susceptible to the killing action of X rays than dry spores (Stapleton and Hollaender, 1952). X rays kill spores of A. terreus more efficiently in the presence of oxygen than in its absence (Stapleton and Hollaender, 1952). A similar observation had earlier been made with the yeast Torulopsis cremoris; viz., in the absence of air a greater dosage was necessary to bring about the same killing action (Anderson and Turkowitz, 1941). It was observed that this yeast, if grown without shaking or aeration and then irradiated, was more resistant than if grown similarly but shaken prior to irradiation. The observation takes on added validity in view of recent results showing that the oxygen effect is general. Concbmons. In summary, it may be said that fungi, in common with other organisms, may be killed or mutated by various radiations and that these effects may be intensified or decreased by adjunctive treatments. The results of some of the adjunctive treatments are most easily inter- preted as influencing the indirect radiation action, e.g., the chemical inter- mediates produced by radiation which subsequently react with the bio- logically significant materials. On the other hand, the differences in effectiveness of ionizing radiations of different quality are most easily interpreted in terms of sharply localized effects. These conditions are not, of course, mutually exclusive. It seems reasoiuible to ascribe a large role to events in the nucleus, based on the obssorvations of haploid and diploid yeast survival curves (Latarjet and Ephrussi, 1949; Tobias, 1952; DeLong and Lindegren, 1951 ; Pomper, unpublished; Lucke and Sarachek, 1953), and on the differences between uni- and multinucleate conidia of Nenrospora (Norman, 1951). The relation, if any, between these nuclear effects and mutation per se RADIATION STUDIES ON FUNGI 449 is not known. From a phenogenetic point of view it is difficult to decide what is meant by dominant lethal mutations in microorganisms. Reces- sive lethal mutations are not, by definition, lethal except in haploid, effectively uninucleate organisms. Furthermore, it is known from studies with various organisms that radiation (especially at higher doses) may cause cytoplasmic changes, and so the role of the cytoplasm, although probably of secondary importance, cannot be disregarded. 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(1940) The radiobiological importance of the energy distribution along ionization tracks. J. Cellular Comp. Physiol., 16: 221-235. Manuscript received by the editor Sept. 6, 1952 CHAPTER 12 Photoreactivation Renato Dulbecco Kerckhoff Laboratories of Biology, California Institute of Technology Pasadena, California Introduction. Historical note. Photoreactivation in bacteriophages: Conditions of inactivation — Methods for studying photoreactivation — Adsorption of phage on bacteria a necessary prerequisite for photoreactivation — Comparison of survival curves after ultraviolet treatment in presence and in absence of photoreactivation — Kinetics of photo- reactivation in phage T2 in condition of single infection — Kinetics of photoreactivation of phage T3 — Kinetics of photoreactivation of phage T2 in condition of multiple infec- tion— Action spectrum of the photoreactivating light — Action of chemical substances on photoreactivation — Photoreactivation of bacteriophages inactivated by agents other than ultraviolet — Photoreactivation of the induction process of phage carried lysogenically. Photoreactivation of plant viruses. Photoreactivation of bacteria: Conditions under ivhich 'photoreactivation occurs — Effect of the growth stage of bacteria on photoreactivation— Comparison of survival curves after ultraviolet treatment in presence and absence of photo- reactivation— Kinetics — Action spectrum of the photoreactivating light — Chemical actions connected with photoreactivation — Action of photoreactivation on the induction of mutation by ultraviolet in bacteria. Photoreactivation in Streptomyces and fungi. Photoreactiva- tion in yea-^t. Photoreactivation in protozoa. Photoreactii>ation in echinoderm zygotes and gametes. Photoreactivation in salamander larvae. Photoreactivation in higher plants. Conclusions and summary. References. 1. INTRODUCTION The term "photoreactivation" designates the phenomenon in which changes produced in different types of organisms, mainly microorganisms, by ultraviolet radiation of wave lengths around 2500 A can be counter- acted if the irradiated organisms, under proper conditions, are exposed to a radiation of longer wave length, in the range between 3300 and 4800 A. The phenomenon received its designation from the fact that it was first n(3ted as a reversal of the inactivation produced by the ultraviolet radiation in some microorganisms (fungi, yeasts, bacteria, bacteriophages) ; how- ever, it includes reversal of effects of ultraviolet radiation other than inac- tivation, such as mutation, delay in time of onset of division in echinoderm eggs and in protozoa, decreased vigor in protozoa, and even morphological changes. Since the outstanding feature of photoreactivation in microorganisms 455 45() HADIXTION BIOLOGY is the ro\orsal of the inaclivation, the incaiiiii^ of iiiactivation iiiuhI he clarilirtl. Tlic clcliiiition is a rolafivc one: a microorganism after treat- ment witli a radiation is inaetixc if in ^iven standard conditions it is not al)le to give rise to inde(initemviltii)lication. 'I'his is tested by determining whether or not indefinite growth can be obtained by placing the organism immediately after irradiation in darkness and at optimum temperature into a nutrient medium. Growth is considered absent if no visil)le colony is formed. A fraction of the organisms judged inactive by this technicjue may be judged active if the technitiue is changed; examples are known in bacteria in which the type of growth medium used or the temperature of incubation afTects the fraction of organisms able to give rise to detectable growth. In bacteriophages the criterion is still more imperfect since an active phage particle can develop a colony only if the first sensitive bac- terium infected by it bursts within a given time of incubation. In spite of this imperfection, a fairly precise method for the study of reactivation phenomena can be developed by strict standardization of techni(iue. 2. HISTORICAL NOTE Photoreactivation was discovered and its generality appreciated by Kelner (1949a), who observed reversal by visible light of ultraviolet inac- tivation in spores of Streptomyces griseua. Previous observations of the phenomenon can be found in the older literature, the most pertinent of which is an observation by Whitaker (1941-42) on the ability of visible light to counteract the action of ultraviolet on Fucus eggs. A review of older observations has been given by Kelner (1950b). At this time the chemical and biological mechanisms involved in ultra- violet effects and in photoreactivation are very imperfectly understood, and the observations that have been reported for various organisms can- not yet be discussed from a unified point of view. Ultraviolet can affect any substances which absorb the radiation. To this class of substances belong the nucleic acids, the proteins, and numerous smaller molecules with specific functions. There have been no reports of photoreactivation of enzymes inactivated by ultraviolet; however, the following significant observation has been published by Shu gar (1951). Crystalline triose- phosphate dehydrogenase, which is firmly combined with diphosphopyri- dine nucleotide (DPN), is partially inactivated during preparation, owing to oxidation of essential SH groups, and can be reactivated by exposure to light of the near-ultraviolet spectrum, with a peak at 3400 A, which is therefore in the absorption band of reduced DPN. The mechanism of reactivation could be a reduction of S — S groups consequent to light absorption in contiguous reduced DPN molecules. Here are found several features which occur in photoreactivation: the spectral range involved is similar; only a fraction of the enzyme activity can be reac- PHOTOREACTIVATION 457 tivated, only the S — S groups contiguous to reduced DPN molecules being reduced; and reactivation is a reaction of approximately first order. Reactivation of triosephosphate dehydrogenase by light may well be a model for photoreactivation. The latter phenomenon differs appar- ently because it affects only functional ultraviolet changes related to growth, the simplest of which is probably the formation of adaptive enzymes (Swenson and Giese, 1950) ; however, this may be so because photoreactivation is an indirect process, involving substances commonly found in the cytoplasm of cells, and because the most outstanding dam- ages produced by ultraviolet are those affecting growth. In this review the observations made on different organisms will be dis- cussed separately. By far the most detailed cjuantitative measurements have been made with the coli phages of the T series. These will be pre- sented first, and some theoretical notions verifying these data will be developed. ]\Ieasurements of photoreactivation in bacteria are almost as detailed as those on phage, and these data, too, can be accounted for on the basis of a few simple assumptions. However, it is remarkable that the formal schemes developed for photoreactivation in phage and bacteria are quite different and seemingly incompatible. Since it does not seem reasonable to assume radically different mechanisms for so obviously similar phenomena, both theories must be viewed with suspicion, and a deeper interpretation q{ all the data must be sought. In the remainder of this review the scattered data which have been reported for other organisms will be briefly summarized. 3. PHOTOREACTIVATION IN BACTERIOPHAGES Photoreactivation has been studied in the bacteriophages of the T group active on Escherichia coli strain B (Dulbecco, 1949, 1950). 3-1. CONDITIONS OF INACTIVATION The bacteriophages used are very stable, and can be kept for months as suspensions in buffered saline solutions. Such suspensions are inac- tivated by exposure to an ultraviolet source, which in the majority of the experiments has been a G.E. germicidal lamp, emitting 80 per cent of its energy in the wave length 2537 A. After irradiation a certain fraction of the particles is inactive. The logarithm of the fraction that is still active after a given ultraviolet dose, plotted versus the dose, gives the so-called "survival curve," whose shape is characteristic for a given phage. 3-2. xMETHODS FOR STUDYING PHOTOREACTIVATION If equal samples from a suspension of phage irradiated with ultraviolet are plated with sensitive bacteria on two equal plates and one is incubated -loS It ADI AIION moLOGY ill ilaikiicss and the other iiiidcr a .source of white hglit, tlie j^hilc; kept in the hf^ht shows after incubation a much greater numl)er of bacteriophage coh)nies; this is the basic experiment of photoreactivation of bacterio- phages. A simihir experiment can be done by mixing bacteria and bac- teriophages in a nonnutrient medium, usually a buffer solution, so that adsorption of tiie bacteriophages onto the sensitive bacteria can take place, but not growth. The mixture is exposed to a white light and then phited with sensitive bacteria on a plate of nutrient agar, which is incu- bated in darkness. lioth these methods are useful. The first has been called the method of photoreactivation on the plate; the second, the method in liquid. The choice of the method to be used depends on the type of information sought. The method on the plate is used for orientation experiments, for testing for c|ualitative effects, and for some quantitative studies, par- ticularly with phages which adsorb slow^ly (e.g., Tl and T5). The method in li(iuid is generally used in quantitative work; it has been suc- cessively applied to bacteriophages T2, T3, T4, and TO. 3-3. ADSORl'TIOX OF I'lIAGJ-: OX BACTERIA A NECESSARY PREREQUISITE FOR PHOTOREACTIVATION Free phages, inactivated with ultraviolet and then exposed to the photoreactivating light in the absence of bacteria, are not reactivated ; for photoreactivation to occur the particles must be adsorbed on the sensitive bacteria. Demonstration that adsorption is required for photoreactiva- tion has been obtained by mixing sensitive bacteria and irradiated bac- teriophages under conditions which do not permit adsorption, exposing the mixture to light, and then plating a sample from the mixture; the fraction of active particles remains unchanged. The condition for non- adsorption can be realized either by suspending phage and sensitive bac- teria in a solution which has an ionic compo.sition unfavorable for adsorp- tion or by mixing a phage with cells of a mutant of E. coli B resistant to the phage (e.g., phage Tl and bacteria B/1). Two types of adsorption of bacteriophage particles on the sensitive bacteria occur, depending on the ionic strength of the medium: when the ionic concentration of the medium is too low, adsorption is reversible — the adsorbed phage can be eluted with distilled water (Puck et al., 1951); at high ionic concentration adsorption becomes irreversible. Photo- reactivation takes place only if the phage has been adsorbed irreversibly (Dulbecco, unpul)lished). Bacteriophages adsorbed on bacteria in a medium which does not per- mit growth remain perfectly photoreactival)le over a period of several hours. The medium may l)e either a hulTcr, in which case the bacteria are taken from a logarithmic pha.se culture, washed and resuspended in the buffer, or a .synthetic medium containing a limited amount of glucose PHOTOKEACTIVATION 459 as the sole carbon source, in which the bacteria have grown to complete exhaustion of the sugar. If, on the contrary, the bacteriophages are adsorl^ed on l)a(^teria in a medium supporting growth, the photoreactiv- ability of the phages decreases with time elapsed between infection and exposure to the photoreactivating light. For example, for bacteriophage T2 adsorbed on E. coli B at 37^C in beef extract, very little or no photo- reactivation can be obtained if exposure to white light is started 30 min after infection. Phage inactivation is obtained also by irradiating bacteria infected with active bacteriophages (Luria and Latarjet, 1947); also in this case photoreactivation can be obtained (S. Benzer, personal communication). Photoreactivation is also obtained if the inactive phage is adsorbed on sensitive bacteria previously irradiated with ultraviolet. 3-4. COMPARISON OF SURVIVAL CURVES AFTER ULTRAVIOLET TREATMENT IN PRESENCE AND IN ABSENCE OF PHOTOREACTIVATION Survival curves (see Sect. 3-1) of different phages in absence of photo- reactivation are of two types: for the large phages (T2, T4, T5, TG) they have an initial curvature with dow^nward concavity and then tend to straight lines; for the small phages (Tl, T3, T7) they are straight near the origin and then decrease in slope, tending again to straight lines (Dulbecco, 1950). Survival curves obtained after the irradiated samples have been exposed to photoreactivation have similar shapes as the curves in dark- ness, but they are shifted in the direction of a higher survival. The comparison of the curves obtained for a given phage in darkness with those obtained after maximum photoreactivation shows that if the ultimate straight parts of the two curves are extrapolated back toward the origins, they intersect in the proximity of the abscissa. For the larger phages, which have a curvature with downward concavity, the inter- section point lies to the right of the origin. For the small phages it lies to its left (Fig. 12-1) (Dulbecco, unpublished). The reason for this behavior is not yet clear. In the section on bacterial photoreactivation (Sect. 5-3) it will be shown that in bacteria a different relation between survival curves in dark and after photoreactivation has been observed, a relation which has been called the "principle of constant ultraviolet dose reduction." It should be emphasized here that the same relation does not hold in bacteriophage reactivation. The methods for determining the existence of a constant dose reduction will be discussed in Sect. 5-3, where the explanation of Fig. 12-1, in which the two tests for such a determination have been applied to phage, both with negative results, is also given. For every phage the survival curves in darkness and after maximum 460 RADIATION BIOLOGY photoreactivatioii tend to striiight lines with ditTerent slopes. The slope is an important (luantitative datum. In fact, if a survival curve of an irradiated population is a straight line, its slope determines the cross section for the radiation of the individual elements. In multiple-hit curves the slope of the straight line to which the curve tends at high doses of the radiation has the same meaning; in an unhomogeneous population the final slope measures the cross section of the individuals of the class ULTRAVIOLET DOSE , seC 0 100 200 300 ULTRAVIOLET DOSE , seC 400 10 20 30 40 50 60 10" ; io'2 10" \ \ '^ \ "-- \ \ ^M__j;>- \ ^ o Q > lOO 50 ^ 1 "s^ \, \ \ ^ ^ \ V K^ X j _^,^ \ in^ T \ k o o o ^ 30 r 20 10 >- I o 0 100 200 300 400 DARK- ULTRAVIOLET DOSE.sec (a) 60 0 10 20 30 40 50 OARK-ULTRAVIOLE T DOSE , sec (b) Fig. 12-1. Survival curves in darkness and after maximum photoreactivation for (a) phage Tl and (b) phage T2. Curves I are for darkness; II, after maximum photoreac- tivation; III, curve giving the ultraviolet dose corresponding to a given survival after photoreactivation versus the dose corresponding to the same survival in darkness (see Sect. 5-3). For survival curves, log survival (left-hand scale) is plotted versus the ultraviolet dose in seconds of exposure (upper scale). For lower curves, light-ultra- violet dose in seconds of exposure (right-hand scale) is plotted versus dark-ultraviolet dose (bottom scale). with least cross section. The fact that in phages the survival curves in darkness and after maximum photoreactivation have different final slopes can be taken as a demonstration that the cross section of the particles to ultraviolet is reduced after photoreactivation. This means that the damage produced by ultraviolet in bacteriophages can be divided into two classes, one completely photoreactivable and the other nonphotoreac- tivable, with a constant ratio, independent of the ultraviolet dose. Exist- ence of these two classes of ultraviolet damages can be explained under two different assumptions : either ultraviolet light produces two different types of chemical effects, of which only one is photoreactivable; or only PHOTOKEACTIVATION 461 one type of damage is produced but not all the bacteriophage particles are exposed to the photoreactivating mechanism. The cross section of phage particles to ultraviolet can thus be sub- divided formally into two sectors, only one of which is photoreactivable. The nonphotoreactivable sector is measured by the ratio slope of straight part of survival curve after photoreactivation ~ slope of straight part of survival curve in darkness The photoreactivable sector a may be taken as a measure of the photo- reactivability of different phages. Data for the seven phages of the T group adsorbed on E. coli B are given in Table 12-1. It is interesting to Table 12-1. Photoreactivity of the Phages of the T Group Photoreactivable Sector a Phage of Cross Section Tl 0.68 T2 0.56 T3 0.39 T4 0.20 T5 0.20 T6 0.44 T7 0.35 note that among the related phages T2, T4, and T6, phage T4, which is much less photoreactivable, apparently contains less nucleic acid than the other two (Luria et al., 1951). A similar correlation occurs also in plant viruses (Sect. 4). The ratio of the two sectors of the total cross section is not a charac- teristic of the phage alone, but is somewhat affected by the host bacterium in which photoreactivation occurs. Phage T2 has a slightly higher photo- reactivable sector if adsorbed on E. coli B/1 than if adsorbed on E. coli B (Dulbecco, unpublished). 3-5. KINETICS OF PHOTOREACTIVATION IN PHAGE T2 IN CONDITION OF SINGLE INFECTION 3-5a. Effect of Different Doses of Photoreactivating Light at Constant Intensity. A sample of ultraviolet-irradiated phage is adsorbed on sensi- tive bacteria in darkness and then exposed to a photoreactivating light of constant intensity; at regular time intervals samples are taken and assayed for active phage. The number of active particles, p{t), increases with the time of illumination; if exposure is continued for a long time a maximum number of active particles, p(=c) is reached, after which any further illumination is without effect. In experiments made with the same ultraviolet-irradiated samples with reactivating light of different intensities, approximately the same maximum is reached, as shown in 402 KADIATIUN UIOLOCJY Fig. V2-2, Init in tiilTereiit times, shorter times being required iit higher intensities, within certain hmits (see also Seet. 3-5b). If the Hght intensity is very high, continuation of tiic illuininalion aftci- the maximum is readied results in loss of infective centers; this elTect is not specific for the photoreactivated particles, since it can be obtained also when bacteria infected with active particles are exposed to a photo- reactivating light of high intensity. Therefore a damaging effect of the photoreactivating light on the phage-bacterium complex interferes with the process of photoreactivation. This damage of the phage is not due to 6 X 10^2 0 10 20 30 40 50 60 70 EXPOSURE TO REACTIVATING LIGHT, min Fk;. 12-2. Fraction of active particles in phaj^e sample as a function of the time of illumination and of the light intensity. Phage T2 was irradiated for 20 sec with the germicidal lamp, adsorbed on resting bacteria, and illuminated in liquid at 37°C. Curve I was obtained with a light of intensity 10 (in arbitrary units), curve II with a light of intensity 2.9, and curve III with a light of intensity O.G. {Dulhecco, 1950.) the light inactivation of the bacteria (IloUaender, 1943). This re(iuires still higher doses (Dulbecco, unpublished). The damaging effect can be reduced by reducing the intensity of the reactivating light ; this, as shown later, affects photoreactivation only to a small extent, but decreases the damaging effect. The logarithm of the fraction of photoreactivable particles that have not been reactivated, log fl — p(/)/p(=o)], plotted versus the time of illumination with a light of constant intensity is called the "photoreacti- vation curve." In the case of phage T2 this curve is very nearly a perfectly straight line (Fig. 12-8). Curves determined with the greatest accuracy show one and sometimes two slight deviations from linearity. The first deviation always occurs at very short periods of illumination, and is due to a relatively higher effi- PHOTOUEACTIVATION 463 ciency of the first short period of iUumiiiatioii. It can he eliminated by usinji; a fiashinfj; instead of fontinuous li^ht, ;is will be shown in the section on the effect of interrupted liftht (Sect. 3-3(1). A second deviation may occur at hiiih intensities and long exposures, owing to the inactivation phenomenon mentioned previously. These two deviations find an expla- nation in accessory phenomena and do not af!"ect the linearity of the curve in principle. The linearity of the curve shows that a photoreactivable particle is reactivated by one hit of the reactivating light, or, in other words, that the transformation of photoreactivable into photoreactivated particles follows a first-order reaction. The slope of the photoreactivation curve is called 0 5- I 0.1 20 120 140 40 60 80 100 TIME OF ILLUMINATION ,min Fig. 12-3. The logarithm of the fraction of photoreactivable particles that has not been reactivated after a given time of illumination, 1 — p(0/p( «= ), plotted against the time of illumination in minutes. Phage T2 was irradiated for 20 sec with the germicidal lamp, adsorbed on bacteria in buffer, and ilhuninated in liquid at 37°C. (Dulhecco, 1950.) the "rate of photoreactivation." It measures the probability that a given photoreactivable phage particle is photoreactivated in unit time. The fact that T2 phage particles which have received several ultraviolet hits are photoreactivated by one photoreactivating hit is an important item of information. To explain this some particular mechanism either in the inactivation or in the reactivation might be conceived. It might be considered, for example, that ultraviolet inactivation of phages consists in formation of an inhibitor on a given essential nucleotide, and that photo- reactivation consists in the removal of the inhibitor. However, a T2 particle contains approximately 5 X 10^ nucleotides, and it adsorbs 10^ ultraviolet quanta for each hit (M. R. Zelle, personal communication). Therefore, for each hit only one-fiftieth of the nucleotides have absorbed a light fiuantum. This absorption would affect the given essential nucleo- 4()4 R \i)i \'n()\ liioi.ocY tide only if oiuM-f2;y can be transmitted to it from a group of 50 neighboring nucleotides or if an inhibitor localized on any nucleotide of this group can alTect the essential one, for example, steiically, as well as pre\'ent other inhibitors from localizing on the same group. Both these assumptions seem rather artificial. Another explanation of the one-hit character of the T2 photoreacti- vation is to assume that photoreactivable inactivation consists of a luimber of independent damages, at least one per photoreactivable hit, and that one quantum of the reactivating light is able to affect them all, by some kind of trigger mechanism producing numerous molecules capable of photoreactivating. This interpretation is also very artificial. Owing to the difficulty in interpreting T2 photoreactivation as a reversal of ultraviolet damage, a different position may be taken, and it may be considered that photoreactivation does not affect the ultraviolet damage itself, but it avoids its consecjuences by making available a sub- stance normally produced in the infected bacterium, which is no longer produced as the consequence of ultraviolet inactivation. As a model, the possibility might be considered that a phage enzyme, E, acting upon a bacterial substrate A, transforms it into B, this step being essential in phage growth, and that the irradiated enzyme is not able to do so any longer. If now the system is exposed to the photoreactivating light, a photochemical product, B*, is produced, which then produces B, and the enzymatic reaction is bypassed. In condition of continuous illumination, in which B* is present in steady concentrations, the probability of pro- ducing B is proportional to the time of illumination, thus giving rise to the one-hit character of photoreactivation. A final remark seems to be pertinent at this point. The one-hit char- acter of the photoreactivation curves of this bacteriophage is derived from data extending to approximately 80 per cent of the maximum photoreacti- vation; in this range a one-hit curve could be produced by an inhomo- geneous population composed of different classes in which the number of hits reciuired varies from one to about ten, if the frequencies of the various classes are properly distributed; the proper distribution can be easily calculated. 3-5b. Effect of Different Light Intensities. Increasing the intensity of the photoreactivating light has the consequence of increasing propor- tionately the photoreactivation rate when the intensity is low; at higher intensities there is less increase in rate, and for very high intensities the rate tends to a maximum value (saturation). A rate-intensity plot gives a hyperbolic curve (Fig. 12-4), which follows the equation h + cl Such a dependence shows that the rate of photoreactivation is determined PHOTOREACTIVATION 465 h}^ an tMiuilibrium condition involving a dark reaction. The substance involved in such a dark reaction might be the pigment that absorbs the light, or the product of the photochemical reaction. This point will be developed further in the treatment of the effect of interrupted light (Sect. 3-5d). 3-5c. Effect oj Temperature. Photoreactivation is considerably affected by temperature; this gives further evidence that dark reactions are involved. The temperature coefficient of the process (Qio) is near 2 at temperatures around 37°C and near 8 for temperatures near 0°C. This considerable variation of Qio with temperature shows that more than one dark reaction is involved in the process. 1. 4' X I0"3 O o. 2 X 10"" 2 3 4 5 RELATIVE LIGHT INTENSITY, 10 6 7 8 9 arbitrary units Pig. 12-4. Photoreactivation rate as a function of the intensity of the reactivating light. The photoreactivation rate is expressed in reciprocal seconds and the light intensity in arbitrary units. Phage T2 was irradiated with the germicidal lamp, adsorbed on resting bacteria, and illuminated in liquid at 37°C. 3-5d. Effect of Interrupted Light. The analysis of the effect of inter- rupted light has been carried out by Bo wen (1953). One of his most significant observations was that with a fixed dose of light given as long flashes (longer than 1 min) and long dark intervals, the fraction of reactivated particles is similar to that obtained with continuous illumination; by making the flashes shorter, without changing the total dose, a point is reached at which the reactivated fraction increases and tends to a maximum when the length of the flashes tends to zero. Another observation was that a fixed dose of light given as short flashes of constant length has a different effect according to the length of the dark period interposed between the flashes. The number of reacti- vated particles increases by increasing the length of the dark periods, and for very long intervals it tends asymptotically to a limit value. A practical consequence of these findings is that if the number of par- ticles photoreactivated is determined as a function of the time of exposure 466 ItADIATlON JJIULOUY to a light oi coufstant intensity, for short exposures the number is rela- tively higher than for longer exposures, so that the photoreactivation curve shows a deviation from linearity (Sect. .3-5a). If the same curve is obtained by giving all doses of light as series of short flashes separated by long dark periods, the deviation from linearity disappears. The two types of experiments just described show that the amount of photoreactivation obtained with a given light dose depends not only on the light reaction, but also on dark reactions connected with it. Bowcn was able to demonstrate that at least one dark reaction involved in photo- reactivation must precede the light reaction, because photoreactivability of bacteria infected with ina('ti\'e phage is affected by a temperature change from 40° to 0°C' if the temperature of the system is changed acvcral minuU'fs before a short light flash (of o sec length). If the temperature is changed immediately after the flash, photoreactivation is not affected. The results of these experiments can be understood on the basis of the following model. The probability for a photoreactivable particle to be reactivated is proportional to the time integral of the concentration of a photoproduct X*. Production and destruction of X* are determined by the following reactions. M - -* N rate A'l N - ^ M k2 N - -> N* hi N*- -Q k. N*- ^ active phage k. The first two reactions establish an ecjuilibrium between two bacterial components M and X"^ which may or may not be combined with the infecting phage; they are both temperature dependent. The third reac- tion is the photochemical reaction, the fourth is the reaction by which X* is destroyed and eliminated, and the fifth is the reaction of X* with phage, which constitutes photoreactivation. After a long dark period, N has an e(iuilibrium concentration [X] = ^^. (12-1) and X* has concentration zero. After a long period of contiinious light. X has an e(iuilil>rium con- centration and X*, which satisfies the hyperbolic i-elation found experimentally PHOTOUEACTIVATION 4G7 After a long dark pciiod, X is piesent at maximum conceiitratioii, iiixcii hy Eq. (12-1); if a \'ery intense light is tui'ncd on, X will l)e very rapidly and almost entirely transformed into X*, which consecjuently will reaeh a high concentration; during further illumination at the same intensity the concentration of N will remain at a lower steady level [given by Eq. (12-2)] and production of N* will continue at a constant rate, lower than the initial one. The photoreactivation curve is, therefore, divided hito two parts: a very short initial part, whose slope is propor- tional to A's/, and therefore not saturated at high light intensity. It measures the velocity of the light reaction; a second longer part, whose slope is proportional to ksl/(k2 + kj). This shows saturation at high light intensities and measures the competition between the dark reaction (12-2) and the light reaction. The regeneration of N after a flash of very-high-intensity reactions (12-1) and (12-2) can be studied by exposing the infected bacteria to a series of light flashes of equal length and intensity, separated by dark intervals of various length. According to the model, regeneration of N in darkness should follow the relation IN, = MM] (1 - .-.-..) where time t is measured from the end of the flash. The experimental data fit this relation very satisfactorily, and determine for reaction (12-2) a time constant of 35 sec at 37°C. Bowen has also studied the effect of temperature on reaction (12-2) by determining /.:2 with three independent methods. The results obtained here are not easily interpretable, because the slope of the Arrhenius plot, as determined with two methods, is not constant ; for temperatures above 20°C the slope corresponds to an activa- tion energy of approximately 9000 cal/mole, and below 20°C to an acti- v^ation energy of approximately 17,000 cal/mole. These experiments have therefore proved that both a light and a dark reaction are involved in photoreactivation, have established their sequence, and have measured the time constant of the reverse dark reac- tion from X" to M. .3-6. KINETICS OF PHOTOREACTIVATION OF PHAGE T3 Phage T3 represents the opposite extreme to phage T2 in the T series of phages. It belongs to the smallest group, and has a very short tail. Its radiobiology is in most respects much simpler than that of the larger phages (Dulbecco, unpublished). Its resistance to ultraviolet is about eight times higher than that of free T2, i.e., it is about as high as that found for T2 several minutes after infection (Luria and Latarjet, 1947). Possibly it lacks a special structure which in T2 is more sensitive to ultra- 408 RADIATION HIOLOr.V violet aiul is tlKM'cforo rosponsihle for the phenomena of multij)hcity reac- tivation. Tlie pli()t()re;i('ti\'ation curve of T3 has amultiple-hitcharacter, with the iHimher of hits proportional to the ultraviolet dose. The mimher of activ(^ particles .S aft(M- a tim(> i follows strictly the e(juation S = Be ne-f > where B is the total number of photoreactivahle particles, /; the average number of photoreactivahle hits per phage particle, and / the photo- reactivation rate. I'nder the assumption that both inactivating and photoreactivating events are distributed at random in the phage popula- tion, this relation shows that a photoreactivahle particle of T3 recjuires, to be reactivated, the occurrence of a number of efTective events equal to the number of inactivating events. 3-7. KINETICS OF PHOTOREACTIVATION OF PHAGE T2 IN CONDITION OF MULTIPLE INFECTION A bacterium infected with more than one particle of phage T2 inacti- vated with ultraviolet has a much higher probability of yielding active phage than if infected with one particle only (Luria, 1947; Luria and Dulbeeco, 1949). The photoreactivation curve for such multiple- infected bacteria (multicomplexes) is similar to that observed in T3, but does not follow strictly the equation realized in T3, although it tends to it for increasing multiplicity and increasing ultraviolet dose (Dulbeeco. unpublished). These findings are taken as indication that phage T2 upon ultraviolet irradiation receives damages in two different structures, of which one, more sensitive to ultraviolet, is photoreactivated by one photo- reactivating event ; the other, less sensitive to ultraviolet, is photoreacti- vated like T3. In single infection the damage produced in the more sensitive structure is predominating; in multiple infection the phage can partly dispen.se with the sensitive part, the more completely so the higher the multiplicity, so that the ultraviolet damage in the other structure becomes predominant. 3-8. ACTION SPECTRUM OF THE PHOTOREACTIVATING LIGHT Bacteriophage T2 adsorbed on E. coli B is an excellent material for the determination of the action spectrum of the reactivating light, because the rate of photoreactivation, experimentally determinable, is proportional to the concentration of the immediate photoproducts of N* [Sect. 3-5d. Eq. (12-3)]. If the intensity of the photoreactivating light is sufficiently low, we can write: Photoreactivation rate = k-ilC, where C = ^'li^I] (A'4 + A- 5), a constant at constant temperature for a given bacterial population. If the light intensity is measured in quanta per unit time, A-3 is proper- PHOTOREACTIVATION 469 tional to the product of the al)sorption coefficient of the photosensitive pigment by the quantum yield. The determination of the photoreactivation rate and light intensit}^ yields, therefore, the relative values of the absorption coefficient of the pigment, provided the quantum yield is constant at all wave lengths. On this basis a series of points in an action spectrum for phage T2 in single infection have been obtained, by measuring the intensity reciuired for each wave length to produce a standard low rate of photoreactivation, far from the saturation point (Sect. 3-5b). The light used was reasonably mono- chromatic, having been obtained by isolating lines of the mercury spec- trum by glass and liquid filters. The action spectrum is constituted by a single band, extending from about 3100 up to nearly 4800 A, wdth a maxi- mum between 3600 and 4000 A (Fig. 12-5). The unirradiated phage does not absorb light in this region ; the ultraviolet-irradiated phage has an ab- sorption band between 3100 and 3700 A, with a peak at 3300 A. It is very doubtful that light ab- sorption in this band may be re- sponsible for photo-reactivation; more likely the effective light is not absorbed in the phage. 3-9. ACTION OF CHEMICAL SUBSTANCES ON PHOTOREACTIVATION Reactivation of irradiated bac- teriophages has not been obtained with chemical means, either in the presence or in the absence of light. Extracts obtained from sensitive bacteria cannot replace the bacteria in photoreactivation; samples of ultraviolet-treated phages kept in darkness or in light in the presence of yeast extract, or catalase, are not reactivated. Photoreactiva- tion occurs ec}ually well in the pres- ence and in the absence of oxygen; rate of the process. 4000 6000 V»AVE LENGTH, fl Fig. 12-5. The action spectrum of photo- reactivation of phage T2. The activity of each wave length, proportional to the reciprocal of the dose of light expressed in ergs per unit area required for obtaining a standard amount of photoreactivation, is plotted versus wave length. The phage was irradiated with the germicidal lamp, adsorbed on bacteria in buffer, and illu- minated in liquid at 37°C. 0.01 M cyanide does not affect the 3-10. PHOTOREACTIVATION OF BACTERIOPHAGES INACTIVATED BY AGENTS OTHER THAN ULTRAVIOLET A small amount of photoreactivation has been found in phages irradi- ated with X rays (Dulbecco, 1950; Watson, 1950). 470 RADIATION UIOLOGY 3-11. lMI()T()!{i;A("ii\ A'i'iOX OK TIIK INDITTIOX I'ROCESS ()!•■ iMiAci'; cAiuui;!) l.vs()(;l•:^•I(•Al-l.^■ As tliscoxcicd l)y liWolt tt al. (!•.).")()) cells of Bdcillii.s ntcydHwriiini carryiiifj; lysofrciiic phafj;es can he induced to lyse by ultraviolet irradia- tion. This cITcct of ultraviolet can also be photoreactivated, i.e., the carrying bacteria will not liberate the phage if exposed to the photo- reactivating light after the inducing exposure to ultraviolet (Jacob. 1950). Latarjet (19") lb) has shown that induction of the same organism is also produced by X rays, and has announced (1951a, b) that the inducing activity of X rays could be counteracted by photoreactivation; this result is surprising since in all other cases X-ray damage has been found to be at most very slight l}- photoreactivable. There is a reason to suspect that the apparent reversal of X-ray induc- tion may not be photoreactivation. It has been found that E. coli B cells irradiated with a dose of the photoreactivating light which does not kill the bacteria and then infected with active phage T2 lose the ability to produce phage. The effect observed by Latarjet (and possibly also by Jacob) might be similar, and might consist in the loss of the ability of the induced phage to grow in the illuminated bacteria rather than in a photo- reversal of the induction (Dulbecco and Weigle, 1952). 4. PHOTOREACTIVATION OF PLANT VIRUSES Bawden and Kleczkowski (1952) observed photoreactivation of tobacco necrosis virus in French bean and of tomato bushy stunt virus in Nico- tiana glutinosa. As with bacteriophages, the effect was obtained only when the ultraviolet-irradiated virus was inoculated into the leaves, which were in turn illuminated with white light. .\ virus preparation whose activity was reduced to about 1 per cent could be restored by the light to 4-10 per cent. No effect was observed in tobacco mosaic virus inoculated into leaves of A'', glutinosa. The different photoreactivability of these three viruses may suggest a correlation with their relative content in nucleic acid, which in tobacco mosaic virus is about one-third of that present in the other two viruses (see also Sect. 3-4). 5. PHOTOREACTIVATION OF BACTERIA Extensive observations (Kelner, 1949b, c; 1950a; Novick and Szilard, 1949) have been performed on photoreactivation of bacteria, attention having been focused almost exclusively on the bacterium E. coli B/r (Witkin, 1946); a few observations have been made on the strains B and Kr2 of the same species. PHOTOREACTIVATION 471 5-1. CONDITIONS UNDER WHICH PHOTOREACTIVATION OCCURS Photoreactivation occurs when the bacteria are .siLspended in a liquid medium or when they are plated on a solid medium. For quantitative work the first method has been used exclusively. Kelner used bacteria grown for 48 hr with aeration in a glucose-ammonium chloride medium, and then diluted with saline by a factor of 2; Novick and Szilard used resting cells obtained from cultures grown in a lactate-ammonium phos- phate medium to approximately 10* cells/ml, transferred into saline, and incubated in this medium under continuous aeration for 14—18 hr to completely exhaust utilizable reserves; these bacteria were kept in the icebox at 6°C and could be successfully used with reproducible results for about one week. 5-2. EFFECT OF THE GROWTH STAGE OF BACTERIA ON PHOTOREACTIVATION The characteristics of photoreactivation of growing and resting cells of E. coll B are very different. This point will be discussed later. Particularly important for the photoreactivability is the growth condi- tion of the bacteria ajter they have been irradiated with ultraviolet. Kelner has found that if the bacteria, prepared as previously specified, are chilled immediately after irradiation, they can be kept chilled for at least 8 hr without significant effect on subsequent photoreactivation. On the contrary, if they are kept at 37°C after irradiation, their photoreactiv- ability decreases, the decrease being very strong if the bacteria are in a nutrient medium (a decrease by a factor of 1000 in 2 hr) ; a considerable decrease occurs also in saline, although at a lower rate (Kelner, 1949c; Novick and Szilard, 1949). 5-3. COMPARISON OF SURVIVAL CURVES AFTER ULTRAVIOLET TREATMENT IN PRESENCE AND ABSENCE OF PHOTOREACTIVATION Kelner (1949c) and, independently, Novick and Szilard (1949) have established that these tw^o curves have a simple relation : the ratio between the ultraviolet dose required for obtaining a given survival in the dark and after maximum photoreactivation is constant for any survival. Formally, this phenomenon can be described by saying that the effect of the light is to reduce by a constant factor the ultraviolet dose given to the sample; hence the name of "constant ultraviolet dose reduction" is applied to the observed relation. The demonstration of the principle can be obtained in two different ways (Novick and Szilard, 1949). The first method is to l^lot the dose required for obtaining a given survival after photoreacti- vation (L) versus the dose observed at the same survival in darkness (/)); the slope of this line measures the fraction to which the ultra\'iolet dose is 472 RADIATION HIOLOGY appaixMilly roduccd. The second method is to draw tangents to the two survival curves, in darkness and after photoreactivation, at points of same survival; the pair of tangents drawn to the two curves for any value of the survival must intersect on the ordinate. Novick and Szilard have shown that both conditions are fulfilled in their experiments with E. coli B r (Fig. 12-6). 200 4 00 500 300 D , sec Fig. 12-6. Survival curves for Escherichia coli B/r in darkness (curve I) and after max- imum photoreactivation (curve II). Curve III gives the ultraviolet dose correspond- ing to a given survival after photoreactivation versus the dose corresponding to the same survival in darkness. The dotted lines are a pair of tangents to the survival curves at two points of same survival (see text). For survival curves, the logarithm of surviving bacteria (left-hand scale) is plotted versus the idtraviolet dose in seconds of exposure (bottom scale). For the lower curve, the light-vdtraviolet dose in seconds of exposure (right-hand scale) is plotted versus the dark-ultraviolet dose (bottom scale). {From Novick and Szilard, 1949.) In E. coli B the relation between the survival in the dark and after photoreactivation depends on the physiological conditions of the bacteria (Xovick, personal communication). In resting bacteria, obtained with the method previously described, multiple-hit type survival curves are observed both in darkness and after maximum photoreactivation; these curves show constant dose reduction by the light. The survi\-al curve of bacteria from a growing culture shows a first period of rapid inactivation, the slope of the curve then decreasing; after maximum photoreactivation a curve of multiple-hit type is obtained. Novick suggests that in growing bacteria the .survival curve in darkness is the result of two types of inacti- vation, of which the first, affecting all but a small fraction of the popula- PHOTOREACTIVATION 473 tion, is completely reactivated by the light; the second is similar to that observed in resting cells, and shows the constant ultraviolet dose reduc- tion by light. Novick and Szilard interpret the constant dose-reduction phenomenon in the following way. They assume that inactivation by ultraviolet is due to formation of a poisonous chemical compound in the cells, produced in amount proportional to the ultraviolet dose. The poison is produced in two forms; one sensitive, the other insensitive to light, the ratio between the amounts of the two forms in individual bacteria being inde- pendent of the ultraviolet dose. The fraction of survivors after a given ultraviolet dose is determined by the amount of poison of both types present in the cells at the time they are incubated with nutrient medium and permitted to multiply. If the bacterium is exposed to light before this moment, the photosensitive poison is destroyed, and only the photo- insensitive one remains. The formal reduction of the ultraviolet dose is due to reduction in the amount of poison. This theory can explain well the observed relations of the survival curves in E. coli B/r and many other features of photoreactivation in this organism (see Sect. 5-4). In those microorganisms, in which the survival curve approaches the one-hit type, as in bacteriophages, the amount of the poison able to pro- duce inactivation in the majority of the organisms reduces to a small number of molecules per organism, so that the poison theory of inacti- vation becomes a "hit" theory. 5-4. KINETICS The kinetics of photoreactivation of E. coli B/r has been worked out by Novick and Szilard (1949) as a development of the poison theory. It is assumed that the photosensitive poison is destroyed by the light in a first- order reaction. As a result, the curve of log [1 — p(t)/p{ oo )] versus time (see Sect. 3-5) has a multiple-hit character; this theory could not there- fore explain a type of photoreactivation like that found in phage T2, in which this curve is a straight line. In E. coli B/r the expectation of the theory is fulfilled. The action of the light in the first minutes of illumina- tion is somewhat less than expected on the basis of the theory, so that a latent period of several minutes is assumed. The rate of destruction of the poison, calculated according to the theory, is independent of the ultraviolet dose. The calculated rate depends on the intensity of the reactivating light and increases with its intensity but without strict proportionality. The latent period increases when the light intensity decreases. If photoreactivation is carried out with a dose which does not give maximum photoreactivation, the survival curves still follow the constant dose reduction principle. 474 llADIATION HIOLOGY Novick (personal communication) has checked the poison theory with the followinji; experiment : A sample of E. coli B/r was iria ol' nil i:i\i()l('t and then pholoreactivated. Ailci I his opcrnt ion the sur\'i\;il corresponded to a dose Ij\{L\ < D) ot ultraviolet ji;i\('ii in t he- dark. According to the poison theory, there should ha\(! been no ditTer- ence between these photoreactivated bacteria and bacteria which received oi-i«i;iiuUly a dose />i of ultraviolet and were photoreactivated. If now a new ultraviolet dose is given to the pliotoreactivated bacteria, their survival curve should be ecjual to the original survival curve in absence of photoreactivation, starting at the ultraviolet dose Li. This was actually found. After a second stage of photoreactivation the survival of the bacteria became equivalent to a dark ultraviolet dose L2; the twice- photoreactivated sample was exposed to ultraviolet for the third time, and again the survival cui\e was similar to the original one, starting at a dose L-i. 5-5. .\CTI()X SPECTRUM OF Tin<: FHOTOREACTIVATING LIGHT Preliminary results on the shape of the action spectrum for bacterial photoreactivation have been published by Kelner (1950b), who has described in E. coli a peak near 4000 A, and by Knowles and Taylor (1950), who determined that the greatest effect occurs for wave lengths between 3500 and 4500 A, with a maximum at 3600 A. A more extensive determination of the action spectra of FJ. coli B/r and Streptomijces griseus spores has been published by Kelner (1951). The relative efficiency of lights of different wave lengths was determined by comparing the amount of light energy re(iviired to produce a standard "degree of photoreacti- vation "(the fraction of inactivated cells that have been photoreactivated) after a standard ultraxiolet irradiation. For values of the degree of photoreactivation near the standard \'alue, the degree of photoreacti- vation is a linear function of the logarithm of the light energy, and inter- polation of the data is possible. In the range of light intensities used, the reciprocity law was found to hold for exposures varying between 1.6 and 75 min for S. griseus and between 5 and 52 min for E. coli. However, long exposures should be relatively less effective in E. coli owing to the decay of photoreactivability with the time of sojourn of this organism at 37°(' in saline (Kelner, 1949c; Novick and Szilard, 1949); this decay might be masked by saturation occurring at high light intensity. This may affect somewhat the shape of the action spectrum. The spectral region examined consisted partly of lines of the mercury spectrum isolated by glass filters and partly of bands separated out of the continuous spectrum of an incandescent bulb by interference filters. The spectral regions of the second type are not well defined because the inter- ference filters do not cut off very sharply. This introduces some uncer- tainty for the points so determined. PHOTOREACTIVATION 475 The action spectrum for E. coli (Fig. 12-7) is similar to that of photo- reactivation of phage T2 adsorbed on the same bacterium (see Sect. 3-8), the peak being more accurately determined at 3750 A. An unexpected result is the finding that the action spectrum for S. griseus spores extends considerably farther into the longer wave lengths, the peak being at 4360 A. The difference between the two spectra is undoubtedly substantial; possible artifacts are discussed by the author, who also discusses the possi- bihty that the photosensitive pigment of S. griseus spores is a porphyrin. 6 - X) o >- o 2 _i_L I I . 3600 4000 4500 5000 5500 6000 6500 7000 WAVE LENGTH , A Fig. 12-7. The action spectra of photoreactivation of Streptomyces griseus and of Escherichia coli B/r. The activity of each wave length, proportional to the reciprocal of the dose of light, expressed in quanta per unit area, required for obtaining a stand- ard degree of photoreactivation is plotted versus wave length. (From Kelner, 1951.) 5-6. CHEMICAL ACTIONS CONNECTED WITH PHOTOREACTIVATION Monod d al. (1949) have observed that bacteria of the strain E. coli K12 irradiated with ultraviolet in citrate buffer and plated on a synthetic agar medium are not photoreactivable, and that photoreactivability appears, together with a considerable dark reactivation, if catalase or ferrous sul- fate is added to the medium. Latarjet and Caldas (1952) showed that the phenomenon is lather erratic and presumably finely dependent on the physiological state of the bacteria. They found that catalase restoi'ation can take place occasionally in th(> absence of light, but usually re(iuires illumination; that it is absent in other strains of the same species, E. coli B 476 HADIATIOX MIOLOGY and B r; that it is absent after X-ray irradiation; and that it takes place in H. nrnjathcrium 899, which, like E. roli K12 is lysogenic, whereas E. coli B and B/r arc not so. Therefore some relation with lysogenicity is indicated. The catalase eiVect seems to \)v. an important feature; it is piobahiy not identical with photoreactivation hut related to it. Johnson d (il. (19r)()) have found that oxygen is not required for bac- terial photoreactivation. 5-7. ACTION OF PIIOTOIUOACTIVATION ON THE INDTCTION OF MUTATION BY ULTRAVIOLET IN liACTEUIA This problem is of great importance for understanding the relation between inactivation and induction of mutations. Ob.servations have been carried out mainly on the induction of the mutation to resistance to bacteriophages in E. coli B/r (Kelner, 19-19b, c; 1950a; Novick and Szilard, 1949). Two different technicjues have been used for determining the extent of the mutagenic action and the effect of photoreactivation. Kelner has determined the number of mutations present under various experimental conditions according to the spray technicjue of Demerec (1946). Novick and Szilard have determined the number of mutants present in a culture arising from a treated bacterial suspension after the bacteria in the sample have been allowed to undergo on the average about ten divisions. With regard to this method, it must be observed that bacteria treated with ultraviolet show a variable lag period before starting to divide, and that mutations induced by ultraviolet become phenotypic after an additional lag (Newcombe and Scott, 1949); the fraction of mutants in the population after growth might therefore be affected by variations in the lag values. Kelner studied the behavior of zero-point mutations (mutations arising before any division of the ultraviolet-treated cells has taken place) and of delayed mutations (manifested after several cell generations). The zero- point mutations appear to be completely suppressed if the bacteria after the ultraviolet treatment are exposed to the photoreactivating light; the fraction of delayed mutations, on the contrary, is little or not at all affected by the light. Novick and Szilard found that the fraction of mutants in the total popu- lation is considerably decreased by the action of the light, and that the fraction of mutants observable after photoreactivation is comparable with the amount o})tainable with a lower ultraviolet dose in the absence of photoreactivation (Fig. 12-8). Induction of mutations by ultraviolet would therefore be affected by photoreactivation in a similar way as the reactivation, with a constant ultraviolet dose reduction produced by light. The difference in the results obtained l)y Kelner and by Novick and Szilard cannot be accounted for by the difference in the method used, since PHOTOREACTIVATION 477 Kelner's data show that the effect of photoreactivation on the delay in appearance of mutations is, if any, a reduction of the lag period. This would affect the fraction of mutants in the method used by Novick and Szilard in the direction of increasing it — in a direction, therefore, which is opposite to the observed difference. Kelner points out that the curve giving the frequency of delayed muta- tions among survivors as a function of ultraviolet dose for E. coli B/r rises 10" q: UJ a. en 5 -.3 _ 100 D , sec Fig. 12-8. Logarithm of number of phage-resistant mutants per 10* bacteria as a function of the logarithm of the ultraviolet dose D in darkness and after maximum photoreactivation for (a) phage T4, (h) phage T6, and (r), phage Tl. The bacteria had passed through 10 generations in liquid culture prior to being assayed for the mutants. {From Novick and Szilard, 1949.) sharply at low ultraviolet doses and less at high ultraviolet doses (Demerec and Latarjet, 1946); therefore, if the light causes a constant reduction of the ultraviolet dose also for the mutagenic effect, the relation between the fraction of mutants before and after photoreactivation should vary according to the ultraviolet dose used. At a low ultraviolet dose, where the curve giving the fraction of mutations versus the ultraviolet dose has a steep slope, the fraction of mutants should considerably decrease after photoreactivation, whereas at a high dose, where the slope is less, the frac- tion of mutants should be less affected. This can account for part of the difference in the results obtained by Kelner and by Novick and Szilard, 478 KADIATIOX UIOLOGY since llif latter authors used lower (loses in their experiments. This explanation does not account for all the dilTerences, however, since the fractions of mutants anions survivors in points correspondinfj; to the sur- vival vahies observed by Kelner in the absence and in the presence of photoreactivation differ, according to Demerec and Latarjet (1946), by a factor of 10 or more, less mutations being present at the lower dose. The etTect of photoreactivation on the so-called " zero-point mutations" seems not sufficiently documented in view of the fact that detection of these mutations iii\-ol\-es infection with phage of a very large number of bacteria inactivated with ultraviolet but able to adsorb the phage. Under these conditions the multiplicity of phage infection may become too low, so that a considerable fraction of bacteria may divide before infection. Newcombe (1950) and Newcombe and Whitehead (1950) have observed that the mutagenic effect of ultraviolet on E. coli B/r (streptomycin- resistance mutants and color-response mutants on mannitol-tetrazolium agar) is particularly strong at low doses of ultraviolet. At these low doses the reversal of the mutagenic effect by photoreactivation is also strong. At a dose of 500 ergs/mm- more than 90 per cent of the potential mutants fail to appear after photoreactivation. At larger doses the mutations produced by ultraviolet alone do not increase with dose, and their photo- reactivability tends to zero. The results show that the effect of photoreactivation on these mutations can be approximately described on the basis of the dose-reduction prin- ciple, as in the case of Novick and Szilard (1949), although the mutagenic effect of ultraviolet is reduced by photoreactivation to a greater extent than the killing action as determined by Kelner (1949c). This difference may not be significant since the amount of reduction of the killing action has not been determined inider the same conditions. 6. PHOTOREACTIVATION IN STREPTOMYCES AND FUNGI Photoreactivation was first discovered in spores of Streptomyces griseus irradiated wdth ultraviolet (Kelner, 1949a). Spores suspended in saline or distilled water are exposed to ultraviolet and then treated with visible light Avith the following results: (1) the ratio of the number of active spores after photoreactivation to the number of active spores before photoreacti- vation increases with the ultraviolet dose; (2) with light of constant inten- sity the number of active spores tends to a maximum value with time of illumination ; (3) increasing the intensity of the reactivating light increases proportionately the rapidity of recovery within certain limits; and (4) the rapidity of recovery increases with the temperature existing during light treatment, up to temperatures of 50°C. The action spectrum of photoreactivation of S. griseua spores (Fig. 12-7) PHOTOREACTIVATION 479 has been determined in detail by Kelner (1951); it has ah-eady been dis- cussed (Sect, o-o) together with the bacterial action spectrum. In Neurospora crassa, Goodgal (1950) has observed that the survival of microconidia irradiated with ultraviolet is affected by photoreactivation, which produces an increase in the number of active conidia with a con- stant reduction in ultraviolet dose. The freciuency of morphological mutants among survivors has also been studied and found to be similarly reduced by photoreactivation. The ultraviolet dose reduction is of the same order of magnitude for lethal and mutagenic action, since a given survival corresponds to a constant fraction of mutants, independently of the presence or absence of photoreactivation. This result is therefore similar to that obtained by Xovick and Szilard (1949) in E. coli (Sect. 5-7). Goodgal attributes these results to one of two possibilities: (1) killing of microconidia by ultraviolet is due to a lethal mutation, and light decreases killing by hindering the occurrence of mutations; or (2) killing and muta- tions are produced bj^ a common mediator formed under ultraviolet treatment and destroyed by the light. Brown (1951) studied the effect of photoreactivation on the reversion of the inositol requirement in .V. crassa by irradiating mononucleate micro- conidia with ultraviolet, and found that the fraction of mutated conidia among the survivors is considerabl}^ less after photoreactivation. This result cannot be due to lack of photoreactivation of inactive mutants, since after photoreactivation the absolute number of mutants increases. 7. PHOTOREACTIVATION IN YEAST Photoreactivation in yeast has been demonstrated by Kelner (1949a) and by Swenson and Giese (1950). The latter authors have shown that the mechanism of enzymatic adaptation to galactose fermentation in Saccharomyces cerevisiae is damaged by ultraviolet and that this effect also can be partly undone by exposing the cells irradiated with ultraviolet to a strong white light. Several points in an action spectrum for the ultra- violet destruction of adaptability have given results compatible with the assumption that the radiation acts on nucleic acid (Swenson, 1950). 8. PHOTOREACTIVATION IN PROTOZOA Kimball (1949) and Kimball and Gaither (1950, 1951) have studied photoreactivation in Paramecium aurelia by using monochromatic radi- ation of wave length 2804 and 2650 A in the majority of the experiments. In this organism irradiation with ultraviolet has a number of effects: I'etardation in cell division, killing of a fraction of animals before autog- amy, reduced vigor after autogamy, and morphological changes in the structure of the macronucleus. 480 RADIATION UIOLOGY The delay in the onset of cell cli\i.sion is put on a fiuantitative basis by (ietermininf^ the time at which the sixlh (iJNision takes place; after this (li\ision no further delay occurs. When the time of the sixth division is plotted against the ultraviolet dose, for each wave length used two dilfer- ent curves are obtained — one in darkness, the other after photoreacti- vation (Fig. 12-9). The experimental points have high dispersion, but curves drawn among the points show that the effect of photoreactivation is compatible with a constant ultraviolet dose reduction. z o > I I- o a 0 12 3 4 5 6 7 DOSE, lO' ergs/mm2 Fig. 12-9. Days to the sixth division plotted against dose of 2804 A ultraviolet. Curve I, dark; curve II, light. Each point represents the arithmetical mean for 5 to 54 lines of descent from a single experiment. (From Kimball and Gaither, 1951.) Death before autogamy behaves in a similar way as the retardation of cell division, and the authors consider that the two phenomena are likely interdependent. Reduced vigor after autogamy, on the contrary, is not related to the two effects already described, since it occurs in animals which had com- pletely recovered from previous radiation disease. Since it occurs after homozygosity has been produced, it has very likely to be attributed to gene mutations; however, direct evidence for this has not been presented. Reduced vigor is affected by photoreactivation in a similar way as the genetic effect. PHOTOKEACTIVATION 481 Changes in the structure of the macronucleus after ultraviolet irradi- ation consist in clumping of granular structures with subsecjuent vacuoli- zation (Kimball, 1949). If the animals are exposed to the photoreacti- vating light following ultraviolet irradiation, some clumping occurs, but fusion and vacuolization do not ensue; the clumps produced are subse- (j[uently dissolved. 9. PHOTOREACTIVATION IN ECHINODERM ZYGOTES AND GAMETES Blum and coworkers have extensively studied the delaying effect of ultraviolet on cleavage in Arhacia punctulata eggs, and the effect of photo- reactivation on this delay (Blum . . . Robinson, 1949; Blum . . . Loos, 1949; Blum, Loos, and Robinson, 1950). In fertilized eggs irradiated with a Hanovia intermediate pressure arc before cleavage, with filters absorbing most of the visible spectrum, the time of onset of division is delayed, the delay markedly affecting the first and less the second and third division. In eggs exposed to the light of a fluorescent lamp after ultraviolet irradiation, the delay is greatly reduced. Eggs irradiated with ultraviolet before fertilization show a similar delay in the onset of the first few divisions following fertilization; treatment of the unfertilized eggs with visible light after ultraviolet irradiation decreases the delay. The delay is not affected if the eggs are illuminated with visible light before ultraviolet treatment. Unfertilized eggs can be separated by centrifugation into two parts, one yellow containing all the echinochrome pigment, the other white con- taining the nucleus. The white halves are damaged by ultraviolet and photoreactivated like whole eggs. This shows that the echinochrome pig- ment is not required for this type of photoreactivation. If the enucleated halves are treated with ultraviolet and then fertilized, no delay occurs, suggesting that the site of action of ultraviolet is in the nucleus. The effective spectral range for photoreactivation of division delay is between 3000 and 5000 A. In other experiments the effect of ultraviolet irradiation and photo- reactivation on sperm has been studied (Blum, Robinson, and Loos, 1950, 1951). When ultraviolet-treated sperm is used for fertilization of non- irradiated eggs or egg halves (both nucleated and enucleated ones), delay in the time of cleavage occurs. Illumination of the ultraviolet-irradiated sperm with photoreactivating light does not affect the delay, which is, on the contrary, reduced if the eggs fertilized with ultraviolet-irradiated sperm are exposed to the photoreactivating light. The authors point out the similarity of this interesting observation with the situation found in photoreactivation of bacteriophages in which the bacteriophage can be reactivated only if adsorbed on the sensitive bacterium (see Sect. 3-3). No photoreactivation was observed after X-ray treatment. 482 ItADIATlOX MIOLOGY Similar results have been obtained on the same material by Marshak (1949a, b), who. iio\ve\<'r. could not deteet photoreaetivatioii in unferti- lized e^ifzis. This author explored the possibility of some chemical actions in the ultraviolet effect and in photoreactivation by treating samiMcs and zygotes, before, during, and after irradiation with ultraviolet and visible light, with the following substances: adenine, streptomycin, foli<- acid. 4-amino-//-methyl folic acid (a folic acid antagonist), and riboflavin (on reactivation of zygote only). None had any effect. Wells and Giese (1950) have investigated llic photoreactivation of the cleavage delay subsetiuent to ultraviolet treatment in gametes and zygotes of the sea urchin Strong ylocentrotus purpurahis, with experiments similar to those performed by Blum and coworkers. Wells and Giese used for inactivation monochromatic ultraviolet obtained with a quartz mono- chromator, and compared the photoreacti\ability of delay produced by several wave lengths between 2450 and 3130 A. It appears that photo- reactivation occurs in eggs treated with ultraviolet of all wave lengths tested with similar efficiency except at the shortest wave lengths, 2450 A. where photoreactivation is reduced. This wave length has a strong effect on the surface of the eggs, w^hose membrane is raised. The lower photo- reactivability of eggs damaged by treatment with this wave length is attributed to ultraviolet damage to the surface membrane. Wells and Giese found that sperms are considerably damaged by the photoreactivating light, so that photoreactivation cannot be detected readily. A small amount of photoreactivation could, however, be demon- strated also on the naked sperm, contrary to the findings of Blum. Robinson, and Loos (1950) with Arbacia. The most effective spectral region for photoreactivation in Strongi/lo- cenfrofus gametes and zygotes lies betAveen 3660 and 4300 A. 10. PHOTOREACTIVATION IN SALAMANDER LARVAE Blum and Matthews (1952) studied the effect of photoreactivation on the killing effect of ultraviolet radiation on larvae of Amblystoma macu- latum and -4. opacum. When the larvae were irradiated with a single massive dose of ultraviolet, no photoreactivation took place; but when the larvae were exposed to repeated low ultraviolet doses, each being followed by exposures to the photoreactivating light, the fraction of surviving larvae was much greater in the light-treated group than in that kept in darkness. It is remarkable that the effect of photoreacti\'ation was still present when the larvae were treated with light 20 hr following ultraA'iolet exposure; in all other organisms photoreactivability is generally lost within a few hours. The spectral region in which the photoreactivating light was active is similar to that observed in other organisms (3000- 5000 A). PHOTOKEACTIVATION 483 11. PHOTOKEACTIVATION IN HIGHER PLANTS Bawden and Kleczowski (1952) found that the first-formed leaves of Phaseolus vulgaris, if isolated from the plant and kept in darkness after exposure to ultraviolet light, acciuired a bronze color owing to death of the cells of the upper epidermis. If, following ultraviolet treatment, the leaves were illuminated with daylight, the bronze color was not produced and the majority of the cells appeared intact. 12. CONCLUSIONS AND SUMMARY 1. Photoreactivation is a very widespread phenomenon, affecting changes of seemingly different nature produced by ultraviolet in organisms of different levels of organization. The fact that most of the known bio- logical effects of ultraviolet irradiation have been photoreactivated seems to suggest that they all have a common step. 2. The effect of photoreactivation is almost completely specific for damages produced by ultraviolet radiation. The susceptibility of dam- ages produced by different ultraviolet wave lengths to photoreactivation is little known, and more complete determinations would be desirable. 3. The site at which the photoreactivable damage is produced is a nucleoprotein, as shown by the results obtained with bacteriophages (Sect. 3-3), plant viruses (Sect. 4), echinoderm sperm (Sect. 9) and yeast (Sect. 7) ; it is probably nucleic acid, since Hershey and Chase (1952) have shown that at least most of the protein part of the bacteriophage does not enter the bacterium it infects. In this respect there seems to be a corre- lation between photoreactivability and the nucleic acid content of viruses (Sect. 3-4 and Sect. 4). 4. The mechanism of reactivation requires substances of "cytoplasmic" nature. The action spectra obtained seem to indicate that different pig- ments are responsible for the absorption of the photoreactivating light in different organisms (Sects. 3-8 and 5-5). This result is surprising in view of the universal nature of the phenomenon. However, the action spec- trum of bacteriophage T2, of E. coli B/'r, and of Streptomyces griseus spores could all be due to light absorption in the same substance, for example, in a flavin, provided that the ciuantum yield varied with the wave length in a different wa}^ in different organisms. 5. The type and sequence of reactions involved in photoreactivation are rather clearly understood in bacteriophages (Sect. 3-5), and presum- ably the same results will be found true for other organisms. The light acts on a pigment in efiuilibrium with a nonpigment substance; the prob- ability of photoreactivation is proportional to the concentration of the product of the light action on the pigment. 6. The killing effect of ultraviolet is reverted l)y one (}uantum of the 484 HADIATION HIOLOGY photoreactivatiiij? liglit in hacteriopluige T2 in sinjjjle infection (Sect. 3-5), by a small number of li^lit (luaiita in the same phafi;o in multiple infection (Sect. 3-7), and in pha^e 'r3 in single infection (Sect. 3-0); by a larger luimber of (juanta in bacteria (Sect. 4-4). It is possible that at an ele- mentary le\el of organization photoreactivation is a one-cjuantum phe- nomenon, and that higher numbers of quanta are recjuired at higher levels. More extensive kinetic investigations would be desirable in various types of microorganisms. 7. The photoreactivation of the mutagenic effect generally appears as a constant ultraviolet dose reduction (Sect. 5-7 and Sect. 6), indicating the intimate similarity with the photoreactivation of the killing effect. The two effects could be chemically identical but biologically different owing to a different localization of the changes responsible for them. REFERENCES Bawdcn, F. C, and A. Kleczkowki (1952) Ultraviolet injury to higher plants counteracted by visible light. Nature, 169: 90-91. Blum, H. F., G. M. Loos, J. P. Price, and J. C. Robinson (1949) Enhancement by "visible" light of recovery from ultraviolet irradiation in animal cells. Nature, 164: 1011. Blum, H. F., G. M. Loos, and J. C. Robinson (1950) The accelerating action of illumination in recovery of Arbacia eggs from exposure to ultraviolet radiation. J. Gen. Physiol., 34: 167-181. Blum, H. F., and M. Matthews (1952) Photorecovery from the effects of ultraviolet radiation in salamander larvae. J. Cellular Comp. Physiol., 39: 57. Blum, H. F., J. P. Price, J. C. Robinson, and G. M. Loos (1949) Effect of ultraviolet radiation on the rate of cell division of Arbacia eggs. Biol. Bull., 97: 232. Blum, H. F., J. C. Robinson, and G. M. Loos (1950) The loci of action of ultra-violet and X-radiation, and of photorecovery in the egg and sperm of the sea urchin. Proc. Natl. Acad. Sci. U.S., 36: 623-627. (1951) The loci of action of ultraviolet and X-radiation and of photorecovery in the egg and sperm of the sea urchin Arbacia punctulala. J. Gen. Physiol., 35: 323-342. Bowen, G. H. (1953) Kinetic studies on the mechanism of photoreactivation of bacteriophage T2 inactivated by ultraviolet. Calif. Inst, of Technol., Ph.D. Thesis. Brown, J. S. (1951) The effect of photoreactivation on mutation frequency in Neurospora. J. Bacteriol., 62: 163-167. Demerec, M. (1946) Induced mutations and possible mechanisms of the transmission of heredity in Escherichia coli. Proc. Natl. Acad. Sci. U.S., 32: 36-46. Demerec, M., and R. Latarjet (1946) Mutations in bacteria induced by radiations. Cold Spring Harbor Symposia Quant. Biol., 11: 38-39. Dulbecco, R. (1949) Reactivation of ultraviolet-inactivated bacteriophage by visible light. Nature, 163: 949-950. (1950) Experiments on photoreactivation of bacteriophages inactivated with ultraviolet radiation. J. Bacteriol., 59: 329-347. Dulbecco, R., and J. J. Weigle (1952) Inhibition of bacteriophage development in bacteria illuminated with visible light. Experientia, 8: 386-387. Goodgal, S. H. (1950) The effect of photoreactivation on the frequency of ultra- PHOTOREACTIVATION 485 violet-induced morphological mutations in the microconidial strain of Neurospora crassa. Genetics, 35: 667. Hershey, A. D., and M. Chase (1952) Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol., 36: 39-56. HoUaender, .\. (1943) Effect of long ultraviolet and short visible radiation (3500 to 4900 A) on Esrhcru-hia coli. J. Bacteriol., 46: 531-541. Jacob, F. (1950) Induction de la lyse et de la production dc bacteriophages chez un Pseudomonas pijocyanea lysogene. Compt. rend., 231: 1585-1587. Johnson, F. H., E. A. Flagler, and H. F. Blum (1950) Relation of oxygen to photo- reactivation of bacteria after ultraviolet radiation. Proc. Soc. Exptl. Biol. Med., 74: 32-35. Kelner, A. (1949a) Effect of visible light on the recovery of Streptomyces griseus conidia from ultraviolet irradiation injury. Proc. Natl. Acad. Sci. U.S., 35: 73-79. (1949b) Experiments on light induced recovery of bacteria from ultraviolet irradiation injury. Bacteriol. Proc. P. 14. (1949c) Photoreactivation of ultraviolet-irradiated Escherichia coli, with special reference to the dose reduction principle and to ultraviolet-induced muta- tion. J. Bacteriol., 58: 511-522. (1950a) Light-induced recovery of microorganisms from ultraviolet radiation injury with special reference to Escherichia coli. Bull. N.Y. Acad. Med., 26: 189-199. (1950b) Action spectra for photoreactivation. Bacteriol. Proc. P. 53. (1951) Action spectra for photoreactivation of ultraviolet-irradiated Escher- ichia coli and Streptomyces griseus. J. Gen. Physiol., 34: 835-852. Kimball, R. F. (1949) The effect of ultraviolet light upon the structure of the macro- nucleus of Paramedian aurelia. Anat. Record, 105: 543. Kimball, R. F., and N. T. Gaither (1950) Photorecovery of the effects of ultraviolet radiation on Paramecium aurelia. Genetics, 35: 118. (1951) The influence of light upon the action of ultraviolet on Paramecium aurelia. J. Cellular Comp. Physiol., 37: 211-234. Knowles, T., and A. H. Taylor (1950) Spectral radiation involved in photoreactiva- tion of ultraviolet-irradiated cultures of micro-organisms. Bacteriol. Proc. P. 49. Latarjet, R. (1951a) Induction, par les rayons X, de la production d'un bacterio- phage chez B. megatherium lysogene. Ann. inst. Pasteur, 81: 389-393. (1951b) Photo-restauration apres irradiation X chez une bacterie lysogene. Compt. rend., 232: 1713-1715. Latarjet, R., and L. R. Caldas (1952) Restoration induced by catalase in irradiated microorganisms. J. Gen. Physiol., 35: 455-470. Luria, S. E. (1947) Reactivation of irradiated bacteriophage by transfer of self- reproducing units. Proc. Natl. Acad. Sci. U.S., 33: 253-264. Luria, S. E., and R. Dulbecco (1949) Genetic recombinations leading to production of active bacteriophage from ultraviolet-inactivated bacteriophage particles. Genetics, 34: 93-125. Luria, S. E., and R. Latarjet (1947) Ultraviolet irradiation of bacteriophage during intracellular growth. J. Bacteriol., 53: 149-163. Luria, S. E., R. C. Williams, and R. C. Backus (1951) Electron micrographic counts of bacteriophage particles. J. Bacteriol., 61: 179-188. Lwoff, A., L. Siminovitch, and N. Kjeldgaard (1950) Induction de la production de bacteriophages chez une bacterie lysogene. Ann. inst. Pasteur, 79: 815-860. Marshak, A. (1949a) Recovery from ultraviolet-light-induced delay in cleavage of Arbacia eggs by irradiation with visible light. Biol. Bull. 97: 244. ISC) RADIATION HIOLOGY (1949b) Recovery from ultraviolet-liglit-indufecl di'lay in cleavage of Arbacia ^ eggs by irradiation with visible liglit. liiol. IJulI. 97: 315-322. Monod, J., A. Torriani, aiul M. Jolit ( I'.Mit i Sur la reactivation de bacteries sterilisees par le rayonnement UV. Conipt. rend., 229: 557-559. Xewconibe, H. B. (1950) Photoreversal of the mutagenic effect of ultraviolet light in E. coli. Genetics, 35: 682. Xewcombe, H. B., and G. W. Scott (1949) Factors responsible for the delayed appearance of radiation-induced mutants in Escherichia coli. Genetics, 34: 475- 492. Xewcombe, li. B., and H. A. W hitehead (1950) Photoreversal of ultraviolet-induced nuitagenic and lethal effects in Escherichia coli. J. Bacteriol., 61: 243-251. Novick, A., and L. Szilard (1949) Experiments on light reactivation of ultraviolet- J inactivated bacteria. Proc. Natl. Acad. Sci. U.S., 35: 591-600. Puck, T. T., A. Garen, and J. Cline (1951) The mechanism of virus attachment to - host cells. I. The role of ions in the primary reaction. J. Exptl. Med., 93: 1 65-88. Shugar, D. (1951) Photoreactivation in the near ultraviolet of u-glycerol-iildcliyde- 3-pliosphate dehydrogenase. Experientia, 7: 26-28. Swenson, P. A. (1950) The action spectrum of the inhibition of galactozymas(> pro- duction by ultraviolet light. Proc. Natl. Acad. Sci. U.S., 36: 699-702. Swenson, P. A., and A. C. Giese (1950) Photoreactivation of galactozymase forma- tion in yeast. J. Cellular Comp. Physiol., 36: 369-380. Watson, J. D. (1950) The properties of X-ray-inactivated phage. I. Inactivation by direct effect. J. Bacteriol., 60: 697-718. Wells, P. H., and A. C. Giese (1950) Photoreactivation of ultraviolet light injury in gametes of the sea urchin Strongtjlocentrotus p^trpiiratits. Biol. Bull., 99: 163-172. Whitaker, D. M. (1941-42) Counteracting the retarding and inhibitor}' effect of strong ultraviolet on Fvcus eggs by white light. J. (Jen. Physiol., 25: 391-397. Witkin, E. M. (1946) Inherited differences in sensitivity to radiation in Escherichia coli. Proc. Natl. Acad. Sci. U.S., 32: 59-68. Manuscript received liy the editor June 20, 1952 CHAPTER 13 Sunburn Harold F. Blum^ National Cancer Institute'^ Bethesda, Maryland and Department of Biology, Princeton University Princeton, New Jersey "... I am black; because the sun has looked upon me." — Song of Solomon Erythema. Sitntan. Photosensitization. Protection against sunburn. Mechanisms. Fact and fancy. References. Although sunburn must always have been an obvious nuisance to man, it seems to have received little attention from scientists before the present century. To be sure, the true nature of the phenomenon could not have been understood until the discovery of ultraviolet radiation in 180P but it was another half century before it was recognized that sunburn is caused by this agent and not by heat.^'^ The first recorded statement I have found that sunburn is caused by ultraviolet radiation is in an account by Charcot which appeared in 1858. This describes two cases of burns from electric arcs and cites similar experiences by the physicists Foucault and Despretz. Foucault had found that uranium glass protected against the burning, which was attributed to the ultraviolet or, as they were then called, the " chemical " raj^s. Charcot clearly recognized that these burns were comparable to sunburn. Widmark in 1889 and 1891 made a more complete study, finding that the rays from a carbon arc passing through 1 Present address: Department of Biologj% Princeton University. 2 National Institutes of Health, Public Hejilth Service, Department of Health, Education and Welfare. ' In that year Ritter found that the sun's spectrum beyond the violet caused the blackening of silver chloride (Ritter, 1803). One year earlier Herschel had shown the existence of the infrared. * Actually, imconcentrated sunlight does not heat the skin enough to produce a burn. For a discussion see Blum (1945). '" Historically interesting is a paper by John Davy (1828), who concluded that, in order to elicit a burn, all wave lengths must affect the skin simultaneously. 487 488 1{ \I)I A'lloN lilOLoOY I (juartz elicited siiiil)uiii. wliereas those passing through window glass did ii(»t. Tills would indicate that the sunburn-inducing radiation includes no wave lengths longer than those cut off by window glass, i.e., about 0.32 M, where modern studies show the long-wave-length limit for sunburn to be.^ W'idmark was particularly concerned with sunburn of the eyes, which he showed to be brought about by the same wave lengths as sun- burn of the skin. By the turn of the century ultraviolet radiation .seems to have been generally recognized as the cause of sunl)urn (e.g., Hammer, 1891; Finsen, 1900; Miiller, 1900), although the spectral region was not delimited more exactly. The first attempt to do this was made by Henri and Moycho in 1914 who used the rabbit as the experimental animal; but the first accurate determinations on human skin were not carried out until the 1920's (Hausser and Vahle, 1922; Haus.ser, 1928). Modern studies of sunburn date from about the same time, but, with certain notable exceptions, these have been sporadic in occurrence and inconclusive in character. Perhaps this may be attributed to the com- plexity of the problem and the difficulties of experimentation in this field, as well as to lack of interest among scientific investigators. In contrast to the relative paucity of experiments on sunburn is the plethora of pop- ular fancies, from which scientists are not altogether immune. A good many false ideas find their explanations in unrecognized physical factors— the optics of the skin or the spectral quality of sunlight; others, in the complex nature of the physiological responses. It is hoped that some misunderstandings may be corrected in the course of this chapter, although a host of questions about sunburn will remain unanswered. Sunburn involves a number of tissues of the skin, and can be understood only in terms of the anatomy and physiology of that organ. Frequent reference is made in the following pages to layers of the skin which the reader may identify in Fig. 13-1. The character of sunburn may vary to some extent with the severity of exposure and from person to person, but it alwa3\s presents the same general picture. An hour's exposure to bright midday summer sunlight is usually followed by reddening of the exposed area. This erythema of sunburn is the gross manifestation of dilation of the minute vessels of the dermis. The erythema may be accompanied by slight swelling, which becomes more pronounced if the exposure is pro- longed. Blistering and desquamation may result from severe exposures, and there may be some pain and itching. The erythema is normally replaced after a few days by suntan, the brown color resulting from redis- tribution and increase in the melanin pigment of the epidermis. The sun- tan may persist for months or years. Not grossly observable, but of * Bowles (1889), who was unaware of Widmark's experiments, arrived at the con- chision that ultraviolot radiation is responsible for sunburn on the basis of experiences in the .\lps. The article is of interest as reflecting the generally vague ideas about radiation current at that time. SUNBURN 489 major importance with regard to sensitivity to subsequent exposure, is the hyperplasia of the epidermis which results in thickening of both the horny layer (corneum) and the viable malpighian layer. The hyperplasia is pre- ceded by degenerative changes in epidermal cells, and there is migration of leukocytes out of the vessels of the dermis. Considering these changes, sunburn may be classified as an acute inflammatory process, comparable to that associated with superficial burns of any kind. The term "sun- burn " is used here to include all these changes without regard to the inten- sity of the response, whether there is only a fleeting redness or whether severe blisters develop. The term is applied when the sunburn results EPIDERMIS DERMIS OR -^ CORlUM Fig. 13-1. Diagram of cross section of skin from the human shoulder, showing the various layers to which reference will be made in the text. The epiderrnis is the outer layer consisting of: sc, the stratum corneum or horny layer, and sm, the stratum Mal- pighii. The cells at the bottom of the malpighian layer constitute the basal cell layer b. The "prickle" cells lie superficial to the basal cells. The dermis or corium includes the tissues of the skin lying beneath the basal cell layer of the epidermis. In the papillary layer of the corium are situated the most superficial blood vessels (v). (After Maximov and Bloom, 1940.) from exposure to ultraviolet radiation from an artificial source as well as when it is caused by natural sunlight. Various aspects of sunburn stem from the action of ultraviolet radiation of wave lengths shorter than about 0.32 p on the cells of the malpighian layer of the epidermis. The effects on the cells are no doubt comparable to those of ultraviolet radiation on living cells in general, but they are manifested here in terms of a complex of secondary processes.'^ These may be treated separately for purposes of study so long as their basic origin and interrelations are not lost sight of. In this chapter various responses which follow the primary photochemical reaction in the cells of the malpighian layer will be considered separately. Later, the mechanism as a whole and the factors influencing sunburn under conditions of natural sunlight will be considered. Sunburn of the eyes also occurs and may be ' That ultraviolet radiation may have more than one primary effect on the cell is shown bv Blum et nl. (1954). 490 RADIATION BIOLOGY very paiiil'ul and di.sabliiif^. This .suhjcct will not he discussed here, the reader being referred to an earher review (Blum, 1945).* ERYTHEMA The ciylhcnia. which is the first grossly observable manifestation of sunburn, appears oiilx on the area exposed to the ultraviolet radiation, being (juite sharply delimited from the surrounding normal skin. Unless the dosage is very severe or prolonged, the erythema docs not appear immediately; with moderate doses there is usually an interval of an hour or more. The erythema may persist for a few days, fading imperceptil)ly into the brown color of suntan. Histological examination shows no changes in any laj'cr before the gross appearance of reddening.^ The erythema itself is revealed as the enlargement and engorgement of the minute vessels of the dermis (corium), which lie just below the epidermis (see Fig. 13-1). The red color results from the increased amount of blood in these vessels. Intracellular edema and the migration of leukocytes into the surrounding tissues begin at about the same time as the erythema. Although these first detectable changes involve the vessels of the dermis, this is not the site of the photochemical reaction that initiates them. Only a very small fraction of the incident ultraviolet — virtually restricted to the longer wave lengths — ever reaches the most superficial vessels (see Fig. 13-7), yet shorter wave lengths, e.g., 0.28 ^x, which are completely absorbed in the epidermis, do elicit erythema.'" Ultraviolet radiation that is absorbed in the epidermis must, then, cause changes there that lead in some way to dilation of the vessels in the layer beneath. It seems necessary to conclude that photochemical changes in the epidermis lead directly or indirectly to the elaboration of some mediating substance or substances which move down to the superficial vessels of the dermis and cause their dilation. The time between dosage with ultraviolet and appearance of erythema is presumably consumed in the elaboration of these dilator substances and their penetration into the dermis. The horny layer of the epidermis, the corneum, is a nonliving structure built from the viable cells of the malpighian layer beneath it. There is a tran- sition zone between the two layers whei'c it may bo difficult to distinguish * It has been claimed that ultraviolet radiation raises the threshold of scotopic vision, but this seems to have been definitely ruled out by recent studies by Wald (1952) who shows conclusively that radiation which might damage the retina does not reach it. 9 The lustological studies of Keller (1924a, b), Miescher (1930), and Ilamperl et al. (1939a) may be cited as the most complete. There are points of apparent divergence which probably result from differences in the spectral quality and dosage of radiation and the times at which the biopsies were made, t)ut the picture is similar in all three studies. '" Evidence that the longer wave lengths that iK-netnitc sligiitly to the dermis may have a direct effect there will be presented a little later on. SUNBURN 491 whether cells are living or dead. The corneum acts as a filter, preventing a large proportion of the incident ultraviolet radiation from reaching the malpighian, and until very recently it was thought to take no active part in the sunbiu'n complex. Rottier (1952, 1953) and Rottier and Mullink (1952) have now presented strong evidence, however, that photochemical changes in the corneum may contribute erythema-producing substances. The Eryfhemal Threshold. The most obvious criterion for quantitative studies of sunburn is the appearance of erythema. The erythema! threshold, i.e., the amount of ultraviolet radiation required to produce just perceptible reddening, has been quite generally used, and is up to now the only feasible measure available. As will be seen, however, it is not a very accurate one and should be interpreted with caution. The erythemal threshold has been measured in various waj^s, but all involve more or less the same type of operation. Usually the skin is covered with a template having a series of small holes through which the ultraviolet radiation may reach the skin. A different dose is applied through each of these holes. The threshold is usually determined after some arbitrary period, say, 24 hr. At this time, if the doses have been chosen appropriately, the highest ones will be represented by red areas corresponding to the positions of the holes in the template. The intensity of the redness will fall off with the dose, those areas having received the lowest doses showing no erythema whatsoever. The dose which has elicited a just perceptible erythema, or the average betw^een this and the next lower one which has produced no erythema, is usually chosen as the threshold. The exact value for the threshold will depend, of course, on the time elapsed between the exposure to ultraviolet radiation and the time of observing, since the erythema l)uilds up rather slowly and then falls off. Studies of the rate of increase and decline of erythema have shown that these factors may vary widely in different individuals (e.g., Schall and Alius, 1926). There are obvious sources of error in determining the erythemal threshold. The presence or absence of a very slight reddening is a diffi- cult end point and is subject to considerable uncertainty in reading since the degree of flushing or tanning of the skin affect the contrast. On the whole, photography has not helped to standardize such measurements, the unaided eye being more sensitive and accurate. It is probable, in any case, that inherent variability in the erythemal response is greater than the errors in reading. ^^ The threshold not only varies widely among indi- viduals but also from one area to another. For example, the palm of the hand or the sole of the foot can hardly be sunburned. On the other hand, the skin of the torso is rather uniformly sensitive. The threshold is affected by the amount of previous exposure to ultraviolet radiation. It may vary widely from one observation to another without known cause " For a discussion of errors of measurement of the erythemal threshold, see Blum and Terus (19-46b). 4D2 RADIATION HIOLOGY (Blum and Torus, 1940h), and thoro are regular changes with season and other factors (see Ellinger, 1941). All these factors must he taken into account in (luantitative studies, hut there are more serious difficulties in the interpictation of measurements which will need discussion. The Ertjlhonal Spectrum. The \-alue of action spectra in characterizing photohiological responses — aiding in fav'orahle cases in detecting the light- absorbing sulistance in the primary photochemical process — has been dis- cussed elsewhere in this book (see also Blum, 1950). The action spectrum for sunburn as measured in terms of the erythemal threshold is generally spoken of as the "erythemal spectrum." This has been determined by several workers whose results are in close agreement as far as the general character of the spectrum is concerned (Hausser, 1928; Luckiesh et a/., 1930; Coblentz et al., 1932). All these investigators found a maximum at about 0.295 m and a minimum at about 0.28 m- It is customary to plot the reciprocal of the threshold against wave length, as in Figs. 13-2, 3, and 7.'-' The shape of the erythemal spectrum must be affected by the corneum, which overlies the malpighiaii layer and acts as a semi-opacjue filter absorbing the wave lengths of the erythemal spectrum selectively. The corneum is composed of flakelike elements and, owing to the reflection and refraction of the light at the boundaries of these, the incident beam is scattered very effectively. '^ This scattering greatly enhances the attenu- ation of the radiation and renders measurements of the true absorption difficult.''* Absorption spectra for human epidermis treated in various ways to reduce scattering are shown in Fig. 13-2. Curves III and IV dis- play the least scattering and may be taken as giving the most reliable picture of the true absorption. Proceeding toward shorter wave lengths, we see that absorption begins to increase rapidly in the neighborhood of 0.3 n, reaching a maximum at about 0.28 /x, then falling to a minimum '2 Hamperl et al. (1939b) state that they have found maxhna in the erj'theinal spectrum at 0.295 /n and 0.26 fx with a minimum at 0.275 fx, and, for some skins with a very thin corneum, another maximum at 0.23 n. These findings are described in a brief note with no further detail and I have found no more complete description. 1' Hen.schke (1948) has shown that the erythemal threshold for radiation striking the skin at different angles follows the cosine law, which is characteristic of completely diffusing surfaces. " In a layer which both ab.sorbs and scatters, as does the corneum, the attenuation of a monochromatic parallel beam in passing through matter may be expressed as /, = / exp [-A-(/-x,sx)/| (13-1) where / and /; represent, respectively, the incident intensity and the intensity after passing through thickness /; k is a constant. The absorption function r\ and the scattering function sx are mutuall}* dependent l)ut may vary to different extents with the characteristics of the absorbing layer and with wave length. In such a layer the counterpart of the Bouger-Lambert absorption law is 1 1 = /e--' (13-2) where the attenuation coefficient a = A-(rx,sx). SUNBURN 493 near 0.25 n, after which it rises toward a new maximum. The general shape of the curves, particularly the maximum at 0.28 /x, resembles that of most unconjugated proteins. It may be concluded that the protein component is principally responsible for the absorption. The true attenuation in the epidermis is difficult to estimate because of the high degree of scattering. Different methods have been used for such measurements, those that are optically satisfactory yielding results in 024 026 0 34 0 36 028 030 0 32 WAVE LENGTH.^ Fig. 13-2. Absorption by human epidermis. {After Blum, 1941a.) Ordinate units are chosen for convenience to bring the data into comparison. The curves for epidermal attenuation, Nos. I, II, III, and IV, are plotted in terms of log /o// in the same units, and are directly comparable. The data are from Lucas (1931) for a sample of human epidermis 0.08 mm thick. Curve I is measured in water, incident light parallel; this curve is dominated by scattering. Curve II, cleared in glycerin, incident light parallel. Curve III, cleared in acetic acid, incident light parallel. Curve IV, cleared in acetic acid, incident light scattered. Curve V is a typical protein absorption spectrum plotted in terms of log /o// but with a different unit. Curve VI is the erythemal spectrum plotted as the reciprocal of the erythemal threshold {\/Qt). {After Coblentz et ai, 1932.) good general agreement (e.g., Lucas, 1931 ; Pearson and Gair, 1931 ; Kirby- Smith et al., 1942; for additional references including other regions of the spectrum see Blum, 1945). Most measurements have been made on whole epidermis, including both the corneum and malpighian layer. It is possible by various means to strip off the whole epidermal layer (Baum- berger et al., 1942), but separation of the two layers of the epidermis has not been feasible. Pearson and Gair's measurements of the transmission of bits of corneum separated from sunburned skin indicate that the greater part of the incident erythemal radiation is absorbed in this layer, and the character of the corneum and malpighian layer also suggest this (Kirby- Smith et al., 1942). Therefore, probably only a relatively small fraction 494 HAUIATION lilOLOGY ivaches the mali)if;hiaii layer itself, where the principal photochemical reaction of sunburn takes place. Rottier (1952) arrives at a similar pic- ture from reflection measurements. An exact determination of the absorption in that layer when in aitu would recjuire that its thickness and that of the corneum, as well as the attenuation coefficients, be accurately known. The corneum is formed from dead cells which have been pushed up from the malpighian layer as new cells are formed there; the elements of the corneum which so effectively scatter the radiation represent the casts of these dead cells. There is no very exact line of demarcation i)et\ve(Mi the two layers, the thicknesses of which vary from place to place on the body of a fi;iven person and from person to person. \'arious factors cause thickening of both layers, one of which is exposure to ultraviolet radiation. The effect of this on the erythemal threshold will be discussed later. Of particular interest with regard to the erythemal spectrum is the correspondence of the maximum of absorption at 0.28 m with the sharp minimum in the erythemal spectrum, clearly shown in Fig. 13-2. The strong absorption l)y the corneum in the region of 0.28 m probably accounts to a considerable extent for the minimum of effectiveness of these wave lengths in producing erythema, a point first made by Hauisser in 1928. It is obvious that with a spectrally selective absorbing layer (the corneum) superficial to that in which the photochemical reaction occurs (the malpighian layer) the measured erythemal spectrum does not reflect directly the nature of the absorbing substance which is concerned in that reaction. To reach the true action spectrum at the level of the malpighian layer, it would be necessary to make appropriate corrections for the attenuation of the radiation by the corneum, but this can be done only roughly. Some attempts at this will be discussed later on, but the inexactness of the measurements on which they must be based should not be lost sight of. The interpretation of the erythemal spectrum is fuither complicated by the findings of Rottier (1952, 1953) and Rottier and Mullink (1952), who present evidence that the corneum participates actively in the erythemal response. They suggest that two photochemical reactions are concerned, one in the corneum and one in the malpighian layer. Dilator substance is contributed to the eryth(>mal lesponse by both reactions. })ut, since there appear to be two different light absorbers involved, the dilator substances presvmial)ly differ also. These investigators suggest that the light absorber in the corneum may be a sterol. This active participation of the corneum does not, of course, prevent it from acting also as a spectral filter for the radiation reaching the malpighian layei', so that what has been said here about the effect of absorption by the conieuni is .still pertinent. It simply adds one more complicating factor that must be taken into account. SUNJiUliN 495 Factors Affecting the Erythemal Threshold. There are various instances ill wliirh it would bo coiu'enient to have a standard erythemal threshold that would ai)pl.v to lii(> "average" person. This would be jiarticularly us(>ful in coinp.Miiii^; the effects of differeiil polychi-omatic sources, e.g., 0.23 0 24 0 25 0 26 0 27 0.28 (a) 0 29 0 30 031 0 32 UJ r 75 50 2 5 - UJ I I I .J=^ --3Z; ..^r^ri^ I s n m -i '---1 1 — -"-^v^^ u-r 0 23 0 24 0 25 0 26 0 27 0 28 0 29 0 30 WAVE LENGTH, JU 0 31 1 0 32 ib) Fig. 1.3-.3. Spectrum of a mercury arc (diagrammatic); (a) absolute intensities; (6) "erythemal effective intensities," i.e., the absolute intensities corrected for the erythemal spectrum shown at I in (a). The height of each vertical line represents the intensity of radiant flux of this wave length (intermediate-pressure mercury arc). The points where curve II cuts the vertical lines represent the intensities when a Corex D filter was present. The points where curve III cuts the vertical lines repre- sent the intensities when a pj'rex filter was present. Point V on the line at wave length 0.2537 m represents the intensity of a low-pressure mercury arc, which may be regarded as emitting only this wave length. {After Blum and Tents, 1946a.) natural sunlight and radiation from the mercury arc. On first consider- ation, it might be expected that a reasonable approximation could be made in terms of the erythemal spectrum, and there have been various attempts to set such a standard. Let us represent the total amount of incident radiation within the erythemal spectrum by the symbol Q and the threshold (luantity of such radiation by Qr. Correspondingly, for monochromatic radiation we may use the symbols Qx and Qxt- In Fig. 13-3a is diagramed the spectrum of a mercury arc in the erythemal region. The spectrum is compo>sed of dis- 4Ut) KADIATION IJIOLOGY Crete monochronriMt ic lines. I"'(>r any j:;i\('n line of wave len{i;th X, Qx = It (13-3) wluMC / is the incident intensity ul' the line and / the duration of the exposure.'^ The total incident energy from the mercury arc within the limits of the erythemal spectrum may be ol)tained hy summing the amounts of energy delivered by the individual lines and multij^lying by /: Q = ({f^ + /o + • • • + A) (13-4) where l\, I-i, . . . , li are the intensities of the \arious lines. In Fig. 13-36 the "erythemal effective intensity" of the lines is indi- cated. This is obtained bj- multiplying the intensity of each line by the Table 13-1. Erythemal Threshold in Ter.ms of "Erythemal ICffective Energy"" (Blum and Terus, 194()a.) Effective energy, X 10^ ergs /cm - Subject Intermediate-pressvn-e mercury arc Low-pressure mercury arc, 0.2537 M No filter Corex D filter Pyrex filter 1 56 262 33 2 45 218 141 30 3 45 248 212 28 4 147 525 324 6G 6 64 240 29 6 5G 335 . . . 45 7 118 320 554 62 7() (avg.) 307 308 41 " See Fig. 13-3a and b. erythemal sensitivity S for that wave length relative to the maximum in the erythemal spectrum at 0.2967 /x (see Fig. 13-3a; the relative sensitivity is, of course, measured in terms of the reciprocal of the threshold). We may then express the "erythemal effective energy" Q' as Q' = til.S, + I,S, + • • • + liSi). (13-5) This is a measure which has been variously employed. Let us accept it as an approximation which can be put to test. In Fig. 13-3 are shown spectra of the mercury arc when restricted with spectral filters, and also the intensity of a low-pressure mercury arc which delivers practically all its radiation in the 0.2537-/i line. In an experiment carried out by Blum >•' It is understood that the values of Q and / are referred to unit area of the skin upon which they impinge, but for brevity of discussion this will not be specifically stated in each case. SUNBURN ■497 and Terns (1946a), thresholds were measured on a number of subjects under these different conditions and calculated in terms of E(i. (13-5). The results summarized in Table 13-1 show that the approximation is not a very close one. The threshold values are of the same order of magni- tude, but vary systematically according to the source, being higher when only the longer wave lengths are present. A CORNEUM «f I c Ic i\ MALPIGHIAN LAYER Cm I m Im \ ' Fig. 13-4. Diagrammatic representation of the epidermis. /, /c, and /„ are intensi- ties at the levels indicated; Ic and \m are the thicknesses of the layers indicated; Oe and am are the attenuation coefficients of the layers indicated. {AjUr Blum and Terus, 19466.) A simplified model may help us in inquiring into the causes of such deviation. Let us indicate by D the quantity of dilator substance elabo- rated in the malpighian layer and resulting in grossly observed erythema. Then for a given wave length X, we may write D = At hc/X 7 (13-6) where A represents the amount of radiant energy of wave length X absorbed per unit of time by the light absorber for the photochemical reaction leading to the formation of dilator substance, and t is the dura- tion of the exposure. The (juantity .4/, like Q\, is measured in units of energy, whereas the amount of D formed must be a function of the number of quanta absorbed ; this number is obtained by dividing by the energy of the quantum, hc/X, where h is Planck's constant and c is the velocity of light. The coefficient 7 is the ratio of molecules of dilator substance formed to the number of quanta absorbed, corresponding to the efficiency or quantum yield of any photochemical reaction ; it may or may not vary with wave length. Let us assume for convenience that the corneum and the malpighian are sharply separated homogeneous layers which may be represented as in Fig. 13-4. The intensity at the bottom of the corneum when a beam of 498 UADIATION lUOLOGY intensity / impiiijics upon the skin surface is /f, and /„, is the intensity at the bottom of the malpighian hiyer. The difTerence between Ic and /„, must represent the amount of ladiation absorbed phis the amount scattered per unit time in the nial|)i^liian layer, and of this A is some dehnitc fraction. We may tiierefore write A = (/c - 1^0 (13-7) where (3 is a proportionaHty factor which may be different for different wave lengths. The light absorber is assumed to be present in such large amount that its concentration is virtually' unchanged during exposure. If Ic is the thickness of the corneum and Uc the attenuation coefficient (see footnote 14) of that layer, we may write /^ = /e-«ci., (13.8) Similarly, Ln = 7,e-«"''- (13-9) where a„, is the attenuation coefficient and /„, the thickness of the mal- pighian layer. Combining E(is. (13-8) and (13-9), /„, = le--dc^-<^.j..., (13-10) and substituting from Eqs. (13-8) and (13-10) in Ecj. (13-7), we obtain A = I[c-'-''{l - c-"-''")/i]. (13-11) Combining Eq. (13-11) with Eqs. (13-3) and (13-6), <3x = [g-a.^.(i _ e-'^-'-'-)0]y\' (13-12) Then for polychromatic radiation, ^_, I ^1 , _ D2 V '^c \. „, /, a...u.\o^ .. \ ' r„— a "^ ■ ■ ■ "^ [e-«''•■ '.■*•!'":*•'•■ '•■:=iii ■9' B: C: D: E: F: Fig. 13-6. Result of exposure to the longer wave lengths of mereury arc (filtered through pyrex) (D,E,F), after exposure to wave length 0.2537 fx (.\,li,C). A: 1.1 ergs cm"^ X 10* of "erythemal effective energy" 3.9 ergs cm~- X 10* of "er3^thenial effective energy" 8.3 ergs cm"^ X 10* of "erythemal effective energy" 15.4 ergs cm~^ X 10* of "erythemal effective energy" 35.2 ergs cm"^ X 10* of "erythemal effective energy" 52.2 ergs cm"^ X 10* of "erythemal effective energy" Doses D, E, and F were applied approximately 15 min after A, B, and C. The diagram represents the various degrees of erythema 2^^ hr after the last exposure. Note inhibition of erj'thema in areas 5, 6, 8, and 9. {After Blum and Terus, 1946a.) latter inhibited the erythema ordinarily resulting from the former, as indicated in Fig. 13-6. The whole of the areas that received the heavy doses of the longer-wave-length radiation subsequently underwent blistering and destjuamation. The inhibition of erythema without inhibition of sunburn as a wh()l(\ was interpreted as resulting from a direct effect of the longer wave lengths of the erythemal spec^trum on the minute vessels of the skin, limiting their response to the dilator substance formed in the epidermis. The trans- mission spectra for human epidermis presented in Fig. 13-7 show that the longer wave lengths of the erythemal spectrum do penetrate below the epidermis to a certain extent and could act directly on the mirmte vessels. The inhibition of erythema would seem comparable to the inhibition of SUNBURN 501 flushing after severe superficial burn from heat, when the skin may be blanched in the area of the burn. Although the inhibition of erythema is experimentally demonstrable only with severe doses of these longer wave lengths of the erythemal spectrum, it seems reasonable to assume that they exert some inhibitory effect even at much lower doses and hence may influence the erythemal threshold when determined with polychromatic radiation. Partial inhibition of dilation of the minute vessels by the longer wave lengths would raise the observable erythemal threshold to 0 24 026 028 034 0 36 038 0 30 0 32 WAVE LENGTH,// Fig. 13-7. Transmission of human and of mouse epidermis. Mi is from ear of normal young mouse; M^ from ear of mouse subjected to repeated exposures to ultraviolet radiation. Hi from untanned volar surface of human forearm; Ho from slightly tanned volar surface of the same forearm; H3 from heavily tanned dorsal surface of forearm. All measurements are for transmitted radiation collected at 45°, epidermis not cleared. The transmitted radiation collected at 180° would be somewhat greater. (After Kirhxj-Smith et al, 1942.) these wave lengths. This would in effect negate Eq. (13-14) by making the response to the dilator substance effectively wave-length dependent. Another explanation may now be based on the finding by Rottier (1952) and Rottier and Mullink (1952) that dilator substance is formed in the corneum. Our simple model does not take this into account, but could be extended to do so by making additional assumptions. If the dilator sub- stance formed in the corneum were different from that formed in the mal- pighian layer and its action on the vessels differed quantitatively, or if the rate of formation and diffusion to the vessels were quite different in the two cases, the relation in Eq. (13-14) need not hold. This is another possible explanation of the discrepancy that has been bothering us; per- haps both explanations are pertinent to the complex response studied in sunburn. 502 li \l)l \l H)\ HlOLOflV III I lie model (icsciilx'd. i ccipiocity (/ X / = a constant) has been assumed : it is specilicnily stated in F>(|. (13-3) and is implicit in Im|. (13-()). I"'xperimentally. recipiocity d<)(\s hold over a wide i:iii«i;e of dosr's when monochiomatic radiation is used to determine the threshold, as has heeii shown by Haiissoi' and \'ahle (1922), C'oblentz el al. (li)32j, and Jilum and Terus ( l!)4()b). On the other hand, when polychromatic radiation is used there may be a wide de\iation tVom i-eci))rocity, as shown by Schall and Alius (192(). 1928a) and by Blum and Terus (194()1)). This would seem to reflect complex time relationships in the mechanism of vascular dilation. The existence of more than one factor active in determining the ery- themal spectrum also explains certain of Hausser's findings (1928) that had always remained puzzling. When he studied the erythemal effec- tiveness in terms of the intensity of the erythema, Ilausser found that for shorter wave lengths a proportional increase in dosage produced a rela- tively greater increase in intensity of the erythema than did a comparable increment of the longer wave lengths. This is difficult to luiderstand in terms of a simple model, but is not surprising in a complex system involv- ing processes with different wave-length dependence. \'ari()us factors have been reported to affect the erythemal threshold, but when these reports are based on minor differences they should be accepted with caution. Threshold measurements are of limited accuracy at best, and the great individual variation makes statistical treatment uncertain in view of the obvious complexity of the erythemal mechanism. Heat is one of the factors that might be expected to affect the erythemal response, but, although numerous studies have been made, the results are somewhat conflicting. Schall and Alius (1928a) and Clark (1936) both found that the temperature diu'ing exposure to ultra\'iolet radiation had very little effect on the threshold. Clark found, on the other hand, that the rate of appearance of the erythema increased with temperatiu'e. This is explained if we assume that the primary photochemical reaction is, as might be expected, virtually independent of temperature, but that subse- quent parts of the erythemal process are temperature dependent. Fail- ure to separate these two phases of the response may account for some of the apparent disagreement in results obtained by other investigators. Recently Ilelmke (1948-49) reported that infrared radiation lowers the erythemal threshold and shortens the time to appearance of erythema in a majority of indi\'iduals. SUNTAN Pigment Migration and Formation. Observed grossly, the erythema of sunburn appears to fade almost imperceptibly into svmtan; i.e., there is a gradual color change from red to brown. But this does not represent an immediate relation between these two aspects of sunburn. Whereas the SUNBURN 503 former results from the increased blood flow in the minute vessels of the dermis, the latter represents the change in position and increase in quan- tity of melanin pigment in the epidermis. That the color of suntan is principall}^ due to melanin is indicated by studies of the spectral distri- bution of the radiation reflected from tanned and untanned skin (Edwards and Duntley, 1939a, b). The difference in position of the erythema and tan may be readily demonstrated by pressing a sheet of glass against the skin. This squeezes the blood out of the minute vessels, causing the red color to disappear, but the tan remains visible since the melanin pigment in the epidermis is not affected. In this way the time of disappearance of erythema and the appearance of tan may be judged. In untanned skin the melanin pigment is located chiefly in the basal cell layer (e.g., Masson, 1948). According to histological studies, about the time that tan first becomes grossly observable, this pigment begins to migrate toward the surface, where it should have a greater effect on the spectrum of the reflected light from the skin's surface. The pigment migration begins about 24 hr after the initial exposure to ultraviolet radi- ation. It was first described by Keller (1924a) (Pigmentverschiebung). It was also noted by Peck (1930) in the case of pigmentation brought about by ionizing radiation from thorium (pseudohyperpigmentation) and is suggested in earlier studies by Lutz (1917-18). The mechanism of this migration of melanin does not seem to be clearly explained. Recent spectrophotometric studies by Jansen (1953) suggest that there is early pigment formation preceding the migration. Later there is increase of melanin in the region of the basal cell layer, the amount depending on the severity of the dose and whether repeated (Lutz, 1917-18; Keller, 1924a, b; Hamperl et al., 1939a). While it has been maintained that all the basal cells can form melanin, the point of view that only certain dendritic cells accomplish this gains favor (e.g., Masson, 1948). The pigment may be found in the prickle cells of the malpighian layer and in the corneum of skin that has been subjected to repeated doses of ultraviolet radiation. ^^ Once formed, the pigment persists in the epi- dermis for months and may still be grossly observable after several years. In later discussion the total of changes involving the melanin pigment will be referred to as melanization, pigment migration and pigment formation being distinguished as parts of the complex. Pigment Darkening. Formerly it was thought that tanning is brought about by ultraviolet wave lengths somewhat longer than those which elicit erythema. But in 1939 Henschke and Schultze published a series of studies which showed that, while such wave lengths do cause darkening of the skin, the mechanism is ((uite different from that of the primary melani- '" What has hoeii .said applies to tlie skin of the white races. In Xegro skin the pig- ment is more uniformly distributed throughout the epidermis, including the cornevim, but comparable histological studies are lacking. 504 RADIATION BIOLOGY /atioM of tlu* cpidciinis. Alxmt a yeai" earlier 1. Ilausscr (1988) liad ii'portctl that a dark hiowii t-oloration i)f normal skin is brought about by wave lengths longer than those which produce erythema. Ilenschke and Schultze studied this phenomenon extensively, coming to the conclusion that it represents the darkening of preformed melanin: it will be referred to here as "pigment darkening." This differs in many respects from the primary melanization that is brought about by the erythemal spectrum, i.e., by wave lengths shorter than about 0.32 n. The action spectrum for pigment darkening extends from about 0.8 ^ to about 0.42 n with a l)road maximum near 0.34 ^i, as is represented in Fig. 13-8. In contradistinction to erythema and pigment formation, pigment darkening is readily brought about by sunlight passing through window glass, which by removing the wave lengths shorter than 0.32 /j., prevents both erythema and primary 024 026 028 030 032 0 34 0 36 0 38 040 042 WAVE LENGTH, _/^ Fig. 13-8. PD, action spectrum for pigment darkening. E, erythemal spectrum: S, sunlight. All ordinates are arbitrarily chosen and do not indicate relative magnitudes. (After Henschke and Schultze, 1939a.) melanization of the epidermis.'^ Pigment darkening may appear within the first few minutes of exposure to sunlight and usually reaches its maxi- mum within an hour, whereas pigment migration does not begin for at least 24 hr, and new pigment formation only after a few days. Several- hundredfold greater dosages of radiant energy are recjuired to bring about pigment darkening than are necessary for erythema and primary melani- zation. Pigment darkening is most pronounced in skin that has been previously sunburned and still retains traces of suntan, whereas melani- zation is greatest in skin not previously sunburned. Pigment darkening does not occur when oxygen has been removed by blanching the skin by pressing a quartz plate against it, whereas erythema and melanization are not afTected by this treatment. Histological examination shows that these longer wave lengths do not cause pigment migration or the forma- *" Common window glass, whicli is a fairly standard product, cuts out almost all the wave lengths shorter than 0.32 ^l. \ person therefore does not ordinarily become sim- burned by .sunlight passing through a window pane, although it is possible to elicit an erythema in this way witli intense niidsuinmer sunlight. There have been window glasses on the market in recent years which transmit somewhat more of the erythemal radiation. SUNBURN 505 tion of new melanin (Hamperl et al., 1939a). The failure to differentiate the processes of mclanization and pigment darkening has led to confusion and to the belief that primarj^ melanization is brought about by wave lengths longer than those of the erythemal spectrum. Miescher and Minder (1939) confirmed and extended the findings of Henschke and Schultze, offering an explanation in terms of the Meirowsky effect. Meirowsky in 1909 and later Lignac (1923) had found that dark- ening of the pigment of cadaver skin is brought about by ultraviolet radi- ation or by heat, and that such darkening does not occur in the absence of oxygen. Miescher and Minder pointed out that the pigment darkening in living skin parallels these findings, and presented further evidence of the identity of these effects. They postulated that the darkening repre- sents the oxidation of melanin pigment already present in the skin in a reduced leuko form. The exact nature of this leuko form was not speci- fied, but P'igge (1939) has since shown that melanin may be reversibly bleached in an in vitro oxidation-reduction system. The writer has sug- gested (1945) that the melanoid pigment described in human skin by Edwards and Duntley (1939a, b), as a result of studies of spectral reflec- tion, is a leuko form of melanin, or a mixture of the oxidized and reduced forms. Sharlit (1945) has vehemently attacked the findings and conclu- sions of Miescher and Minder. Unfortunately, he does not give any description of the ultraviolet radiation he used nor does he indicate that he recognized either the difference between pigment formation and pigment darkening or the spectral difference of the radiation bringing about the two effects. It is to be hoped that this discrepancy in results will be explained by further experimental studJ^ Hormones and Tanning. That hormones may influence the tanning of human skin was first shown by Hamilton and Hubert (1938), who found in eunuchoids that previously sunburned areas darkened in color when androsterone was injected. Although other factors are concerned, includ- ing circulatory changes, melanin pigment is involved in this phenomenon (see Hamilton, 1948). Biochemical Aspects. In vitro reactions leading to the formation of melanin have been applied with considerable success to melanin formation in certain species, but until recently there have been objections to inter- preting suntanning in terms of such reactions. There are still uncer- tainties to be explained, but the resolution of some of the difficulties seems to be in sight. The general subject has been recently reviewed by Lerner and Fitzpatrick (1950), and there will be no attempt to do so here. Let us assume, tentatively, that the melanin formation associated with suntan follows the same chemical steps as the in vitro reaction, which may be schematized in abbreviated form suitable to this discussion as follows: tyrosinase Tyrosine + Oa > dihydroxyphenylalanine -^ intermediates -^ (do pa) melanin. ')()(') UADIATIOX ItlOLOOV The amino acid tyrosine is oxidized to dihydroxyphenylalaniiie, com- monly known as "dopa," l)y llio action of the enzyme tyrosinase, a copper protein comj^lex. Molecnlai' ()\y}>;en takes part in the (irst step in vitro and ajiain in later stei)s. There seems no objection to applyinj^ such a scheme to melanin formation in suntanninji;, hut difficulty arises when further attempt is made to represent the melanin formation, let alone the whole process of suntanning, in terms of a photochemical forwarding of this in vitro reaction scheme. In 1933 Frankenburger found that exposure of solutions of tyrosine to ultraviolet radiation resulted in the formation of a brown pigment, pre- sumably melanin, but very large doses of radiation were re(iuired. Later Arnow (1937) found that ultraviolet radiation could bring about oxidation of tyrosine to dopa, but again the amount of radiation recjuired was much greater than would be needed for the in vivo production of suntan. Rothman showed in 1942 that this reaction may be accelerated by the presence of small amounts of ferrous salts and that when these are present the quantities of energy retiuired are nearer to those effective in sun- tanning. The possibility must be considered that ultraviolet radiation acts in vivo to bring about the oxidation of tyrosine to dopa in a manner parallel to the in vitro reactions. After this, the succeeding steps may presumably take place without the intervention of ultraviolet. There seems one objection to all these schemes in that the in vitro production of melanin, and specifically the initial step, are aerobic, whereas in vivo the reduction of the molecular oxygen .supplied by occlusion of the circulation to the skin during the exposure to ultraviolet radiation has no effect on the subsequent melanization (Blum et a/., 1935; Henschke and Schultze, 1939a; Blum, 1941b). It may be objected that by such methods the partial pressure of oxygen in the skin is not reduced sufficiently to inhibit the reaction. Sharlit (1945), as a result of his studies on cadaver skin, suggested that oxygen is obtainable from the cytochrome system. It can only be pointed out that the same method of removing oxygen inhibits pigment darkening and photosensitized oxidations. This point need not be emphasized, however, since there are other difficulties to consider. Rothman et al. (1946) have proposed a scheme which is not based directly on the in vitro reactions and so may avoid the above objection. They found for human skin, as had Ginsberg (1944) for the guinea pig, that there is present a factor which inhibits melanin formation in vitro. This they identified tentatively as water-extractable sulfhydryl com- pounds. They suggest that ultraviolet radiation and other injurious agents cause destruction of these compounds with the result that the formation of melanin can proceed. Fitzpatrick et al. (1949, 1950) include Rothman's scheme to account for the melanin formation of suntan. They also include the oxidation of tyrosine to dopa by direct photo- chemical effect, and add another factor, the acceleration of this reaction SUNBURN 507 by the formation of dopa itself, which they have found to be catalytic in trace amounts. The photochemical formation of dopa meets, of course, the same difficulty mentioned, since, presumably, oxygen must be present during the exposure to ultra\dolet radiation. It may be pointed out that this is not an objection to the in vitro reaction scheme as a whole since oxygen is present during the time when the melanin is actually formed, but only to the acceptance of the photooxidative step as the initial one. But it seems that none of these schemes, except possibly that of Rothman, takes cognizance of what happens in suntanning. None accounts adequately for the delay of several days between the exposure to ultraviolet radiation and the first observable formation of new melanin. Whatever the ultra\'iolet radiation does, it must be thought of as setting off a chain of events that leads to melanin formation at a later time. The removal of inhibiting substances as suggested by Rothman might act in this way, but it is difficult to fit the other schemes into this picture, although the formation of small amounts of dopa, which then catalyze the formation of larger amounts, has attractive aspects. It may also be pointed out that the initial part of the melanization process — migration of pigment already formed — is not accounted for in any way by these schemes. The objection may be le\aed against a good many of the in vitro studies that massive doses have been used which bear no relation to those which produce in vivo effects. There is more and more evidence to indicate that profound changes may be brought about in living cells by very small quantities of ultraviolet radiation. Thus, while ultraviolet may bring about in living skin the formation of a small amount of melanin according to reactions such as have been demonstrated in vitro, the skin might be pretty thoroughly cooked before the quantity reaches the proportions of suntan. On the other hand, a very small amount of ultraviolet radiation might, by eliciting changes in the cell, lead to the ultimate production of a good deal of melanin. In this chapter the point of view is adopted that the primary effect of the ultraviolet radiation is injury to cells of the malpighian layer and that melanization, like other aspects of the sunburn mechanism as a whole, is secondary to this injury. Melanization is a common response of the epi- dermis to injury of any kind, which maj^ represent an over-all reaction similar to or identical with that by which melanin is formed in vitro. But the initiating of such a reaction need not be specifically related to the agent that brings about the injury. In this regard the experiments of Peck (1930) may be cited, which show that melanization brought about by radiation from thorium closely parallels melanization from ultraviolet radiation, there being, in both cases, first pigment migration and then the elaboration of more pigment. There is little reason for believing that the ionizing radiation from thorium would bring about the same kind of 508 RADIATION BIOLOGY photoclu'inical reaction that is postuhittHl by the hiochoinical schomo just discusst'd, hut both ioiii/.iufj; radiation and ultrax'ioh't injure the epidermal cells. Moreover, melanization of human epidermis tollows injur}' by such diverse agents as heat, lubbinji;, photodynamie action (photosensitized oxidation) and wounds. I'nt'oitunatcly, there is no very good explana- tion i'oi- melanization ot the epidermis caused by any of these agents, but, until such matters are more clearly understood, the idea of direct forma- tion of melanin by photochemical reaction, attractive though it may be, should not be accepted without reservation. PHOTOSENSITIZATION Sensitization of the skin to light by exogenous agents cannot })e dis- cussed here at length. It may be pointed out, however, that in human skin there are at least two different types of such action. As examples, sulfanilamide sensitization and the photodynamie action of eosin (the cause of photosensitization by lipstick) may be chosen. vSulfanilamide does not act as a true photosensitize!" — i.e., it does not participate as the light absorber — but seems only to increase the injury to the skin brought about by ultraviolet radiation. Eosin, on the other hand, acts as a photo- sensitizer, absorbing the light and bringing about photochemical oxidation of skin constituents. Photosensitization by eosin is inhibited by occlu- sion of oxygen; photosensitization by sulfanilamide is not (I^lum, 1941b). Photosensitization of human skin is discussed elsewhere by the author (1941a), and other aspects of photooxidation will be treated by Norman Clare in volume III of this series. PROTECTION AGAINST SUNBURN Natural Protection. Following even a mild degree of sunburn the ery- themal threshold rises and may remain above its previous level for about two months. The decrease in sensitivity to ultraviolet radiation takes place at approximately the same time that suntan is developing. This observation and the relative insensitivity of Negro skin to suidight led long ago to the idea that the melanin pigment serves to protect the skin against sunburn by absorbing the sun's rays (e.g., Davy, 1828; Wedding, 1887; Bowles, 1889). The experiment of painting the skin with an opacjue material, e.g., India ink, and so obtaining protection from sun- burn, has been repeatedly interpreted as supporting the idea of protection by the melanin pigment (e.g., Davy, 1828; Finseii, 1900) which persists as one of the most popular misconceptions regarding sunburn. The idea apparently remained unchallenged until about 1920, after which objections were raised by a inimber of workers. Probal)ly the first of these was With (1920) who found that areas of vitiliginous skin, which do not develop melanin i)igment, can be rendered less sensitive to ulti-a- SUNBURN 509 violet radiation by repeated doses of this agent, an observation also made a few years later by Meyer (1924). Keller (1924b) noted that the first darkening of the skin results from migration of the pigment rather than from increase in amount. Others pointed out that sensitivity to ultra- violet radiation returns to normal before the tan disappears (Perthes, 1924; Schall and Alius, 1928,a,b). The first to recognize the major factor contributing to the reduced sensitivity that develops after exposure to ultraviolet radiation was Guillaume. In a short paper in 1926 and in a book published in 1927 he called attention to the thickening of the corneum that results from the active epidermal proliferation which is one of the aspects of sunburn, and pointed out that this must greatly reduce the penetration of ultraviolet radiation to the malpighian layer. Histological studies by Lovisatti (1929) and by Miescher (1930) soon gave strong support to Guillaume's idea. As early as 24 hr after exposure to ultraviolet radiation, degenera- tive changes in the prickle cells of the malpighian layer are detectable. Later the whole layer including the basal cells may be involved, depending on the severity of the dose. When the acute stages of this reaction sub- side both the malpighian layer and the corneum are left thickened. An idea of the difference in penetration that this makes is illustrated in Fig. 13-7. The thickening is eventually reduced and in about two months may have returned to normal if there has been no further exposure to ultraviolet radiation. The erythemal threshold returns to normal at about the same time. As mentioned, vitiliginous areas of skin do not tan, but do increase their erythemal thresholds after exposure to ultraviolet radiation (With, 1920; Meyer, 1924). The same is true for albino skin (Lovisatti, 1929). That decreased epidermal penetration resulting from thickening is the expla- nation in these cases is also supported by the decrease in transmission of the skin of the albino mouse after repeated exposure, which is illustrated in Fig. 13-7 (Kirby-Smith et al, 1942; Blum and Kirby-Smith, 1942). Li none of these cases is there any melanin pigment formed. Such evidence does not, of course, preclude the possibility that increase in melanin plays some part in reducing the amount of ultraviolet radiation reaching the malpighian layer, but this factor may be of only minor importance. It is easy to be misled by the observation that the skin becomes darker to the eye when tanning takes place, because this is not directly dependent on absorption by melanin within the erythemal spectrum. Presumably, absorption in the latter region would be due largely to the phenolic ring, as in the case of protein. It seems doubtful that the increase of absorp- tion due to melanin could be nearly as great as that due to increase of the protein layer by thickening of the corneum, and that the total attenuation of the radiation should be influenced to a much greater extent by the latter. .) 10 RADIATION HKH-OCY ( )l" |);irliiul:ir iiilcicst in this regard is Xcfiro skin. Here melanin is not only nioic plcntilul hut is normally present in large amounts in the eor- ntuin rathci than being concentrated in the neighl)orhood of the basal cell layer as in the unexposed skin of the white races. The Negro character- istically has a high erythemal thi'eshold (Miescher, 1<)32), and it might seem obvious to attribute this to the greater amount of melanin. How- ever, it is generally agreed that the Negro corneum is considerably thicker than that of the white races — although no extensive histological account seems to have been pul)lished — and so the high erythemal threshold may be due in this case to the protection afforded by a thick corneum rather than to high melanin content. Because, as a rule, suntan and thickening of the corneum develop at about the same time, it is natural to associate the former with the pro- tection afforded by the latter. Actually, however, the thickness of the coi-neum and the erythemal threshold both return to normal in about two months if the skin is guarded from exposure to ultraviolet radiation. On the other hand, suntan may persist for months or years, fading away gradually, but at the same time subject to transient phases of bleaching and darkening. While as a general rule a tanned skin is less susceptible to sunburn than is an untanned one, this may be most misleading. There are persons who develop very little tan, yet can expose themselves to sun- light with relative impunity, and some who are fairly dark in complexion are comparatively sensitive to sunburn. In studying the erythemal thresholds of a considerable number of subjects, the author has been unable to find any close correlation between complexion and sensitivity to ultraviolet radiation. The possibility that the epidermal cells develop a local immunity, pre- sumably by the formation of antibodies, was suggested by Perthes (1924) ; who carried out experiments which he interpreted as demonstrating such immunity, but these may have been confused by the inhibition of ery- thema (Blum and Terus, 1946a). The findings subsequent to Perthes' experiments indicate that it would be very difficult to demonstrate such immunity if it occurred. Hausmann and Spiegel- Adolph (1927) sug- gested that changes in the proteins of the corneum might lead to a decrease in transmission. The thickening of the corneum seems so obviously the major factor in limiting the amount of the erythemal ultra- violet reaching the malphigian layer that other contributing factors would seem to be relatively unimportant. Artificial Protection. During World War II the problem of evaluating various agents designed for protection of the skin against sunburn assumed certain minor importance. While the problem seemed at first relatively simple, it proved to be quite difficult, and no completely satisfactory solu- tion was found. Only as the study progressed did its complexity become manifest, and in the end the most important thing gained was a somewhat SUNBURN 511 better understanding of the various factors involved. On first consider- ation the problem appears essentially a physical one. The desired end is to obtain a protective layer of some kind that will absorb the erythemal spectrum but preferably not absorb much of the visible. Obviously, the film cannot be too thick, must be pliable, and should meet certain "cos- metic " requirements. The corneum of the skin, which may be taken as a model for it, seems to be a much better protective agent than any artificial one that has been devised. The corneum is a scattering and absorbing agent with a very high attenuation coefficient for the wave lengths of the erythemal spectrum. The problem becomes more complex when another selectively absorbing layer is put on top of the corneum. i I PREVENTIVE dp h ^P \ J i CORNEUM Uc Ic /c \ MALPIGHIAN LAYER a-m 1 1 Im /m y i Fig. 13-9. Diagrammatic representation of the optical conditions of epidermis with a layer of preventive superposed. /, Ip, I^ and /„ are intensities at the levels indicated; Ip, U, Im are thicknesses of the layers indicated; Up, Uc, and am are attenu- ation coefficients of the layers indicated. (After Blum et al., 1946.) We may extend our previous analysis to cover this situation. Figure 13-9, in which the preventive is indicated as having the thickness Ip and the attenuation coefficient a^, illustrates this situation. The intensity Ip of a monochromatic beam at the bottom of this layer is given by Ip = /e-""'". (13-15) Following the same reasoning as in developing Eqs. (13-9) to (13-12) we may write Dhc Qp\ — ^g-aplpQ-adc^l _ e-«".''")^]^X (13-16) to describe the situation when the preventive is in place. An index of the protection afforded by the prev^entive should be given by the ratio of the thresholds with (Qpr) and without {Qr) the pre\'entive; this ratio we will designate for monochromatic radiation as P\, which is related to the other 512 (limiit it i(\s l)y UADIATION BIOLOGY Dhc Q,n [e-»i>'pe-»'''(l — e-"'-''")/3]7\ Dhc (13-17) [g-ac/c(l _ e-«".'".)|3]'yX Since for monochromatic- radiation the vahie.s of 1) at the threshold should be the same with or without the preventive, we may cancel out to obtain l\ = ('--"'-. (13-18) That is, with monochromatic radiation the ratio P should measure, within the limits of accuracy, the attenuation by the preventive, and 26 '^ 24 - • t • 22 ~ • 20 - • "5^ 18 - • • ■z o Is le LiJ o - • • • • • t • • • 12 ^ • 10 - e - 1 1 1 1 1 1 • • 1 1 1 , 8 12 16 20 24 28 32 36 40 ERYTHEMAL EFFECTIVE ENERGY THRESHOLD fQJ, ergs cm'2 x lO"* Fig. 13-10. Variation of protection P with erythcnuU threshold in 22 suhjeets. All the points were ol)tainP(l with layers of the same sunburn preventive 25 m thick spread on skin of the abdomen. (After Bhnn et ai, 1946.) should be independent of the threshold of the individual. P\ would, of course, vary with wave length acc^ording to the spectral characteristics of the preventive. With polychromatic radiation — e.g., sunlight — the problem is not so simple, and in actual measurements it was found that the ratio P varied systematically with the threshold as illustrated in Fig. 13-10. Let us examine the possible reasons for such an unexpected variation. We may write E(\. (13-13) in a more general form, such as might be used SUNBURN 513 for graphical integration when deaHng with a spectrally continuous source such as sunlight within the limits u, v, of the erythemal spectrum /-i 1 /"" Dhc d\ ,,^ ,^, ^ = '"" ].. [e-Hl - .-~'-)gl7X- ('^-'8' We may WTite the corresponding eciuation from Eq. (13-lG), when the preventive is in place, and describe the ratio of thresholds with and with- out the preventive as follows: , f" DhcdX he Qr ['- DhcdX ■ ^ ^ ^ Quantities which could be canceled out in obtaining Eq. (13-18) now lie within the integral and cannot be canceled. Thus there must be uncer- tainty involved in the use of the ratio P as an index of the protection afforded against sunburn by sunlight or any other polychromatic source. This might account for a considerable amount of the variation found experimentally in the values of P obtained for different persons. The systematic variation with individual threshold, which is illustrated in Fig. 13-10, could be accounted for if we invoke the variation of D with wave length, for which the evidence has been discussed. If it is assumed that with no preventive the threshold is determined principally by the amount of absorption in the corneum, it may be assumed that in persons with high threshold a smaller proportion of the incident longer wave lengths of the erythemal spectrum penetrate to the vessels of the dermis where such radiation inhibits the dilation of the minute vessels. Thus the inhibitory effect should be less in the person with a thick corneum who will also have a high threshold, tending to decrease the difference between the erythemal threshold of high- and low-threshold persons. The same thick- ness of a given preventive should diminish the intensity of the radiation reaching the dermis in the same proportion in the case of the high- and the low-threshold persons, but would have a greater absolute effect on the latter than the former. This could account for the lower values of P found for high-threshold persons, which is illustrated in Fig. 13-10. On the other hand, if the threshold is considerably affected by the amount of dilator substance formed in the corneum, the picture may be changed somewhat. The thing of practical importance is that the amount of protection measured by P varies with the threshold. Radiation from a carbon arc was used in making the measurements shown in Fig. 13-10, but the same systematic variation was found when similar measurements were made 514 RADIATION BIOLOGY ■with the mercury arc. Mcusuromcnts made with the mercury arc gave much higher \ahies of /■• than those obtained on the same persons witlj the carhoii aic The six'cl rum of neither of these sources closely resembles that of sunHght, and it is obvious that, with such wide varia- tions in the ol)tainal)le values of /-*. tests of sunburn preventives with sources other than sunlight can have little practical significance as (|uanti- tative measures. Sunlight itself is a variable (juantity, its intensity and spectral distribution changing radically with latitude, season, and time of day, and the validation of laboratory tests against actual field conditions is therefore beset with many unavoidable difficulties. Moreover, the diflRculties of assembling an adequate number of human subjects under conditions appropriate for such validation may be overwhelming. It may be tempting to choose some simpler method without validation (see Giese and Wells, 194Ga, b), but the dangers inherent in doing so must be obvious from what has just been stated. Eventually, physical methods such as that of Luckiesh et al. (1946) may replace the laborious testing on human skin, but these too will recjuire validation (see Blum c( «/., 194(). for further discussion of methods). It should be obvious that tests with methods chosen uncritically and without validation may be misleading. MECHANISMS Up to this point sunburn has been treated as a complex physiological response further complicated by difficult optics. Only descriptions of various aspects of this complex have been given, with little attempt to explain the more intimate underlying mechanisms. It is now time to attempt to put together a general picture of the whole. In the scheme arranged in Fig. 13-11, it is represented that various physiological responses are mediated by specific substances elaborated as a result of the action of ultraviolet on living cells. In erythema this seems certainly to be the case, since wave lengths completely absorbed in the epidermis pro- duce dilation of vessels in the underlying dermis, and such action seems to be interpretable only in terms of a transportable mediating substance. In other cases the presence of a mediating substance in the scheme can be considered as little more than a guess, and the tentativeness of the whole must be emphasized. In the diagram (Fig. 13-11) the central theme evolves from an initiating photochemical reaction taking place in the malpighian layer, which results in the elaboration of a number of substances by the cells of that layer. Each of these substances induces a specific physiological response in the immediate locus of its elaboration or in a neighboring tissue. Consider first the nature of the initiating photochemical reaction in terms of the light-absorbing substance. As has been shown, the possibility of identi- fying this substance by means of the action spectrum is complicated by a SUNBURN 515 o ^x 5». CM z z < z UJ LiJ < ^ _l _l < z 3 O q: OD UJ LO lO < 5 o ^ /^ 1 2 / y Z < _J z '^ 0 5 / f y- cr / , UJ < Ul 5 Nl Q / J 2 CL < Ul -I UJ u_ / / ^ / / ^r' / / o / s 0 / / ^r / / < u. -I X / / / / / / / / _LULAR NERATION PIDERMIS OWED BY FERATION £ / / / / y s nf / // y^ CE DEGE IN E FOLL PROL m ^UJ ^ 0 DQ / / / tK. <7 c 1 / / ^ OTHE -LAMM UBSTA > / to / y< z UJ n 1- 5^ H If) O UJ q: u z tt y^ 2cn -4^ MIGRATI OF LEUKOCY y^ jQ b \ ZO < UJ ^^ 0 — UJ Q ^ — z ^^ 0 w ^r ' c 3 Q. ^^^^ ^ Ul < z ^ \ V Ul V o is _l -I UJ ^ \ '^ \ 3 Ul -I y- Ul CO ^ k i; \ ^ \ 02 \ \ \ Z a; \ \ ^ > UJ 0 < \ \ °5 t- < 5 Ul -►J \ \ < l- _|00 _ CO Q 3 _j _ -fc_ X 1- >- tr UI c Q 0 CO 0 1 , ... \ CO J > ^ / uj52 ' \ 4 °/ 'T \ / m ^ 1 2 - ^ z z 0 0 p i=u. < / I H O ^ \ 7 — "^ CD 0 ^ \ 2 in ± Ul > — \ / X § \ ^"■--^"yl z Ja - g ^>^ / \ 1 ^(L \ Z - 5 Ul r^\^ u. 0 o?co <«F1 UJ LJ z cc < 1- _l t^ — CD ATION GHBOR ESSEL Q: CI —1 u_ A < o CO ^2> h 1 lU V) HOTO CHE MICA EACTION 1M EDI ATE EFF N CELLS UBSTANCES LABORATED ISSUE ESPONSE 2 I 1- o z Ul -1 UJ 0 in > 0 z < (t < S a. q: £ o CO t J 1- L C Oi 510 KAUIATION BIOLOGY MumlHT of factons. The t'rytlu'mal .spectrum is ohviou.sly domiiiated by the attenuating? properties of the corneum which determine the spectral minimum at t).28 n and must influence the position of the maximum at 0.2U7 M- Some of the attempts to interpret the erythemal spectrum do not take this into account. The most adecjuate attempt to examine the erythemal spectrum in terms of the radiation actually absorbed in the epidermis was that of Mitchell (1938) who used the data of Lucas (1931) to correct for epidermal t ransmission. Mitchell arrived at the conclusion that the light absorber is protein, but sub.se(iuent evidence throws doubt on this. In the first place, neither the inhibition of erythema by longer wave lengths nor the elaboration of dilator substance in the corneum was recognized at the time of Mitchell's analysis. These factors should affect the shape of the erythemal spectrum to an unpredictable extent. It is implicit in Mitchell's analysis that the (juantum efficiency [factor y in Eq. (13-6)] is independent of w^ave length. But, if the inactivation of enzymes by ultraviolet radiation may be taken as characteristic of the action of this agent on proteins in general, this cannot be as.sumed since the inactivation is, in some cases at least, wave-length dependent (e.g., McLaren, 1947). The recent demonstration that dilator substance is formed in the corneum frustrates any attempt at analysis in terms of a single substance. It seems altogether reasonable to draw an analogy between the effects of ultraviolet radiation on the cells of the human malpighian layer and the action of this agent on cells in general, which has been discussed elsewhere in this volume. Evidence seems at present to favor the idea that the light absorber in such effects is, as a rule, nucleic acid, and recent findings of Blum et al. (1950) indicate that, in the case of retardation of cell division in sea urchins' eggs, the locus of action is the nucleoprotein. But in the.se experiments, as is true in general, the ultraviolet radiation may po.ssibly produce changes that are not reflected in the particular criterion studied (e.g.. see Blum, Cook, and Loos, 1954). It must therefore be kept in mind that, although luicleoproteins in the malpighian cell are without doubt affected by the ultraviolet radiation that reaches these cells, this should also be true of the simple proteins in the cells to a greater or less extent. Thus, while it seems reasonable that the various phj\siological responses studied in sunburn are initiated by the action of ultraviolet radiation on nucleoproteins, it is possible that some of them involve other photo- chemical reactions. Other light absorbers have been suggested in con- nection with various aspects of sunburn (e.g., Ellinger, 1930; Rothman and Rubin, 1942; Frankenburger, 1933), but these have various objec- tions which will not be di.scussed at length here (see Blum, 1941a, 1945). Even in systems optically much better suited to the comparison of action and absorption spectra, the distinction between nucleic acid and simple protein may be difficult, and there are other cell constituents absorbing in SUNBURN 517 the same region which, though present in much less amount, have rather similar absorption spectra. Thus attempts to analyze the erythemal spectrum, even when carried out as carefully as Mitchell has done, cannot lead to very definite conclusions. So, while nucleoprotcin has been indi- cated as the light absorber for the photochemical reaction initiating the principal theme schematized in Fig. 13-11, even this very basic postulate must be open to question (particularly in light of recent studies, Blum et al, 1954). In the diagram (Fig. 13-11) the immediate result of the photochemical reaction in the epidermal cells is described rather vaguely as injury. Among other substances elaborated by these cells is a dilator substance which brings about the vasodilation of the minute vessels of the dermis, and is grossly manifested as erythema. An increase in dilator substance has been demonstrated in skin (Nathan and Sack, 1922; Ellinger, 1930) and blood (Laurens and Kolnitz, 1940) of animals that have been exposed to ultraviolet radiation, but the identity of the dilator substance con- cerned in the erythema of sunburn has not been clearly shown. Lewis and Zotterman (1926) suggested that it was histamine or a histamine-like H-substance, and this view was once rather widely accepted. However, Percival and Scott (1931) found that the response of the skin to histamine pricks is not altered by the presence of the erythema of sunburn, and this is also true for skin which has been exposed to ultraviolet radiation but has not yet developed erythema (see Blum and Terus, 1946a). While the initial erythema of sunburn is rigidly restricted to the area exposed, after severe doses of ultraviolet radiation a red flare may develop which extends outward from the exposed area. This usually appears only after about 24 hr. The flare is irregular and differs in general appearance from the initial sharply limited erythema. Lewis and Zotterman (1926) called attention to the resemblance of this flare to that which results from prick- ing histamine into the skin, but, in the latter case, the flare appears almost immediately, and they do not adequatel}^ explain the delay in the case of sunburn. According to Lewis' hypothesis (1927), the histamine flare is the result of antidromic impulses in afferent nerves and in the case of sunburn it seems likely that stimulation of these nerves might result from injury to the fibers themselves or to surrounding tissues. The flare is most pronounced when the erythema is induced by longer wave lengths of the erythemal spectrum, suggesting that the dermis itself, which is reached by those wave lengths, may be the locus of this effect. Krogh (1929) concluded that there must be at least two dilator substances con- cerned in sunburn, and, if erythema and flare are separate entities medi- ated by different substances, this should be true. The whole (luestion of the nature of the dilator substance is complicated by the finding that a dilator substance is produced by photochemical changes in the corneum (Rottier, 1952, 1953; Rottier and Mullink, 1952). 518 RADIATION HIOLOGY This has boon iiichulcil in the schcinc without any commit incut as to the nature of the Hj;ht al)sorher or tlie dilator substance. On the left of the scheme, injury to the minute vessels of the dermis is indicated as resultinjj; in inhibition of their dilation. It may be supposed, in :icct)rilance with the aij^unAciit ahcady jjresented, that the underlying photochemical changes are essentially the same as those which result in (>rythema but that they occur at a different locus. It is suggested that injury to epidermal cells and to the vessels of the dermis may both be con- cerned in the dilation of vessels immediately adjacent to the area exposed to ultraviolet radiation, which manifests itself grossly as the delayed flare. The various aspects of sunburn are essentially similar to those of inflam- matory responses in general. Menkin (1940, 1942, 1943a, b, 1944) has succeeded in isolating from inflammatory exudates several substances which he concludes act specifically in bringing about certain of the tissue responses characteristic of inflammation. It seems not unlikely that the complicated picture presented by inflammation may ultimately be analyzed in terms of such "inflammation substances," and this general thesis has been follow^ed in the scheme shown in Fig. 13-1 1 where the elab- oration of a number of such substances is indicated as resulting from injury to the epidermal cells. According to Menkin, the increase in capil- lary permeability which occurs in inflammation is not due to histamine as supposed by Lew'is and others but to a substance which he calls "leuko- taxine." This substance also exerts a chemotactic effect, and, by virtue of both these actions, brings about the migration of the leukocytes from the capillaries into the surrounding tissues. In the present scheme leuko- taxine is credited with bringing about the migration of leukocytes and with tissue edema, the latter being a direct result of increased capillary permeability. Indicated by a dotted line in the diagram is the probable participation of vasodilation as a lesser factor in the edema. Cellular degeneration in the epidermis, followed by proliferation, is indi- cated as resulting directly from injury to the epidermal cells by the pri- mary photochemical reaction. Alternatively, there is indicated the formation of other intermediary substances which may cause such cellular degeneration and proliferation. The substance necrosin, w^hich Menkin (1943b) finds to cause the degeneration of cells, may be responsible in part for such changes in the epidermis and w^ould be included in this category. It should be emphasized that none of these mediating substances has as yet been isolated from sunburned skin. Consistent with the rest of the scheme, there has been introduced, as one of the products of the injury to epidermal cells, a " melanotactic " factor which contributes a part of the melanization of the epidermis that becomes manifest as suntan. This melanotactic factor presumably accounts for the migration of the melanin i)igment which is observed in the early stage of the melanization process. The existence of such a sub- SUNBURN 519 stance is cut iivly liypothetical. Also indicated as a result of the injury to epidermal cells is the formation of new melanin which is the other factor contributing to melanization of the epidermis. A dotted line suggests the direct participation of ultraviolet radiation in the formation of melanin as a possible minor factor in melanization. Indicated on the extreme right of the diagram is the darkening of bleached melanin which is brought about by the longer wave lengths of the ultraviolet and the near-visible spectrum, approximately 0.3-0.42 n. This is an oxidation of bleached melanin by molecular oxygen as shown by ]\Iiescher and Minder (1939). 1.0 I.I 1.2 1.3 1.4 WAVE LENGTH, ^ Fig. 13-12. Spectral distribution of sunlight. 0, outside the atmosphere (air mass 0); 1, with the sun at zenith (air mass 1); 2. with the sun 60° from zenith (air mass 2). Curves R and C indicate, respectively, the sensitivity of the human rods and cones; the ordinate units are arbitrarily chosen. {After Blum, 1945; data from Moon, 1941.) Sunburn may logically be classed with other types of burn. There is a ciuantitative aspect to be recognized in that the primary damage in sun- burn is very superficial because of the low penetration of ultraviolet radi- ation. As in the case of other superficial burns, some systemic involve- ment may be expected when the burned area is extensive enough. Studies of such systemic effects will not be discussed here, nor will the various claims for therapeutic effects of ultraviolet radiation. For an account of some of these, and of other effects of sunlight on man, the reader is referred to an earlier review (Blum, 1945). Sunburn by Natural Sunlight. Many misconceptions about sunburn arise from failure to take into consideration the character of sunlight as an environmental factor. It is natural to evaluate the intensity of sunlight in terms of perception by the human eye, but this may be very misleading as regards the intensity of the sunburn-producing portion. The situation is best illustrated by referring to the diagram in Fig. 13-12. which shows 520 RADIATION HIOLOGY the spectral distribution of sunlight iiiidcr different conditions. Curve 0 describes sunlight outside the earth's atmosphere as determined by extra- l)olation, showing a maximum at about 0.48 m- As the sunlight passes through the atmosphere, its spectrum is modified by the absorption and scattering of some wave lengths to greater extent than others, as indicated in curves 1 and 2 of the figure. Curves R and C are, respectively, spectral sensitivity curves for the rods and cones of the human eye. It may be seen that the sensitivity of the eye becomes virtually nil before the long- wave-length limit of sunburn is reached at 0.32 m- It is obvious, there- fore, that the eye gives no direct information as to the amount of ery- themal radiation in sunlight. Neither does it give much indirect infor- mation, because some of the factors that greatly influence the intensity of the erythemal portion of sunlight have little effect on the portion which the eye sees. The amount of erythemal radiation in sunlight varies much more widely with time of day, season, and other factors than does the visible portion of the spectrum. This is because of differences in absorption and scattering by the atmosphere. The intensity of the erythemal region of the spec- trum of sunlight is greatly diminished by absorption by ozone, which cuts off virtually all wave lengths shorter than 0.285 fx.^^ The wdde variation in the intensity of the erythemal portion of the spectrum with season, lati- tude, and time of day also depends on absorption by this gas. For example, when the sun moves from zenith to G0° from zenith — the condi- tions represented by curves 1 and 2 — the erythemal spectrum is greatly reduced. But the change in intensity registered by the human eye is rela- tively small because the gases of the atmosphere absorb the visible light only slightly. Sixty degrees in zenith angle corresponds to 4 hr in time, so even on a bright midsummer day one is not likely to be sunburned before 8 o'clock in the morning, or after 4 o'clock in the afternoon, although at both times the sun is shining brightly so far as the eye can discern. Obviously, chance of sunburn should be judged in terms of time of day rather than visual impression. Of course, it must be remembered to correct for daylight saving and for the difference between the two extremes of an official time zone, where the deviation from true time may be over an hour. Variation in ultraviolet radiation with season is anothei- matter that tends to be left out of account. In the north temperate zone the maximum insolation occurs on June 21, the summer solstice. One unfamiliar with the problem may be surprised to learn that the erythemal radiation may be more intense at the beginning of May than it is at the end of August. This is likely to lead to severe sunburn in the springtinn^ when, moreover, the subject is not likely to have as thick a corneum as at " For a discussion of solar radiation and the factors dotorininin^ its intensity and spectral di.stribution at the earth's surface see Chap. ,i of this volume by Sanderson and Hulburt. SUNBURN 521 the end of the summer. There is a tendency to think of the tropics as receiving much more sunlight than the temperate zones and therefore to anticipate greater danger of sunburn in the tropics. This is again a (juestion of season. At the time of the simimer solstice the sun is over the Tropic of Cancer, 23°27' north of the e(iuator. On that date there is about the same sunlight at 47° north — roughly the latitude of Seattle, Washington; St. John's, Newfoundland; and Paris, France — as at the equator. On June 21, other things being ecjual (the ozone layer probably would not be), there should be about as much danger of sunburning at the one latitude as at the other. Another less obvious factor likely to mislead the unwary is the high proportion of erythemal radiation that is contributed by reflection from the sky. Sky radiation is sunlight that has been scattered, principally by the gas molecules of the atmosphere. The shorter wave lengths are scattered to a greater extent than the longer ones, and hence the sky radi- ation is richer in the erythemal radiation than in visible radiation. ^^ At noon on a very clear day in temperate latitudes, the sky radiation may constitute only 10 to 15 per cent of the visible component falling on a hori- zontal surface, whereas for wave lengths shorter than 0.32 /x, the direct and sky radiation are about ecjual under the same conditions (Pettit, 1932; Luckiesh et at., 1944). Obviously one need not be directly exposed to the sun to receive a sunburn if he is sufficiently exposed to the sky. This explains why, for instance, one may sit under a beach umbrella pro- tected from the direct rays of the sun and yet receive a severe sunbiuni from sky radiation which the umbrella does not cut off. On a lightly overcast day, particularly in a fog, the scattered erythemal radiation may be many times the direct radiation and may cause a severe sunburn. At high latitudes at midday the sunburn-producing component of the sky radiation is also greater relative to that of the radiation coming directly from the sun than it is at low latitudes^^ (Coblentz et al., 1942). Dust and smoke absorb the erythemal wave lengths very strongly, and a slight haze that is barely perceptible to the eye may completely wipe out this part of the spectrum. Hence sunburn is more likely in rural regions than in the neighborhood of industrial cities. The seashore with an onshore wind carrying away all traces of smoke or dust may be a particu- larly favorable place for sunburning, as may be the high mountains. Snow and ice reflect the erythemal spectrum to a high degree, explaining in part at least the coining of the terms "snow burn" and "glacier burn." Water reflects less than is commonly believed (Coblentz et al., 1933). Although sunburn is primarily produced by wave lengths at the short 2" The sky is blue for the same reason, bhie and violet being scattered to a greater extent than the longer wave lengths. 2' DeLong (1884) reported in tlie log of the ill-fated Jeanetle the difficulties from sunburn during the arctic summer. 522 RADIATION BIOLOGY end of the solar spectrum — shorter than 0.32 /x it should not be forgotten that pigment darkening is hiought ai)out by somewhat longer \va\'e lengths extending into the visible at about 0.42 /x. Henschke and Schultze (1939b) showed that when suntan is produced by natural sun- light a good deal of the color may be due to pigment darkening rather than the production of new pigment, because of the high intensity of the wave lengths between 0.32 and 0.42 /x as compared to those shorter than 0.32 /j (see Fig. 13-8). This should be particularly true when the sun is far from zenith as in the later afternoon and early morning, and at noon in late fall or early spring in temperate latitudes. Differences in the pigment- darkening effect may explain why some persons retain a rather dark sun- tan throughout the winter months while others do not. In the early spring, when there is relatively little of the erythemal radiation in sunlight but a considerable amount of the pigment-darkening radiation, darkening of the skin by the latter may be mistaken for new suntan. The belief that this darkening is accompanied by immunity to sunburn may lead to over- exposure when the erythemal radiation increases with the progress of the season. FACT AND FANCY The convincing fact that one has been severely sunburned Avhen he thought he was adequately protected from exposure looms large against any explanation that may be offered in terms of such a complex of factors as has been discussed. In the past, when still less was known about the subject, it was a natural tendency to invent factors — sometimes with little respect for physics or physiology — which would seem to explain such puzzling occurrences. In a book published in 1905 b}^ Giles it is suggested that there are penetrating "Y" and "Z" rays in sunlight which are analogous to X rays and which account for the alleged injurious effects of tropical sunlight. While we ma}^ smile today at this naive invention, we may wonder how much influence this book had in generating popular fear of tropical sunlight and in establishing such practices as the wearing of red spine pads in the tropics, which continued at least up to the time of World War II.-'- Another wholly uncritical book reflecting this fear was that of Woodruff" (1905), on which Jack London based his diagnosis of his own breakdown during the Cruise of the Snark.-^ I recall too, what difficulty I had only a few years ago in convincing a practitioner of medicine that the "actinic" rays of the sun would not pass through the metal roof of an automobile. Certainly some of the difficulty one encoun- ters in this respect comes from failure to recognize the importance of indirect radiation from the sky. 2^ An ainiisiiifz; account of field tosts in the Philip)pint>s to dotoiinine the value of oranf!;c-r('(l underwear (IMialen, I'.tlOj shows how .seriou.sly such ideas were taken at one time in some quarters. ^^ The problem of tropical sunlight has l)eeii discussed elsewhere (Blum, 1945). SUNBURN 523 There is a widely accepted opinion that wet or perspiring skin is more susceptible to sunburn than dry skin. This was investigated by Blum and Terus (194()b), but no significant difference was found in the ery- themal threshold for skin wet with water from that for dry skin. Simi- larly, subjects showed no significant difference in threshold before and after profuse sw^eating in a "hot" room. I find a clue to the origin of this particular idea in a personal experience. A number of years ago I received a severe sunbiu'n after an excursion in a small boat, during the whole of which I thought myself protected by a white shirt I was wearing. There were intermittent show^ers with bright sunlight between, and the sea w^as choppy so that my shirt and skin were drenched during most of the day, either from rain or spray. I remember that some additional pro- tection was offered by my undershirt which was also wet but under which the sunburn was less severe. I might have concluded that wetting the skin had lowered the erythemal threshold so that I sunburned through the shirt which I knew would have afforded adequate protection under other circumstances. The alternative answer was, of course, that the trans- mission of the erythemal radiation by the shirt was increased by wetting. Support for the latter idea came some years later from tests of the pro- tection afforded against sunburn by fabrics (Blum and Terus, 1946b), when it was found that various kinds of white shirtings became much more transparent to the erythemal radiation when wet, presumably as a result of diminished scattering by the wet fibers. The wide differences in individual erythemal thresholds, the variation of the threshold in a given individual, and the failure to evaluate correctly the amount of exposure to erythemal radiation in sunlight, are factors which no doubt contribute to the variety of opinions encountered regard- ing the efficiency of a given sunburn preventive. These factors, and the misconceptions regarding tanning, may account for many apparent vagaries of sunburn. In these pages I hope I have explained away a number of false ideas, but I have also taken the risk of introducing a few more by some of my speculations regarding the mechanism of sunburn (particularly in the scheme in Fig. 13-11). So, in ending, I should call attention to the need for further study of the sunburn process, always with proper regard for the knowai physical and physiological aspects of the problem lest we be led further into the realm of fancy. REFERENCES Arnow, L. E. (1937) The formation of dopa by the exposure of tyrosine solutions to ultraviolet radiation. J. Biol. Chem., 120: 151-153. Baumberger, J. P., V. Suntzeff, and E. V. Cowdry (1942) Methods for the separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis. J. Natl. Cancer Inst., 2: 413-423. Blum, H. F. (1941a) Photodynamic action diseases caused by light. Reinhold Publishing Company, New York. ;j2-i UADIATION lUOLOCJY (1941b) Studios of photosensitivity duo to sulfanilamide. J. Invost. Dermatol., 4: 15!» \7.i. — (11)45) Tho piiysioloKioal offoots of suulinlit on iiiuii. IMiysiol. Revs., 25: 483-530. (1950) Aotion spootra and ahs()r|)tioii spoctra. In, liiophysical rosoarch methods, ed. ¥. M. I'hor. Interscionco Publishers, Inc., Now York. Vp. 417-449. Blum, H. F., J. S. Cook, and G. M. Loos (1954) A comparison of five effects of ultra- violet li^ht on the Arbacia eg^- J- Gen. Physiol., 37: 313-324. Blum, 11. F., M. Kic'hor, and W. S. Torus (1940) Evaluation of proteotive measures against sunburn. Am. J. Physiol., 140: 118-125. Blum, H. F., and J. S. Kirby-Smith (1942) Natural protection against sunburn. Science, 96: 203-204. Blum, II. F., and W. S. Terus (1940a) Inhibition of tho erythema of .sunburn by large doses of ultraviolet radiation. Am. J. Physiol., 140: 97-100. (1946b) The erythemal threshold for sunburn. .\m. J. Physiol., 146: 107- 117. Blum, H. F., W. G. Watrous, and R. J. West (1935) On the mechanism of photo- sensitization in man. Am. J. Physiol., 113: 350-353. Bowles, R. L. (1889) Sunburn. Alpine J., 14: 122-127. Charcot (1858) Erytheme de la face et opthalmie produits par Paction de lumi^re ^lectrique. Compt. rend. soc. biol., 10: 03. Clark, J. H. (1936) The temperature coefficient of the production of erythema by ultraviolet radiation. Ain. J. Hyg., 24: 334-342. Coblentz, W. W., F. R. Gracely, and R. Stair (1942) Measurements of ultraviolet solar- and sky-radiation intensities in high latitudes. J. Research Natl. Bur. Standards, 28: 581-591. Coblentz, W. W., R. Stair, and J. M. Hogue (1932) The spectral erythemic reaction of the untanned human skin to ultraviolet radiation. J. Rosoarch Natl. Bur. Standards, 8: 541-547. (1933) Measurements of ultraviolet solar radiation in various latitudes. J. Research Natl. Bur. Standards, 10: 79-88. Davy, J. (1828) Observations on tho effect of the sun's rays on tho human tiody. Trans. Med. Chir. Soc. Edinburgh, 250 273. DeLong (1884) The voyage of the Jeanette. Houghton Mifflin Company, Boston. Edwards, E. A., and S. Q. Duntley (1939a) Tho pigments and color of living human skin. Am. J. Anat., 65: 1-33. (1939b) An analysis of skin pigment changes after exposure to sunlight. Science, 90: 235-237. EUinger, F. (1930) tJber die Entstehung eines Korpors mit histaminahnlichen Wirkungen aus Histidin unter Ultraviolettbestrahlung und die Bedeutung dieses Vorganges fiir Lichtorythem. Strahlentherapie, 38: 521-542. (1941) Biologic fundamentals of radiation therapy. Elsevier Press, Inc., New York. Figge, F. H. J. (1939) Melanin: a natural reversible oxidation-reduction system and indicator. Proc. Soc. Exptl. Biol. Med., 41: 127. Finsen, N. R. (1900) Neue Untersuchungen iiber die Einwirkung des Lichtes auf die Haut. Mitt. Finsons Lysinstittit, 1:8-34. Fitzpatrick, T. B., S. W. Becker, Jr., A. B. Lerner, and 11. Montgomery (1950) Tyrosina.se in human skin : demonstration of its presence and of its role in human melanin formation. Science, 112:223-225. Fitzpatrick, T. B., A. B. Lorn(>r, E. Calkins, and \V. H. Summerson (1949) Mam- malian tyrosinase: melanin formation by ultraviolet radiation. Arch. Dermatol, and Syphilol., 59: 620-625. SUNBURN 525 Frankenburger, W. (1933) Photochemische Betrachtungen zur Wirkung ultra- violetter Strahlen auf die menschliche Haut. Naturwissenschaften, 21:1 16-124. Ciieso, A. C, and J. M. Wells (1946a) Sunburn protection, natural and artificial. Sci. Monthly, 62: 458-464. (1946b) Sweat and water-resistant sunburn preparations. J. Am. Phann. Assoc, Sci. Ed., 35: 208-212. Giles, G. M. (1905) Climate and health. William Wood & Company, Baltimore. Ginsburg, B. (1944) The effects of the major genes controlling coat color in the guinea pig on the dopa oxidase activity of skin extracts. Genetics, 29: 176-198. Guillaume, A. C. (1926) Le pigment epidermique, la penetration des rayons u.v. et la mecanisme de protection de I'organisme vis-a-vis de ces radiations. Bull. mem. soc. med. hop. Paris, 50 (3d series): 1133-1135. (1927) Les radiations lumineuse en physiologic et en therapeutique. Masson et Cie, Paris. Hamilton, J. B. (1948) Influence of the endocrine status upon pigmentation in man and in mammals. In, Biology of melanomas, ed. M. Gordon. Spec. Publ. N.Y. Acad. Sci., 4: 341-357. Hamilton, J. B., and G. Hubert (1938) Photographic nature of tanning of the human skin by studies of male hormone therapy. Science, 88: 481. Hammer, F. (1891) Uber die Einfluss des Lichtes auf die Haut. F. Enke, Stuttgart. Hamperl, H., U. Henschke, and R. Schulze (1939a) Vergleich der Hautreaktionen beim Bestrahlungserythem und bei der direkten Pigmentierung. Virchow's Arch, pathol. Anat. u. Physiol., 304: 19-33. (1939b) tjber den Primarvorgang bei der Erythemerzeugung durch ultra- violette Strahlung. Naturwissenschaften, 27: 486. Hausmann, W., and M. Spiegel-Adolf (1927) Uber Lichtschutz durch vorbestrahlte Eiweisslosungen. Klin. Wochschr., 6: 2182-2184. Hausser, I. (1938) tJber spezifische W^irkungen des langwelligen ultravioletten Lichts auf die menschliche Haut. Strahlentherapie, 62: 315-322. Hausser, K. W. (1928) Einfluss der Wellenlange in der Strahlenbiologie. Strahlen- therapie, 28: 25-39. Hausser, K. W., and W. Vahle (1922) Die Abhangigkeit des Lichterythems und der Pigmentbildung von der Schwingungszahl (Wellenlange) der erregenden Strah- lung. Strahlentherapie, 13: 41-71. Helmke, R. (1948-49) tJber die Beeinflussung des Uitraviolett-B durch gleichzeitige Bestrahlung mit Infrarotlicht, gepriift an der Erythemschwelle und Latenzzeit. Strahlentherapie, 78: 145-148, and various earlier papers in the same journal. Henri, V., and V. Moycho (1914) Action des rayons ultraviolets monochromatiques sur les tissus. Mesure de I'cnergie de rayonnement correspondant au coup de soleil. Compt. rend., 158: 1509-1511. Henschke, G. (1948) Die Winkelabhiingigkeit des Ultravioletterythems und ihre Bedeutung flir die Dosimetric. Strahlentherapie, 77: 297-299. Henschke, U., and R. Schulze (1939a) Untersuchungen zum Problem der Ultra- violett-Dosimetrie. III. Uber Pigmentierung durch langwelliges Ultraviolett. Strahlentherapie, 64: 14-42. (1939b) Untersuchungen zum Problem der Ultra vaolett-Dosimetrie. IV. Wirkung der Sonnenstrahlung auf die Haut. Strahlentherapie, 64: 43-58. Jansen, M. T. (1953) A reflection spectrophotometric study of ultraviolet erythema. J. Clin. Invest., 32: 1053-1060. Keller, P. (1924a) Uber die Wirkung des ultravioletten Lichtes auf die Haut unter besonderer Beriicksichtigung der Dosierung. III. Histologic der Lichtent- ziindung. Strahlentherapie, 16: 537-553. (1924b) Uber die Wirkung des ultravioletten Lichtes a>if die Haut unter 026 IIAUIATIOX mOLOGY bosoiidonT ncriicksiclitimiiiK dor DosioruiiK. IV. F.ichtgewohnunR und Pig- inoiiti(>ning. Stnihlciitlicrapie, 10: 824-8;i5. Kirby-Sinith, J. S., H. F. liluiii, and II. (1. Grady (1942) Pciii-tratioii of ultraviolet radiation into skin, as a factor in carcinogonosis. J. \atl. Cancer Inst., 2: 403- 412. Krogli, A. (1929) Tlic anatomy and piiysiology ol the capillaries. Rev. cd. Vale University Press, New Haven, Conn. Laurens, H., and II. Kolnitz (1940) The effects of carbon arc radiation on blood pres- sure and blood liistaniine. Med. Record, 152: 209 212. Lerner, .\. H., and T. B. Fitzpatrick (1950) Biochemistry of melanin formation. Physiol. Revs., 30: 91-126. Lewis, T. (1927) The blood vessels of the human skin and their responses. Shaw and Sons, London. Lewis, T., and V. Zotterman (192(5) Vascular reactions of the skin to injury. IV. Some effects of ultraviolet light. Heart, 13: 203-217. Lignac, G. O. E. (1923) tjber den Chemisnuis und die Biologic des men.schlichen Hautpigments. Virchow's Arch, pathoi. .\nat. u. Physiol., 240: 383-4 Hi. Lovisatti, N. (1929) L'assuefazione della cute al radiazioni ultraviolette. Arch. radiol., 5: 958-966. Lucas, N. S. (1931) The permeability of human epidermis to ultraviolet irradiation. Biochem. J., 25: 57-70. Luckiesch, M., L. L. Ilolladay, and A. 11. Taylor (1930) Reaction of untanned human skin to ultraviolet radiation. J. Opt. Soc. .\mer., 20: 423-432. Luckiesh, M., A. H. Taylor, H. N. Cole, and T. Sollmann (1946) Protective skin coatings for the prevention of sunburn. J. Am. Med. Assoc, 130: 1-6. Luckiesh, M., A. H. Taylor, and G. P. Kerr (1944) Seasonal variations of ultraviolet energy in daylight. J. Franklin Inst., 238: 1-7. Lutz, W. (1917-18) Zur Kenntnis der biologi.schen Wirkung der Strahlen auf die Haut, mit speziellen Beriicksichtigung der Pigmentl)ildung. Arch. Dermatol, u. Syphilis, 124: 233-296. McLaren, A. D. (1947) Photochemistry of proteins. J. Polymer Research, 2: 107- 109. Masson, P. (1948) Pigment cells in man. In, The biology of melanomas, ed. M. Gordon. Spec. Publ. N.Y. Acad. Sci., 4: 15-51. Maximov, A. A., and W. Bloom (1940) A textbook of histology. 3d ed., W. B. Saunders Company, Philadelphia. Meirowsky, E. (1909) tJber Pigmentbildung in voin Kcirper losgeloster Haut. Frank- furt. Z. Pathol., 2: 438. .Menkin, V. (1940) Dynamics of inflammation. The Macmillan Company, New York. (1942) Further studies on effect of adrenal cortex extract and of various steroids on capillary permeability. Proc. Soc. Exptl. Biol. Med., 51: 39-41. (1943a) On mechanism of fever production with inflammation. Proc. Soc. Exptl. Biol. Med., 54: 184-186. (1943b) Chemical basis of injury in inflammation. Arch. Pathol., 36: 269- 288. (1944) Chemical basis of fever. Science, 100: 337-338. Meyer, P. S. (1924) Gewoluuing vif ilitjrioscr Ilautsfcilen an ultraviolettes Licht und andere Reize. Arch. Deriuatoi. u. Syphilis, 147: 238-241. Miescher, G. (1930) Das Probleni des Lichtschutzcs und der Lichtgewohnung. Strahlentherapie, 35: 403-443. (1932) Untersuchungen liber die Bedeutung des Pigments fur den UV.-Licht- schutz der Haut. Strahlentherapie, 45: 201-216. SUNKURN 527 Mioscher, G., and H. Minder (1!)39) Untcrsuchunscn iihcr die diirch l;ing\velliges Ultraviolett hervorgerufpno Pigmontdunkoluiig. Strahlenthorapie, 66: 6-2)5. Mitchell, J. S. (1938) The origin of the erythema curve and the pharmacological action of ultraviolet radiation. Proc. Roy. Soc. London, B126: 241-261. MoUer, M. (19U0) Der Einfluss des Lichte.s auf die Haut. Erwin Xiigele, Stuttgart. Moon, P. (1941) Proposed standard solar-radiation curves for engineering use. J. Franklin Inst., 230: 583-617. Nathan and Sack (1922) Uber entziindungserregende Wirkung von Extrakten aus normaler und pathalogisch verJinderter Haut bei Meerschweinchen. Arch. Dermatol, u. Syphilis, 138: 391. Pearson, A. R., and C. J. B. Gair (1931) Penetration of radiation into animal tissues. Brit. J. Phys. Med., 6: 27-30. Peck, S. M. (1930) Pigment (melanin) studies of the human skin after application of thorium X, with special reference to the origin and function of dendritic cells. Arch. Dermatol, and Syphilol., 21: 916. Percival, G. H., and C. M. Scott (1931) A study of the skin vessels in some forms of inflammation of the skin. J. Pharmacol. Exptl. Therap., 41: 147-163. Perthes, G. (1924) Ueber Strahlenimmunitat. Miinch. med. Wochschr., 71: 1301. Pettit, E. (1932) Measurements of ultraviolet .solar radiation. Astrophys. J., 75: 185-221. Phalen, J. M. (1910) An experiment with orange-red underwear. Philippine J. Sci., Med. Sec, 5: 525-546. Ritter, J. W. (1803) Versuche liber das Sonnenlicht. Ann. Physik, 12: 409-415. Rothman, S. (1942) In vitro studies of pigmentation. II. Influence of ascorbic acid on oxidation of tyrosine by ultraviolet radiation. J. Invest. Dermatol., 5: 61-75. Rothman, S., H. F. Krupa, and H. M. Smiljanic (1946) Inhibitory action of human epidermis on melanin formation. Proc. Soc. Exptl. Biol. Med., 62: 208-209. Rothman, S., and J. Rubin (1942) Sunburn and para-aminobenzoic acid. J. Invest. Dermatol., 5: 445-457. Rottier, P. B. (1952) Sur I'interpretation du spectre d'action de la lumiere ultra- violette par I'intermediaire de deux substances-meres photolysables, avec local- ization differente dans I'epiderme. Synthese de semeiologie et therapeutique, 21: 14-17. (1953) Theerythematousactionof ultraviolet light on human skin. I. Some measurements of the spectral response with continuous and intermittent light. J. Clin. Invest., 32: 681-689. Rottier, P. B., and J. A. M. Mullink (1952) Localization of erythemal processes caused by ultraviolet light in human skin. Nature, 170: 574-575. Schall, L., and H. J. Alius (1926) Zur Biologic des Ultraviolettlichts. III. Die Reaktion der menschlichen Haut auf die Ultraviolettbestrahlung (Erythem- blauf). Strahlentherapie, 23: 161-180. (1928a) Zur Biologic des Ultraviolettlichts. IV. Die Reaktion der mensch- lichen Haut auf wiederholte XHtraviolettlichtbestrahlung (Lichtschutz). Strahlentherapie, 27: 769-783. — (1928b) Zur Biologic des Ultraviolettlichts. Bruns' Beitr. klin. Chir., 143: 721. Sharlit, H. (1945) Melanin production in skin. II. Further histochemical observa- tions. Arch. Dermatol, and Syphilol., 51: 376-383. Wald, G. (1952) Alleged effects of the near ultraviolet on human vision. J. Opt. Soc. Amer., 42: 171-177. Wedding, M. (1887) Z. Ethnol., 19: 67. Widmark, E. J. (1889) tJber den Einfluss des Lichtes auf die vorderen Medien des Auges. Skand. Arch. Physiol., 1: 264-330. •,2S RADIATION' inOLOOV (1891) tTlier den lOinfhiss cles Lichtes auf d'w. Haut. Hcitr. OpthaliTiol., Wit ami Coiiii)any, Lcipzin. Pp. JiW 151) (also llynira: Fcslhaiid Xr. '.i, 188'.»). Witli, C (li)20) Stiuiies cm tliccfTcct of li^lit on vitiligo, lirit..). Dermatol. Syphilis, ;J2: 145-155. Woodruff, C. E. (1905) The effects of troijical liKlit on white men. Rebinan Com- pany, Now ^■ork. Manuscript received by the editor July 2(), 11)51. ADDENDUM Recent experiments in this laboratory indicate that the erythemal threshold, primary pigmentation, and thickening of the epidermis, do not exhibit photorecovery. That is, illumination with "visible" Hght after exposure to ultraviolet does not affect these manifestations of sunburn. CHAPTER 14 Ultraviolet Radiation and Cancer Harold F. Blum^ National Cancer Institute'^ Bethesda, Maryland and Department of Biology, Princeton University Princeton, New Jersey "... sans ttre holiophobe a outrance, je crois opportun d'arreter I'attention de ceux qui abusent des bains de soleil sans aucun controle." —A. H. Roffo Experimental studies: The carcinogenic wave lengths — Tumor types and penetration of the radiation — Quantitative aspects. Theoretical. The role of sunlight in cancer of the skin of man: Topographical distribution — Complexion — Occupation — Distribution. Prevention. References. In 1928, G. M. Findlay of Edinburgh published in The Lancet a brief paper describing the production of cancer in the skin of mice by repeated exposure to mercury arc radiation. He had been interested in the mech- anism by which crude tar induces cancer, and this observation was more or less accidental. Within the next few years, three other groups of workers announced similar results. Putschar and Holtz (1930) in Gottingen and Roffo (1933) in Buenos Aires used rats as the experimental animals; Herlitz et al. (1931) in Stockholm used mice. It appears that none of these three groups was aware, when beginning its experiments, of the others' activity or of Findlay's previous paper, so all their results may he regarded as independent findings. Only Findlay and Roffo were directly interested in the problem of cancer, the latter undertaking the experiments because of ideas regarding the role of cholesterol in carcino- genesis. Herlitz et al. and Putschar and Holtz were interested in the effects of excessive dosage of ultraviolet on vitamin D, and did not antici- pate the induction of cancers. Within the next decade a number of other workers carried out this type of experiment, and it became recognized 1 Present address: Department of Biology, Princeton University. 2 National Institutes of Health, Public Health Service, Department of Health, Education and Welfare. 529 530 UADIATION BIOLOGY generally that ultraviolet radiation is a carcinogenic agent for mice and rats. These experimental studies revived interest in the idea, already over thirty years old at the time, that sunlight is a cause of cancer of human skin. The earliest to suggest this were Unna (1894), Dubreuilh (1896), and Sheild (1899). The most extensive observations were those of Dubreuilh on the workers in the vineyards of the Bordeaux region, among whom he found more cutaneous cancer than among the urban population. He called particular attention to the limitation of skin cancer to the face and hands, remarking that the position on the face seemed to accord with the area exposed by the peasant headdress. His studies were reported at length in 1907. There were also extensive reviews by Hyde in 1906 and by Bellini in 1909. Four principal lines of evidence supporting the idea that sunlight is a cause of cancer of the skin of man were based on these early clinical observations: (1) cancer of the skin occurs principally on parts exposed to sunlight; (2) cancer of the skin is more prevalent in out- door workers than in sedentary workers; (3) the incidence of cancer of the skin is greater in regions of the earth that receive the greatest insolation; and (4) cancer of the skin occurs more often in light-complexioned persons than in dark. At the time these arguments were outlined, basic informa- tion that was essential to support them was lacking, and this information could not be supplied until experimental studies on animals had been initiated. The various lines of evidence will be examined in some detail in this chapter. The laboratory studies have also provided a tool for studying the process of carcinogenesis by ultraviolet radiation. The successes and difficulties encountered will be discussed a little later. But the experi- ments themselves, what they show, and what their limitations are must be discussed before either the mechanism of carcinogenesis or the etiology of human cutaneous cancer is considered. EXPERIMENTAL STUDIES The Carcinogenic Wave Lengths. Obviously, one of the first things to be done in the laboratory was to delimit the wave lengths that induce the cancers. The first attempt to do this was made by the late A. H. KotTo, whose findings appear extensively and rather diffusely described in a bulletin publi.shed in Spanish from his institute in Buenos Aires. Many of th(> more important ones are, however, briefly summarized in an article published in French in 1934. Roffo exposed rats to mercury arc radiation passing through various colored glasses. These filters are not accurately described, but one which is designated as "verre transparent" may l)e assumed to be common window glass. Whereas rats exposed directly to the mercury arc radiation developed cancers, those protected by this glass, ULTRAVIOLET RADIATION AND CANCER 531 or by any of the colored ones, did not. In addition, Rolfo carried out similar experiments with sunlight; he is, in fact, the only experimenter to have induced cancers with natural sunlight. h\ this case, too, "verre transparent" filters prevented the formation of tumors. If the assump- tion is correct that Roffo's "verre transparent" was ordinary window glass, he may be credited with having discovered that the carcinogenic wave lengths are those shorter than about 0.32 /x,^ i.e., that the carcino- genic ultraA'iolet has the same long-wa\'e-length limit as the erythemal spectrum. The question was definitely settled a little later. Funding et al. of Copenhagen in 1936 reported extensive experiments on the car- cinogenic wave lengths before the Comite International de la Lumiere at Wiesbaden. They found that a filter which transmitted wave lengths 0.28 fx and longer if placed in front of a mercury arc permitted induction of cancer, but that window glass cutting off the 0.313 n and all lines of shorter wave length prevented induction of the tumors.^ In 1941, Rusch et al. of Wisconsin reported similar experiments with like results. They were unaware of the earlier work of Funding et al., which was not readily available. The experimental evidence serving to establish the long-wav^e- length limit of the carcinogenic radiation is summarized in Table 14-1, which includes a few additional observations. Up to the present, no studies have been reported in which monochro- matic radiation was used to induce cancer, other than some by Rusch et al. (1941) that seem inconclusive and some by Blum and Lippincott (1942) who used the 0.2537-m line from a low-pressure mercury arc and showed that this wave length is weakly effective in inducing cancer in albino mice (see Table 14-1). The idea seems current that studies with monochromatic radiation should show much more than is already known about the carcinogenic process, and that studies with polychromatic radi- ation are faulty in some way not clearly specified. The difficulties of working with monochromatic radiation should become obvious when it is realized that groups of identically treated mice as large as 40 in number are needed if reasonably good comparisons are to be made. The difficulty of exposing such numbers of animals to monochromatic radiation must be obvious. But, besides the experimental infeasibility, there are appar- ently insurmountable difficulties of interpretation that would arise once the studies with monochromatic radiation were completed. An analysis by the writer (1943a), based on a few experiments with filters, indicates the uncertainty and the apparent futility of more exact measurements with monochromatic radiation. The situation may be compared with that of sunburn, discussed in Chap. 13. The erythemal spectrum has been determined with monochromatic radiation, and a smooth curve may be drawn to describe it, whereas we have only a rough idea of the shape of ' Approximately the shortest wave lengths transmitted by ordinary window glass. *See Fig. 13-3, Chap. 13, for the position of the mercury lines. 532 TJAniATIOX lUOT.OGV TaBLK 14-1. I'XI'KRIMENTS DkTEKMININO TIIK OaHCI \n(iK\ IC WaVK I.KNT.TIIS (From IMum, 1048.) Source Investigators Year Animal Filter Shortest wave length reaching skin in appreci- able amount, Tumor induc- tion Merc my arc" in quartz (intermedi- UolTc. 1934 Rat Rat None Window glass'' 0 2302* 0.3341'' + ate pressure) Funding et nl. 1936 Mouse None S&G BG3 Window glass S&G BG8 0.2302!- 0.2820'' 0.334H 0.4108'' + + Rusch el al. 1941 Mouse None Corning 970 Window glass Corning 352 0 2302'' 0 292.J'' 0.3341'' 0 . 4040'' + + Blum 1943a Mouse None Pyrex glass Corex D Window glass' 0 2302 0.29()7 0.2699 0.3341 + + + Bain and Rusch 1943 Mouse Special 0.2925'' + Mercury arc-*^ (low pressure) Riisch et al. Blum and Lippincott 1941 1942 Mouse Mouse None None 0 . 2537" 0.2537 + Sunlight Roffo 1934 Rat Rat None Window gluss'^ 0.2900 0.3200'' + Tungsten filament Findlay Roffo Rusch et al. 1928 1934 1941 Rat Rat Mouse None None None 0 . 4000'- 0 4000'' 0.4000'' — SI* Beard et al. 1936 Rat 0.2699'' + ■• The emission of such lamps is similar to that illustrated in Fig. 13-3. Other papers demonstrating carcinogenesis by such arcs are not included since they provide no further evidence regarding wave- length dependence. >> Based on the present author's estimate. The characteristics of the source as well as the transmis- sion of the filter have been considered in making these estimates. <= " Verre transparent," which is assumed to be ordinary window glass, prevented carcinogenesis as did also several colored glasses which should have longer wave length cutoffs. «' Based on the original author's statement. The characteristics of the so\irce as well as the transmis-* sion of the filter have been considered in making these estimates. ' Uni)ublished result. / Nearly all the radiation emitted within the carcinogenic range is in the 2537 .\ line. "The dosage used was apparently iti:iilc(niate (compare with Blum and Lippincott. 1942). '' This is a combination of a tungsten filament and a mercury arc in special glass envelope. ULTRAVIOLET RADIATION AND CANCER 533 the carcinogenic spectrum. The erythemal mechanism involves at least two light-absorbing substances, whereas the carcinogenesis process prob- ably involves only one since reciprocity is obeyed with polychromatic light. Tumor Types and Penetration of the Radiation. Those cutaneous tumors of man in which sunlight may have an etiological role are car- cinomas of either the squamous cell or basal cell type. All originate in the epidermis. When experimental tumors were produced in rodents with ultraviolet radiation, it was generally expected that they would be of these same types. Indeed, the earlier reports indicated that this was generally true. Only Roffo (1934) claimed that a considerable proportion of the tumors induced by ultraviolet radiation were sarcomas, i.e., tumors of tissues underlying the epidermis. There remained a certain skepticism regarding this point when in 1940 experimental studies were initiated at The National Cancer Institute, but it was then found that the incidence of sarcomas induced in the ears of albino mice by ultraviolet radiation was very high — over 90 per cent (Grady et al., 1941, 1943). This apparent disparity between human cutaneous cancers and the tumors experimentally induced in the skin of mice needed to be resolved. It was thought that the differences in distribution of tumor types in the two cases might be explained by difference of penetration of the ultra- violet radiation, since mouse epidermis is considerably thinner than human epidermis. In order to settle this question, the transmission of ultraviolet radiation was measured for mouse skin and for human epi- dermis (Kirby-Smith et al, 1942). Figure 13-7, in the preceding chapter, compares the spectral transmissions of samples of mouse and of human epidermis. In the mouse, as in man, the transmission decreases markedly after repeated exposures to ultraviolet radiation because of thickening of the corneum, but mouse skin always remains more transparent to the carcinogenic wave lengths than does human skin, even when the latter has not been exposed to ultraviolet radiation. On the basis of these measurements of physical penetration of the carcinogenic agent, it is altogether plausible that, if ultraviolet radiation is the common carcino- genic agent, tumors of human skin should occur almost entirely in the epidermis, whereas in the mouse deeper tissues are involved. Differences in response of tissues to the action of the ultraviolet radi- ation might also be expected, and there is evidence of such differences. Table 14-2 shows the incidence of tumor types obtained under different experimental conditions. The size of the dose had little influence on the distribution of tumor types, and there is not much evidence of any relation to the time from the first dose to the appearance of the tumor. ^ On the other hand, the ratio of carcinomas to sarcomas differed markedly with the frequency with which the doses were given. When the dose was ■' The "development time" defined in the next section. ■)34 KADIAIION ltl()I.()(!Y irpeati'd tlnily, or 5 days ii week, there was a relatively hifi;h percentafje of eareinomas. When there was only one dose per week, sarcomas pre- domiiiatetl. This dilTerenec obviously cannot be attributed to penetra- tion of the radiation. Tablk 14-2. Distribution ok Types of Tumor Induckd by Ultraviolkt Radiation in the Ears of .\lbino Mice (Strain A) (After CJrady H al., H)43.) No. of Tumor typ cs Carcinoma/ animals Carcinoma Sarcoma sarcoma ratio Dose (5 exposures Weekly dose per week) (ergs /cm 2): 43.0 15 5 15 0.333 16.5 62 9 59 0.153 13.0 95 27 90 0.300 9.9 77 12 75 0.160 7.9 70 20 67 0.298 5.3 66 20 62 0.323 3.6 76 Total 461 19 72 0.264 Individual induc- td (days): tion time, td (5 100-150 112 20 106 0.189 exposures per 150-200 186 45 177 0.254 week) 200-250 88 23 86 0.267 250-300 42 12 40 0.300 300 -1- 33 Total 461 12 31 0.387 Schedule of expo- Xo. of expo- sures sures per week : 1 60 3 (iO 0.050 5 461 112 -140 0.255 7 77 Total 598 32 68 0.470 In total, about 95 per cent of the tumors were pure sarcomas or sarcoma mixed with carcinoma. About 25 per cent of the tumors contained car- cinoma, a few of these being pure carcinomas, but for the most part mixed with .sarcoma.^ The sarcomas were predominately spindle-cell types, most of them presumably arising from connective tissue, but some from muscular elements. There were a few hemangiomas, one sebaceous * These figures are for tumors of the ear only, the eye and other sites not being included. ULTRAVIOLET RADIATION AND CANCER 535 j^laiid tumor, and one osteochondrosarcoma. The small number of car- cinomas fits with the thinness of the malpighian layer in the mouse ear, which never becomes as thick as it does in human epidermis and hence never absorbs as large a proportion of the incident radiation. When the attempt is made to compare the incidence of occurrence of tumors or of the type of tumors induced by ultraviolet radiation in different species of animals, this question of penetration must be taken into account. An interesting experiment in this regard was made by Hueper (1941), who elected to study the effects of ultraviolet radiation on congenitally hairless rats with the expectation that he would obtain more tumors than with haired mice. To his surprise, only one of the hairless rats developed a tumor and this was a carcinoma. On examination he found that the corneum of the hairless rats was very much thicker than the corneum of the haired animals, which could account for his findings. The generally greater incidence of carcinomas in rats than in mice (Putschar and Holtz, 1930; Roffo, 1934; Beard et al., 1936) may be explainable in similar terms. The morphological character of these tumors leave little doubt as to their malignanc3^ Metastasis is relatively rare, but it does occur. This is also characteristic of most cutaneous tumors of man, the malignant melanomas being an outstanding exception. Transplantation has been successfully carried out in some instances. For a more complete dis- cussion of tumor types and other aspects of their pathology, the original papers (Grady et al, 1941, 1943) should be consulted. Quantitative Aspects. In discussing the quantitative aspects of the induction of tumors by ultraviolet radiation reference is made principally to a series of studies carried on at The National Cancer Institute, since they provide the most extensive data collected thus far and since, because of the method used, they are quantitatively intercomparable. Precau- tions were taken in these experiments to assure that the data would be reproducible. In the first place a genetically homogeneous strain of mice, strain A, was used. This is an albino mouse which tends to develop tumors of the lung but which does not spontaneously develop cutaneous tumors. The females of this strain also develop cancer of the breast, but the males do not; therefore, in order to reduce the number of variables, only the latter sex was used. The diet and other treatment of the animals was made as uniform as feasible so as to reduce biological variability to a minimum. The mice were subjected to measured doses of ultraviolet radiation from a mercury arc given at regular intervals. After a considerable number of doses — in only a very few cases was the dosage period less than 1 00 days — tumors appeared on the ears of the mice, the time of appearance depending on the size and the freciuency of the dose. The time from the application of the first dose of ultraviolet radiation to the appearance of such a tumor in a given mouse is here called the "development time," and in the fol- 530 RADIATION HIOLOGY lowing discussion is syniholi/od us /,,. Foi' piaclical reasons, Ihc appcai- aiu'c of a tumor was taken as the time at which a tumor of a fi;i\'en esti- mated size, approximately 100 mm' in volume and consisting of about 10" cells, was present on the ear. At this time the tumors were usually doubling their volume every few days, so that the error in estimation of td was relatively small. Since the ears of these mice are almost hairless, the skin was exposed directly to the ultraviolet radiation. No tumors 100 -0.2 -0.1 0 O.l TUMOR DEVELOPMENT TIME C/ I- < _] tij q: ^ ^ ^ ^ y ^ ^ \ ■* — (' -^ ^ ^ ^ ^ ^ kB ^ ^ ^ ^ .y- TIME [i) Fig. 14-5. Diagram to illustrate progrossivo accolcratioii of relative growth rates. thus far to postulate any intimate mechanism for carcinogenesis, but suggests a frame wdthin which such a mechanism needs to be fitted. The theory is based on the postulate that successive doses of radiation progressively accelerate the relative rate of cell proliferation, each dose bringing about an increase in rate. In formulating an approximate model to describe this progressive acceleration of growth rate, \ve begin by rewriting Eq. (14-2): ^ = G- (14-9) The left-hand member of this ecjuation represents the relative instan- taneous growth rate, which can be plotted against time, as in Fig. 14-5. If the rate remained constant, i.e., if Eq. (14-9) were obeyed strictly, the line representing the growth rate would be parallel to the abscissa. Let it now be assumed that, on receiving each dose of ultraviolet radiation, the tumor grows at a rate which is increased by an amount roughly propor- tional to the dose, where the factor of proportionality may depend on the past history of the course of exposures but does not change rapidly from ' This criticism appears to extend to the hypothesis of Iversen and Arley (1953), who seem to have overlooked our argument. ULTRAVIOLET RADIATION AND CANCER 545 day to day. This is illustrated in Fig. 14-5, where the growth rate is shown as rising abruptly to a new level with each dose, the new rate being maintained until the next dose is received. Without assuming that this diagram pictures the exact happenings in a tumor, it may be accepted as an approximation which may be put to test. The dotted line in the figure represents a smooth acceleration, which for a long series of doses would very closely approximate the stepwise curve we have drawn. This line is described by dV kDt V dt i which, when D and i are constant, may be integrated to (14-10) where Fo and V are the volumes of the tumor at the beginning and end of the time t, D is the dose of ultraviolet radiation, i is the interval between successive doses, and /c is a proportionality constant.^'' The rate of growth is assumed to be negligible at the time of the first dose. The development time td has already been defined as the time from the first exposure until the tumor reaches a given volume designated as Vd. For this case, Eq. (14-11) may be rewritten as If, in addition, the assumption is made that Fo is the same in all cases, this is equivalent to saying that Vd/Vo is a constant at the time td.^^ For any series of experiments in which the interval i is maintained constant, td should therefore vary inversely as the square root of the dose D, since by rearrangement we obtain td = 2i , 1 d ¥^"ro D-y^ (14-13) and all the values within the brackets are constants. Figure 14-C illus- trates what happens when this relation is applied to the data. In this figure values of td are plotted against D, on log-log coordinates. The values of td plotted are based on the time to 50 per cent incidence of tumors within groups of identically treated mice. Examination of the " The equation may be used to describe tumor growth when t has high values, but does not hold exactly for short periods. A treatment applicable to the latter which has to be used in interpreting some of the data is developed more completely in the original paper, but Eq. (14-11) serves the present purpose. ^' This is a tentative assumption. The data are better fitted if Fo is considered to vary (unpublished analysis). 546 ItADIATION HIOLOflY curves in Figs. 1 1-1 and 2 will show that this is a justifiable procedure since, because of the luiture of the incidence curves, the same relation holds for any other percentage incidence. In 11 of the experiments descrilx'd in Fig. 14-0 the doses were applied 5 days per week, the interval being taken as 7/5 days and the curve so labeled in the figure. Above a certain value of D, designated /;„„ t.i does not decrease with increase in D, i.e., the curve is a horizontal line. Below D„, the points are quite well fitted by the drawn curve which has the slope ->2, representing the con- dition, explicit in Eq. (14-13), that ta varies inversely as the square root of the dose. The reason for the flattening of the curve at D„. will be dis- cussed later. Figure 14-6 also shows data for dose intervals of 1 day and -0.5 -0.4 -03 -0.2 0 6 0 7 -0.1 0 01 0 2 0.3 0.4 0 5 DOSE (D), log of values Fig. 14-6. Relation between dose D and development time tj for three different intervals i (days). The relative accuracy of the points is indicated by the vertical lines, which represent the limits which should be exceeded only once in 20 times on the basis of chance alone. The symbol /)„, indicates the point beyond which td does not decrease with increase in dose. (From Blirm, 1950.) of 7 days. The drawn curves resemble the curve for the 5-day-per-week schedule. There are insufficient points on the curves for the 1- and 7-day intervals to establish the shape assigned, but the agreement with the curve of the 5-day-per-week schedule is obvious. The fit of the equation to the data is also shown in Fig. 14-7, where the curves are plotted on numerical ordinates. The graphs in Figs. 14-6 and 7 show that the data support the theory within the limits of accuracy as well as c-ould be expected. E(iuations (14-12) and (14-13) do not hold exactly for different values of i, necessitating the following correction : 1 ^'^ 2{i — a) where a is a constant having the value 0.52 however, affect the general argument. Most of the tumors grow rapidly once they appear (14-14) This modification does not, In some cases ULTRAVIOLET RADIATION AND CANCER 547 they double in volume in as little as 2 or 3 days. Curves illustrating certain aspects of progressively accelerated growth are shown in Fig. 14-46. Curve I is an accelerated growth curve following Eq. (14-12) in which Vd/Vo is taken as 10** and td as 150 days. The value 10** represents the change from a single cell of volume 10~^ mm'^ to a tumor of 100 mm^, that is, Vd- As the curve shows, such a tumor would first be grossly detectable about 20 days before it reached 100 mm* volume. This agrees in general with observation, since, as a rule, these tumors are not grossly manifest for more than two or three weeks before thev reach this volume. 3 4 5 6 7 DOSE PER EXPOSURE, D Fig. 14-7. Relation between dose D and development time td for three different intervals i. The drawn curves follow the theory of progressive acceleration of tumor growth discussed in the text. The data and curves are the same as those plotted in a different manner in Fig. 14-6. (From Bhini, 1950.) To illustrate another aspect of the problem, let us assume for purposes of discussion that every tumor develops from foci of cells, the growth of which foci is progressively accelerated by successive doses of radiation. Let us imagine that there are two foci, one growing thus at a "fast" rate and the other at a "slow" rate. If the growth rate of the fast focus exceeds that of the slow focus by only a very little, the ultimate tumor would arise almost exclusively from the fast focus. As shown in Fig. 14-46, the broken curve IV is based on the same value of Vd/Vo as is curve I, but is calculated for a slightly lower acceleration. At 150 days, when curve I arrives at volume 100 mm'*, curve IV is only about 10 per cent as high, yet curve IV if continued reaches 100 mm* only 10 days later than I. The slow focus represented by curve IV would contribute only 10 per cent to the total tumor volume at time td- If it is assumed that there is some 548 RADIATION BIOLOGY sort of distribution of the rates of growth of the tumor cells, only those growiii{>; near the maximum rate would contribute appreciai)ly to the total tumor volume. If there were many foci of tumor cells in close proximity, those which proliferated most rapidly might so far outstrip the others that the tumor when finally formed would be composed almost entirely of cells arising from fast-growing foci. We have not yet accounted for the maximum limit of growth rate repre- sented in the curves by the failure of increase in dose to decrease the time to appearance of tumors after a certain value D„, is reached (see Figs. 14-0 and 7). It must be obvious that there is a maximum rate of growth which otioo r o s 5 80 o O UJ o < 60 UJ u UJ a. UJ > < _j o u 40 20 END OF - EXPOSURES CD CE CF 1,9 2.0 2.1 2.2 2.3 2.4 TUMOR DEVELOPMENT TIME fi'^j, log days Fig. 14-8. Effect on development time of discontinuation of dose. In series CF, CE, and CD the exposures were discontinued at the times indicated. In series CH the dosage continued until the appearance of tumors. {From Blum, 1950.) a tumor cannot exceed. Physiological factors, for example, the rate at which materials for growth can be supplied, must set an upper limit. So progressive acceleration of growth, which is the essence of this theory, could be pushed only to a certain point, and, acceleration being directly related to dose, a maximum should be reached beyond which increase in dose would not increase the rate of development. This is what appears to happen. It seems probable that the minimum value of ta in the three curves in the figures corresponds to a common maximum rate of prolifer- ation which cannot for physiological reasons be exceeded. Numerically this condition is satisfied. According to the model illustrated in Fig. 14-5, if the exposures were stopped, a tumor should continue to grow at the rate that was established at the time the exposures ceased. In a certain number of experiments the doses of ultraviolet radiation were stopped before tumors appeared, with a resultant increase in /,/. This is illustrated in Fig. 14-8, where it is shown that the earlier the doses were discontinued, the longer was td. In Table ULTRAVIOLET RADIATION AND CANCER 549 Table 14-4. Effect of Discontinuing Schedule of Doses (From Blum, 1950b.) For all pxperimonts, data arc for 50 per cent tumor incidence; £) = 1.8; i = 1.4. This is the experiment described in Fig. 14-8. All the series were run simultaneously. Series U U* td (observed) td (calculated) CH CF CE 166 95 88 74 100 126 238 166 195 214 312 164 185 192 CD 212 * Period without exposure. 14-4, observed values for td are compared with values calculated on the basis of the equations describing progressive acceleration of tumor growth. It is seen that the observed values of td are greater than the calculated, indicating that the rate of tumor growth is slower at some point than is 2 1 2 2 2.3 2.4 TUMOR DEVELOPMENT TIME (td), lOg days Fig. 14-9. Effect on development time of the interruption of doses. In experiments DA, DC, DE, DG, and DI the exposures were discontinued for 30 days and then resumed. The arrangement of the periods of respite are indicated in Table 14-5. The control, DK, consisted of a smaller number of animals; the curve drawn is based on pooled data. {From Blum, 1950.) predicted by the equation. The discrepancy between the observed and calculated values of td increases systematically with increase in td- Another type of experiment is illustrated in Fig. 14-9.'- In this, an '2 Similar experiments have been described by Rusch and Kline (1946), but the data are not applicable to the present analysis and therefore direct comparison cannot be made. Their interpretation does not take growth rates into accovmt and is subject to general criticisms already made in this chapter. :>.■)() RADIATION BIOLOGY initial period of exposures was followed by a rest period of 30 days, after which the exposures were resumed for another period. In some cases there was another terminal rest period, because it became necessary to discontinue the whole experiment. A^ain the calculated values of td gi\en in Table 14-5 dilTer somewhat from the ob.scrved values, the observed being generally greater than the calculated. The discrepancies seem to be in the same direction as those that result when comparisons of the data for different dosage intervals are considered. All in all, these discrepancies do not seem too important when it is realized that our treatment has been based on a crude model. Figure 14-5, which describes this model, certainlj^ does not accurately describe actual tumor growth; it is hardly to be believed that the growth rate rises immediately on the application of ultraviolet radiation as is indicated in that diagram. Table 14-5. Effect of Interrupting Schedule ok Doses (From Blum, 1950b.) For all experiments, data are for 50 per cent incidence; D = 2.0; / = 1.4. This is the experiment described in Fig. 14-9. All series were run simultaneously. Series DK DI. DG DE. DC. DA. ii t'Z* h U* 155 75 30 64 47 30 103 33 30 117 9 19 30 131 11 5 30 145 20 td (observed; td (calculated) 155 169 180 189 191 200 155 170 173 176 178 181 * Periods without exposures. As regards the more intimate aspects of carcinogenesis by ultraviolet radiation there is relatively little to say. The active hyperplasia which results from sunburn, and which is also very marked in mouse skin sub- jected to ultraviolet radiation (Grady et al., 1941), attracts attention and suggests that the same or a similar primary mechanism is involved. It is obvious that some acceleration of cell proliferation is involved in cancer development whether or not it is of the progressive type suggested. In this respect it is interesting that there is no real evidence of acceleration of cell division due to the direct effect of ultraviolet radiation on the cell, but only retardation (see Giese, 1947; Blum and Price, 1950). Such evi- dence does not eliminate the possibility of indirect effects (see Hollaender and Duggar, 1938; Loofl)ourow and Morgan, 1940). The idea that a steroid is changed by ultraviolet radiation into a car- cinogen was for a time popular. Roffo, who thought cholesterol was the precursor, seems to have been the first to propose this. Various investi- gators, using a variety of approaches, failed, however, to find evidence to support this idea (see Blum, 1940, for numerous references). Studies on ULTRAVIOLET RADIATION AND CANCER 551 vitamin D suggest that the activation of the provitamin takes place in the corneum, or at least very superficially (see Blum, 1950a), and it appears that most of the steroids of the skin are found in the corneum rather than in the deeper tissues. Yet the site of carcinogenesis in the case of the mice is deeper than this. There is other evidence against this hypothesis. The polycyclic hydrocarbon carcinogens, which are the type we should expect to find formed under these circumstances, are quite diffusible. If strain A mice are treated with carcinogens either by injection or by paint- ing on the skin, in which case the carcinogen is of course licked off and gets into the alimentary canal, there is a great increase in the incidence of cancers of the lung (Shimkin, 1940). Yet the incidence of lung tumors in this same strain was not found to be greater among mice in which cancer had been induced by ultraviolet radiation from that in untreated con- trols.'^ Thus it appears that no diffusible carcinogen is formed by the ultraviolet radiation, and this is strong evidence against the participation of a polycyclic hydrocarbon carcinogen in this type of carcinogenesis.'^ THE ROLE OF SUNLIGHT IN CANCER OF THE SKIN OF MAN After a discussion of the results of animal experiments, the clinical findings bearing on the question of the etiological role of the ultraviolet radiation of sunlight in cancer of the skin of man may be better evaluated. In our introduction four general lines of evidence were mentioned, and these may now be considered. Topographical Distribution. All authorities agree that cutaneous cancer occurs in the white race predominantly on the face ; for example, in 91 per cent of the 1626 cases reviewed by Lacassagne (1933) the tumors were on that area. Basal-cell and squamous-cell cancers predominate among skin cancers, and it is these types which display the strong predi- lection for the face. The other principal site of these tumors is the back of the hand. This evidence alone strongly supports the idea that sunlight, which reaches other areas of the body to a very small extent, has a role in the etiology of basal-cell and sciuamous-cell cancers. The occasional appearance of one of these tumors on an unexposed area of the body would not negate this conclusion, since no doubt there are other agents which ^^ Actually there was a slight decrease in the treated animals, the probable causes of which have been discussed by Blum (1944). The experiments also indicated a differ- ence in general susceptibility to the development of cancer within tlic genetically homogeneous strain. ^* There have been various experiments showing that exposure to ultraviolet radia- tion decreases the incidence of cancers in mice painted with carcinogenic hydrocarbons. Recently Engelbreth-Holm and Iversen (1947) offered evidence that this is due to photooxidation of the hydrocarbon. This explanation was offered by the writer as early as 1940. Other possible factors may be involved (e.g., Blum, 1943b), but in any event there seems no reason to connect photochemical reactions of these carcinogens with the induction of cancer by ultraviolet radiation. 552 RADIATION BIOLOGY cause such cancers. We ma^^ cite, for example, the kaiigri cancers result- ing from heal, and cancers caused by carcinogenic chemical agents, such as the cancer in chimney sweeps and in workers in crude petroleum. On the other hand, the much more malignant, although fortunately much rarer, malignant melanomas cannot be attributed to the action of sun- light; these tumors parallel in distribution the pigmented nevi (Pa(;k, 1948), and, altliough a good many melanomas occur on the face, there are other sites of predilection, particularly the genitals and the feet, where sunlight would be least likely to play a part. The melanomas will be excluded for the present discussion; references to cancer of the skin will mean basal-cell or squamous-cell cancers. There have been attempts to relate the distribution of the cancers of the face to the incidence of sunlight on different areas, but, as pointed out in an earlier paper (Blum, 1940), these are not very convincing. Different writers use different methods of describing the position of the tumors, and this leads to confusion. The analysis of Magnusson (1935) who related the distribution of tumors to the thickness of the epidermis and the consequent penetration of light in these areas is obviously in error because it fails to take into account the factor of diffusion of radiation by the corneum (see Chap. 13). Altogether, such attempts seem to contribute very little support to the argument that cancer of the skin is caused by sunlight. But the basic finding that these tumors are limited in very large proportion to the face is itself sufficient evidence to indict sunlight as a causal factor, particularly when supported by the other lines of evi- dence which will be discussed. There have been attempts to explain the distribution of cutaneous cancer on other grounds, which do not seem too convincing. Some of these have been summarized earlier (Blum, 1940). Recently Corson et al. (1949) have pointed out that tumors may occur in persons wearing spectacles, where these focus the light rays. They attribute this to heating of the skin, but the evidence is not convincing. Some glasses used for spectacle lenses transmit the carcinogenic wave lengths of sunlight to a considerable extent. Complexion. "A commonplace in dermatological lore is that skin cancer occurs more frequently in blonds than in brunets." This state- ment is found in an article by Taussig and Williams (1940) in which they attempt to characterize skin color and to associate it with cancer of the skin. There have been numerous other attempts of this kind, but on the whole such a correlation is uncertain. It is, of course, very difficult to know what the terms "blond" and "brunet" mean. The idea that pig- mented skin is less liable to sunburn than unpigmented skin — the fallacy connected with which was discussed in the last chapter — may have had considerable influence on thinking along this line. Among the white races it is difficult to correlate the threshold for the erythema of sunburn with complexion. ULTRAVIOLET RADIATION AND CANCER 553 On the other hand, the threshold for sunburn is much higher in Negroes than in the white races. It is, therefore, of interest to find that cancer of the skin is very rare in members of the Negro race in this country (Dorn, 1944), although there seems to be no evidence of a general immunity to cancer in that race. Moreover, it appears that when cancer of the skin does occur in Negroes it has a very different distribution, being located about as frequently on unexposed as on exposed parts (Schrek, 1944a, b). Here is evidence to support the idea that among the white races sunlight is a principal cause of cancer of the skin. Vint (1935) reports a high inci- dence of squamous-cell cancers among the natives of Kenya, accounting for 36 per cent of the tumors among this Negro population. He points out, however, that these tumors are associated with tropical ulcers of the leg. Basal-cell cancers are rare. This evidence seems to support rather than conflict with the findings on Negroes in the United States, as regards etiology. Roffo (1939) states that in the Argentine all the cases of skin cancer that he observed occurred in foreigners or immigrant families, none in Indians or Negroes. Occupation. It is commonly believed that cancer of the skin is more frequent in outdoor workers than in indoor workers. This is reflected in such terms as "seaman's skin," "peasant's skin," and "farmer's skin," which are applied to allegedly precancerous changes when they occur on the exposed parts. It is obviously difficult to obtain statistics on this. Perhaps the most convincing are those of Peller and Stephenson (1937) and Peller and Souder (1940) who point out that mortality from cancer of the skin and lip is about three times as high for the United States Army and Navy as for the average population for the same age group. Other evidence, some conflicting, has been discussed by the writer elsewhere (1940). All in all, this part of the evidence seems the least conclusive at the present time. Distribution. There have been many statements that the incidence of cutaneous cancer is greater in those regions of the earth which receive most sunlight, but there was for a long time no basis for a critical analysis. In 1944, however, Dorn published extensive statistics which seem to bear out this idea. He examined the incidence of various types of cancer in three groups of urban areas in the United States: (1) Chicago, Detroit, Pittsburgh, and Philadelphia; (2) San Francisco and Almeda County, California, and Denver; (3) Birmingham, Atlanta, New Orleans, Dallas, and Fort Worth. He designates these groups for his purposes as northern, western, and southern, respectively, but actually they form a series of nonoverlapping latitude groups. Their mean latitudes weighted accord- ing to populations of the respective areas are 40°, 38°, and 32°. In Fig. 14-10 Dorn's figures for incidence of cancer are plotted against latitude. A clear-cut north-south distribution of cutaneous cancer incidence is indi- cated for both sexes of the white populations, with greater incidence in the 55-1 RADIATION niOLOGY south than in the north. In tliese data the category " cancer of the buccal ca\ity" inchides some tumors of the exposed parts of the lip, which prob- ably accounts for the distinct north-south distribution of the incidence of such tumors. Cancer of all other sites does not show this relation to lati- tude. A small increase with increasing latitude among males is opposed by a small decrease among females. These statistics suggest that some factor which varies with latitude markedly affects the incidence of cancer of the exposed parts of the body. 120 2 O 100 3 a o c- > 80 - 60 40 to o 20 - \ \ I ^-— \-— -_._ -"-■ . ..^ - ^^^ \ ^\. \ - '^■-^ ^\^ ^i jH ^ - — - ^"^"^'C ^^\^ X ^~~-^ — . ■On """^ ~-~>:», 1 1 1 i 1 1 1 "n — -- 3000 q: UJ I - 2000 1000 32 34 36 38 40 NORTH LATITUDE, degrees Fig. 14-10. Distribution of cancer with latitude. Curve I, skin, male; II, skin, female; III, buccal cavity, male; IV, buccal cavity, female; V, all other sites, male; VI, all other sites, female. {From Blum, 1948, based on data of H. P\ Dorn.) We may examine the possibility that this factor is sunlight, but to do so we must know the extent of variation with latitude of the particular com- ponents of sunlight that we accuse of inducing cancer. The animal experiments indicate that the wave lengths concerned are the same as those that cause sunburn. Total annual sunlight does not vary with latitude to nearly so great an extent as would be required to explain the distribution of cancer incidence shown by Dorn's data; but the carcino- genic wave lengths do show a considerable difference in north-south distri- bution.'^ In order to make an exact comparison between the incidence of carcinogenic radiation in sunlight at various latitudes and the corre- sponding incidence of cutaneous cancer, it would be necessary to know the action spectrum of carcinogenesis as well as to have complete data on the shorter wave lengths of sunlight, Init it is not feasil)le to obtain the former '•"■The factors determining the variation of .sunlight with latitude are discussed briefly in Chap. 13 and extensively by Sanderson and Ilulburt in Chap. 3. ULTRAVIOLET RADIATION AND CANCER 555 data for reasons that have already been discussed. Even if an action spectrum could be obtained experimentally for the mouse, this would not apply quantitatively to human skin because of the difference in the trans- mission of the ultraviolet radiation. Direct measurements of the inci- dence of carcinogenic ultraviolet wave lengths have not been made at a sufficient number of points on the earth to give a complete picture of this distribution in detail, and it would be extremely laborious to do so. Such measurements would, moreover, be of uncertain value in the solution of the present problem, because their interpretation would depend on the missing information regarding wave-length dependence of carcinogenesis in human skin. Some general ideas can be obtained, however, from data that are available. For the purpose, data calculated by O'Brien (1943) for antirachitic action (bg,sed on the absorption spectrum of provitamin D) , which has the same long-wave-length limit as carcinogenesis, are used here. There is no reason to believe that the action spectrum for carcinogenesis in man is related to that for antirachitic action, and the action spectrum for the erythema of sunburn might be thought more closely representative. But there are inherent objections to using an accepted erythemal spec- trum for this purpose, as was pointed out in Chap. 13; in any case O'Brien's calculations indicate that about the same relative values would be obtained if the erythemal spectrum was used in place of the antirachitic spectrum. Such estimates must obviously be very rough, at best being influenced by numerous factors which cannot be readily taken into account, for example, cloudiness and dust. However, they provide as satisfactory an index as is now available. The variation of cancer incidence and carcinogenic radiation with lati- tude is indicated in Fig. 14-11. In order to have a relative basis of com- parison, the value for the lowest latitude, 32°, has been taken as 100 per cent for each variable. When the data are plotted in this way, the inci- dence of cutaneous cancer shows about the same magnitude of change with latitude for both sexes (curves III and IV). Two curves for annual incidence for carcinogenic radiation are plotted, one for 2.0 mm of ozone in the atmosphere (curve I) and one for 2.8 mm of ozone (curve II). Neither of these curves shows as great variation with latitude as do the cancer incidence curves. Ozone, which is the principal limiting factor with regard to the shorter wave lengths of sunlight, varies with latitude, and the two curves represent approximate extremes for the latitudes covered by the cancer incidence data, 2.8 mm representing the northern- most and 2.0 mm the southernmost condition. Thus the true range of variation with latitude should be greater than is indicated by either curve alone. In curve V, the annual ultraviolet radiation corresponding to 2.0 mm of ozone is used for latitude 32° and that for 2.8 mm of ozone is used for latitude 41°. The resulting curve which should represent more 556 RADIATION' HIOI.OGY nearly the riiiifijc of carcinofioiiie radiation has a slope similar to that of the cancer-iiu'ideiK'c curves althoiitrh not quite so steep. The aj^reement is certainly as good as might he expected, considering the numerous assumptions that had to l)e made in order to carry out the analysis. The least that can be said is that the expected variation in the amount of carcinogenic radiation with latitude probably is sufficient to account for the variation of cancer-incidence data with latitude that is indicated by Dorn's (hita. Such fa(^tors as the amount of smoke, cloudiness, and dust 100 Pi 80 60 •a u a 20 32 34 36 38 40 NORTH LATITUDE, degrees Fig. 14-11. Distribution of carcinogenic radiation and cutaneous cancer with latitude. Curve I, 2.0 mm ozone; II, 2.8 mm ozone; III, incidence of cutaneous chancer, female; IV, incidence of cutaneous cancer, male; V, maximum difference. (Frorn Blum. 1948, based on data of H. F. Dorn and Bryan O'Brien.) are left out of consideration, and these may limit the amount of carcino- genic radiation in specific areas, but there are no means for estimating their importance. PREVENTION When we consider how the lines of evidence converge, it seems difficult to reach any conclusion other than that the ultraviolet radiation of sun- light is a major causal factor in cancer of the skin in the white population of the United States. The evidence is against applying this conclusion to the Negro population, where the incidence of skin cancer is small in any event. If such a conclusion is to be accepted even tentatively, the question of possible means of prevention of these cancers arises. Even if it were feasible, it would not be desirable to counsel the complete avoidance of sunlight. This is true particularly since skin cancers of the squamous- and basal-cell types are among the least dangerous of malignant growths, and their incidence is relatively low even under conditions which seem most favorable for their occurrence, for example in the southern United ULTRAVIOLET RADIATION AND CANCER 557 States. Certainly it is not desirable to instill fear of exposure to sunlight in a large part of the population, but certain prophylactic measures may be suggested. Continuous regular exposures to artificial sources of ultra- violet radiation, even in moderate doses, would seem unwise, particularly when these are used during the winter months to supplement summer exposure, either for cosmetic or therapeutic reasons. Probably a large proportion of the white population could practice such regular exposures without accident, but an unfortunate few might be expected to develop cutaneous cancer. Those individuals who show changes in the skin which the dermatologist recognizes grossly as "precancerous," for instance, the appearance of keratoses, or those w^ho have already had one cancer of the skin of the squamous-cell or basal-cell type, might well be cautioned against even relatively mild continued exposure to ultra- violet radiation. But they should be informed of the character of the carcinogenic radiation and the facts regarding sunlight which have been discussed in the previous chapter. They should know that they are relatively safe in exposing themselves in the early morning or late after- noon, but should avoid the midday sun. They should also be informed that window glass offers good protection against carcinogenic radiation. The use of sunburn preventives when going outdoors may also be recom- mended, although the uncertainty of evaluating these has already been discussed in the previous chapter. REFERENCES Bain, J. A., and H. P. Rusch (1943) Carcinogenesis with ultraviolet radiation of wave length 2800-3400 A. Cancer Research, 3: 425-430. Beard, H. H., T. S. Boggess, and E. v. Haam (1936) Experimental production of malignant tumors in the albino rat by means of ultraviolet rays. Am. J. Cancer, 27: 257-266. Bellini, A. (1909) Dell' influenza degli agenti fisici e piu particolaramente della luce nella eziologia dell' epithelioma cutaneo. Giorn. ital. malattie venerei e pelle, 50: 732. Blum, H. F. (1940) Sunlight and cancer of the skin. J. Natl. Cancer Inst., 1 : 397- 421. (1943a) Wavelength dependence of tumor induction by ultraviolet radiation. J. Natl. Cancer Inst., 3: 533-537. (1943b) Physiological and pathological effects of ultraviolet radiation. Ann. Rev. Physiol., V: 1-16. (1944) Relationships between spontaneous tumors of the lung and cutaneous tumors induced with ultraviolet radiation in strain A mice. J. Natl. Cancer Inst., 5: 89-97. (1948) Sunlight as a causal factor in cancer of the skin of man. J. Natl. Cancer Inst., 9: 247-258. (1950a) Radiation: non-ionizing; photophysiology and photopathology. In, Medical physics, ed. O. Glasser. Year Book Publishers, Inc., Chicago. Vol. II. Pp. 753-766. (1950b) On the mechanism of cancer induction by ultraviolet radiation. J. Natl. Cancer Inst., 11: 463-495. 558 HADIATION lUOLOGY Blum, 11. l'., J. S. Kirby-Smith, and 11. L',. Cinul}' (I'Jll j t^uaiititutivc induction of tumors in mice with ultraviolet radiation. J. Xatl. Cancer In.st., 2: 25!J-2G8. Blum, H. F., and S. \V. Lippincott (n)42) Carcinogenic effectiveness of ultraviolet radiation of wavolonKth 25:i7 A. J. Natl. Cancer In.st., :i: 211 2\(). liluin, H. v.. and ,J. 1*. Price (1950) Delay of cleavage of tlie Arhtina egg hy uitra- vK.lcl r.'idiation. J. Cien. Physiol., :«: 285-:}(ll. Corson, E. F., G. M. Knoll, H. A. Lusconihe, and H. B. Decker (1949) Role of spectacle lenses in production of cutaneous changes, especially epithelioma. Arch. Dermatol, and Syj)lulol., 59: 435-488. Dorn, H. F. (1944) Illness from cancer in the United States. U.S. Public iicaith Kepts., 59: 33-48, 65-77, 97-115. l)ul)reuilh, \V. (1896) Des hyperkeratoses circoiiscrites. Ann. dermatol. .sypliilig., 3d series, 7: 1158-1204. (1907) Epith^liomatose d'origine solaire. Ann. dermatol. .syphilig., 4th series, 8: 387. Engelbreth-IIolm, J., and S. Iversen (1947) The effect of ultraviolet irradiation on the carcinogenic potency of certain hydrocarbons. Cancer Research, 7: 372-378. Findlay, G. M. (1928) Ultra-violet light and skin cancer. Lancet, 215: 1070-1073. Funding, G., O. M. Henriques, and Iv Rekling (1936) Uber Lichtcancer. Iller luternationaler Kongress fiir Lichtforschung, Wiesbaden. Pp. 166 168. Giese, A. C. (1947) Radiations and cell division. Quart. Rev. Biol., 22: 253-282. Grady, H. G., H. F. Blum, and J. S. Kirby-Smith (1941) Pathology of tumors of the external ear in mice induced by ultraviolet radiation. J. Xatl. Cancer Inst., 2: 269-276. • (1943) Types of tumor induced bj' ultraviolet radiation and factors influ- encing their relative incidence. J. Natl. Cancer Inst., 3: 371-378. Herlitz, C. W., I. Jundell, and F. Wahlgren (1931) Durch Ultraviolettbe.strahlung erzeugte maligne Neubildungen bei weissen Miiusen. Acta Paediat., 10: 321- 352. Hollaender, A., and B. M. Duggar (1938) The effects of sublethal doses of mono- chromatic ultraviolet radiation on the growth properties of bacteria. J. Bac- teriol., 36: 17-37. Hueper, W. C. (1941) Cutaneous neoplastic responses elicited by ultraviolet rays in hairless rats and in haired litter mates. Cancer Research, 1: 402-406. Hyde, J.N. (1906) On the influence of light in the production of cancer of the skin. Am. J. Med. Sci., 131: 1-22. Iversen, S., and N. .\. Arley (1953) Application of quantum hit theory to tumor induction by ultra-violet radiation. Nature, 171: 257-258. Kirby-Smith, J. S., H. F. Blum, and H. G. Grady (1942) Penetration of ultraviolet radiation into skin, as a factor in carcinogenesis. J. Natl. Cancer Inst., 2: 403-412. Lacassagne, .\. (1933) Repartitions des differentes varietes histologiques d'epi- thclionuis de la peau (plus particulierement ceux de la tete) suivantes Ics regions anatomiques, le sexe, et Page. .Vnn. dermatol. syphilig., 4: 497, 613, 722. Lippincott, S. W., and H. F. Blum (1943) Neoplasms and other lesions of the eye induced by ultraviolet radiation in strain .\ mice. J. Xatl. Cancer Inst., 3: 545-554. Loofbourow, J. R., and M. N. Morgan (1940) Investigation of the production of growth-promoting and growth-inhibiting factors hy ultra-violet irradiated micro- organisms. J. Bacteriol., 39: 437 453. Magnusson, .\. N. \V. (1935) Skin cancer. .\ clinical study with special reference to radium treatment. Acta Radiol., Suppl. 22: 1-287. ULTKAVIOLET RADIATION AND CANCEK 559 O'Brien, B. (1943) Some biological offccts of solnr radiation. Ann. Hojit. Rmifh- sonian Inst., pp. 1()!)-134. Pack, G. T. (1948) A clinical study of pigmented nc\ i and nielanonias. In, The biology of melanoma. Special I'nbl. N.Y. .\cad. Sci., 4: 52-70. Teller, S., and C. G. Souder (1940) (dancer in the United States Army. Army Med. Bull., No. 41, January. Peller, S., and C. S. Stephenson (1937) Skin irritation and cancer in the U.S. Navy. .\m. J. Med. Sci., 194: 326-333. Putschar, W., and F. Holtz (1930) Erzeugung von Hautkrebsen bei Ratten durch langdauernde Ultraviolettbestrahlung. Z. Krebsfor.sch., 33: 219-260. Roffo, A. H. (1933) Cdncer y .sol. Bol. inst. incd. exptl. estud. cdncer, Buenos Aires, 10: 417-439. (1934) Cancer et soleil. Carcinomes et sarcomes provoques par Taction du soleil in toto. Bull, assoc. franc, etude cancer, 23: 590-616. (1939) ilber die physikalisch-chemische Atiologie der Krebskrankheit. Strahlentherapie, 66: 328-350. Rusch, H. P., and B. E. Kline (1946) The influence of a rest period on the carcino- genicity of ultraviolet irradiation applied in interrupted doses. Cancer Research, 6: 486. Rusch, H. P., B. E. Kline, and C. A. Baumann (1941) Carcinogenesis by ultraviolet rays with reference to wave length and energy-. Arch. Pathol., 31: 135-146. Schrek, R. (1944a) Cutaneous carcinoma. IV. Analysis of 20 cases in Negroes. Cancer Research, 4: 119-127. (1944b) The racial distribution of cancer. I. Epithelial tumors of the skin, lip, and breast. Cancer Research, 4: 433-437. Sheild, A. M. (1899) A remarkable case of multiple growths of the skin caused by exposure to the sun. Lancet, i, 22-23. Shimkin, M. B. (1940) Induced pulmonary tumors in mice. II. Reaction of lungs of Strain A mice to carcinogenic hydrocarbons. Arch. Pathol., 29: 239-255. Strong, L. C. (1949) The induction of mutations by a carcinogen. Brit. J. Cancer, 3: 97-108. Tau.ssig, J., and G. D. Williams (1940) Skin color and skin cancer. Arch. Pathol., 30: 721-730. Unna, P. G. (1894) Die Histopathologie der Mauskrankheiten. A. Hirschwald, Berlin. P. 725. Vint, F. W. (1935) Malignant disease in the natives of Kenya. Lancet, 229: 628- 630. Manuscript received by the editor ]\far. 12, 1951 NAME INDEX Page numbers in boldface type denote bibliographical references A Abbott, C. G., 92, 102 Abelson, P. H., 400, 421 Abetti, G., 95 Acree, S. ¥., 160 Acton, A. P., 143, 153 Adler, E., 192, 196 Agnew, J. T., 142. 153 Aickin, R. G., 153 Aird, R. B., 193, 201 Albanese, A. A., 319, 323 Aldington, J. N., 153 Aldous, E., 366, 371, 391, 393, 415, 419- 420, 429 Alfert, M., 208, 233, 239, 243 Alius, H. J., 491, 502, 509, 527 Allen, A. J., 153, 185, 199 Allen, A. O., 381, 421 Alpatov, W. W., 304, 323 Alper, T., 336, 360 Alsup, F. W., 315, 322. 323 Altenburg, E., 251-252, 255-256, 267, 281 Altenburg, L., 278, 281-282 Altmann, R., 211, 217. 243 Alyea, H. N., 30, 37 American Medical Association, 53, 65, 75, 91 American Public Health Association. 76, 91 Amstein, E. H., 134, 154 Anderson, E. H., 366, 368, 375, 394, 403- 404, 409, 412, 416, 419, 421, 425 Anderson, J. A., 154 Anderson, R. S., 335, 349. 358, 361, 380, 424, 445, 447, 448, 449 Anderson, T. F., 349, 356, 360, 400, 421, 441, 442, 449 Anderson, W. T., Jr., 154 Andrews, H. L., 137, 154 Ane, J. H., 330 Angier, R. B., 191, 199 Arley, N. A., 544, 558 Arnold, W. A., I(i8. 179, 195, 396. 422, 440. 452 Arnow, L. E., 506, 623 Aronoff, S., 194, 195 Atwood, K. C, 369, 372, 417, 421, 431, 438, 440, 446, 449 B Babcock, H. D., 97, 152, 154 Back, A., 286, 287, 289, 291. 293-295, 297, 303, 320, 323, 326 Backstrom, H. L. J., 30, 37, 143, 154 Backus, R. C., 461 Bacq, Z. M., 436, 449 Bahlke, A. M., 75, 93 Bailey, W. T., Jr., 353, 360 Bain, J. A., 532, 557 Baker, E. B., 131, 154 Baker, R. F., 227, 245 Baker, R. S., 37, 38 Baker, S. L., 374-375, 386, 421, 426 Bamford, C. H., 15, 37 Bank, O., 215, 243 Banning, M., 134, 146, 154 Barbrow, L. E., 164 Barer, R., 148, 154, 22(). 243 Barnes, B. T., 129, 162 Barnett, C. E., 129, 154 Barr, E. E., 131, 154 Barron, E. S. G., 295, 296, 322, 323, 335, 418, 421 Barton, D. W., 265-266, 281 Bass, A. M., 143, 144, 154 Bass, L. W., 182, 198, 216. 246 Bateman, L., 29, 37 Bauch, R., 443, 449 Baumann, C. A., 531, 532. 559 Baumberger, J. P., 493. 523 Bauple, R., 157 Bawden, F. C., 349, 360, 395, 422, 470, 483 Bawn, C. E. H., 30, 37 Baxter, N., 190, 191, 196 Bayliss, N. S., 143, 153, 157, 174w., 181, 195 Bayne- Jones, S., 388-389, 422 Bazarian, A., 153 Beard, D., 363 Beard, H. H., 532, 535, 557 561 562 UADIATlOiN BIOLOGY Ik-ard, J. W., ;«1, 360, 363 Broker, J. A., 131. 154 HiH'kor. S. W., Jr., oin\. 524 Bockhorn, E. J., 395, JU2. 111). 110 117. 422 IVcKS, E. W., 129. 154 Bocso, N. C, 129. 154 Bchrons, M., 244 Bellini, A., 530. 557 liciulich. A., 180, 195 Bciicdotti-Pichlcr. A. A., 209, 243 liciiford, F., 150, 154 B(-nn, R. E.. 142, 154 Bennett, A. H., 213. 243 Bennett, H. S., 218. 243 Bennison, B. E., 291, 319, 323 lienton, J. G., 247 Benz, F., 192. 198 Benzer, S., 344, 347, 356-358. 360, 459 Berger, R. E., 246 Berner, F., 293, 322, 323 Berry, G. P., 340, 358, 363 Bertani, G., 407, 414, 422, 423 Bcrthoud, A., 31, 35. 37 Belts, R. H., 71, 92 Beutler, H. G., 152. 154 Bianc'ini, H.. 324 Bigeleisen, J., 1(5, 39, 107, 173, 198 Billen, D., 366, 379, 382, 400-401. 422, 429 Billings, B. H., 131, 147, 154 Bird, G. R., 155, 243 Bishop, C. J., 254, 281 Bishop, M., 281 Blacet, F. F., 131, 155 Black, W. A., 294. 320, 321, 323 Blank, I. H., 396, 422 Bloom, W., 489, 526 Blout, E. R.. 123, 155, 226, 243 Blum, H. F., 37, 297, 298. 301, 302, 304- 306, 323, 395. 422, 426, 476, 481, 482, 487-523, 524, 526, 529-557, 558 Blunt, T. P., 365. 383, 423 Bodenstein, M., 23, 37 Bodian, D., 235, 245 Boggess, T. S., 532, 535, 557 Bohn, G., 294, 324 Bonet-Maury, P., 338, 340-342. 360 Bonhoeffer, K. F., 21, 37 Boothe, J. H., 191, 199 Bovie, W. T., 317, 324, 39(i, 422 Bowen, E. J., 12, 28, 3(), 37, 133, 143, 144, 155, 165. 176, 196 Bowen, G. H., 465-467 Bowles, R. L., 488. 508, 624 Boyce, J. C, 149, 165, 159 Bniee, K. C., 443, 449 Brachet, J., 213, 215, 217, 243 Brackett, F. P., Jr.. 133, 165 Brackett, F. S.. 76. 91, 159, 225, 244, 450 Biadfield, A. E., 195 l^radsjiaw, B. C., 26, 38 Brandt, C. L., 400, 422 Braude, E. A.. 171. 179. 196 Brawn. W., 402. 422 Breakstone, R., 215, 247 Bretscher, E., 370-371. 373-374. 380, 415, 427 Brink, N. G., 192. 195 Brockman, F. G., 131. 156 Brockman, J. A., Jr., 192. 195 Brode, W. R., 171, 178n., 184, 192, 195, 225, 243 Broquist, II. P., 192, 195 Brown, E. W., 76. 92, 93 Brown, J. S., 410, 422, 411, 449, 479 Brown, M. G., 291, 292, 324, 330 Brumberg, E. M., 226, 233, 243 Bruvnoghe, R., 367, 399. 422, 423 Brvan, J. H. D., 208. 243 Bryan, W. R., 334. 360 Bryson, V., 392, 403, 407. 422 Buc, G. L., 146, 155 Buchbinder, L. M., 50, 91 Biicher, T., 14, 37 Biicker, T., 143, 155 Buisson, H., 106 Bungenberg de Jong, H. G., 215. 243 Bunting, H., 248 Burch, C. R., 226, 243 Burke, A., 376, 378 Burkholder, R. R., 404, 422 Burnett, W. T., Jr., 375-376, 378, 380, 422 Burton, M., 15, 36, 40, 381, 422 Butenandt, A. H., 173, 196 Buttolph, L. J., 68, 70, 73, 91 c Cady, W. M., 146. 155 Cain, C. K, 191, 195 Calcutt, G., 322, 324 Caldas, L. R., 396, 419, 426, 438, 449, 475 Calkins, E., 506, 524 Calnan, D., 310, 330 Calvert, H. R., 34, 38 Calvert, J. G., 133. 156 Calvin, M., 12, 39, 165. 171, 198 Caminita, B. H.. 76, 93 Campbell, W. L., 449 Cannon, C. V., 149, 165 Cario, G., 13, 38 Carlson, J. G., 318 Carter, C. E., 389, 400-401. 428 NAMK INDEX 563 Carter, E., 155 Cartwright, C. H., 150, 155 Casals, J., 350, 363 Cashman, R. J., 137, 155 Casparis, P., 97, 102 Caspersson, T., 42, 91, 184, 195, 206, 208, 211, 215, 217-220, 223, 225- 228, 230-238, 241, 243, 244 Casulik. D. B., 191, 199 Catcheside, D. G., 277, 281 Catsch, A., 277, 283 Cavalieri, L. F., 18(i. 195 Chako, N. Q., 181, 195 Chambers, H., 374, 3i)9, 422 Chapman, L. M., 219, 244 Charcot, 487, 524 Chase, C. T., 154 Chase, H. Y., 300, 324 Chase, M., 483 Cheslev, L. C, 322, 324 Chirgwin, D. H., 174, 196 Christian, W., 190, 192, 201 Christiansen, C, 146, 155 Christiansen, J. A., 23, 38 Clapp, R. H., 129, 155 Clare, N., 508 Clark, C, 252, 281 Clark, F. J., 264, 283 Clark, J. B., 331, 412-413. 430 Clark, J. H., 324, 502, 524 Claus, W. D., 45, 46. 91, 368, 384-387, 389, 393, 422, 425 Cleland, G. H., 413. 423, 449 Cline, J., 458 Cline. J. E., 26. 38 Coatney, G. R., 291, 319. 323 Coblentz, W. W., 109-111, 130, 137, 155, 386, 388, 423, 492-493. 502, 521, 524 Coghill, R. D., 406, 411, 425, 439, 451 Cohen, I., 299, 302, 327 Cohen, S. S., 347, 360 Cole, H. N., 514, 526 Cole, P. A., 225. 244, 450 Commoner, B., 183. 196, 230, 244 Constantin, T., 438, 449 Cook, E. v., 299, 324 Cook, J. S., 489, 516-517, 524 Coolidge, A. S., 155 Cooper, M., 208, 218, 244 Corson, E. F., 552, 558 Coulson, C. A., 173-175, 177, 196, 367, 370-372, 375-376, 380-381, 392, 414, 416, 427 Coulter, C. B., 185, 196 Craig, D. P., 174n., 196 Crammer, J. L., 172, 196 Crane, R. A., 131, 155 Crist, R. H., 155 Crolnnd, R., 404, 423 Grossman, E. B., 317, 318, 325 Crowther, J. A., 292, 293, 324 Crumb, C, 75, 93 Curcio, J. A., 115 Curran, H. R., 396. 423 Gushing, J. E., 452 D Dacey, J. R., 155 Daglish, C, 190, 191, 196 Dale, W. M., 335, 401, 418. 423 Daniels, F., 158 Dannenberg, H., 184, 187, 196, 200 Darby, E. K., 160 Daugherty, K.. 322. 326 Davidson, H., 403, 422 Davidson, J. N., 213, 216, 222, 238. 244 Davies, H. G., 228, 236. 244 Davis, W., Jr., 143, 158 Davy, J., 487, 503, 524 De Boer. K. O., 264-265, 267-268, 281 Decker, H. B., 552, 558 Dejardin, G., 129, 137, 155 Delaporte, B., 416, 423 Delbrlick, M., 347, 350, 353, 360, 362, 402, 420, 427, 430 Del Mundo, F., 71, 74, 91 DeLong, G. W., 521, 524 DeLong, R., 437, 448, 449 DeMent, J. A., 129, 156 Demerec, M., 253, 267, 281-282, 368, 389, 404-407, 411, 414, 416, 419, 423, 451, 452, 477, 478 Deming, L. S., 166, 201 Dempsey, E. W., 218, 244, 248 Denmark, H. S., 146, 155 Dennison, D. M., 145, 157 Deriaz, R. E.. 248 Despretz, C. M., 487 Devi, P., 404, 423 Dewar, M. J. S., 174, 196 Dickey, F. H., 413-414. 423, 432, 449 Dickinson, R. G., 31, 35, 38 Dickman, S., 418, 421 Dickson, H., 441, 443, 446, 447, 449 Dimond, A. E., 433, 434, 437, 441, 442, 449 Dippell, R. v., 312, 324 Di Stefano, H. S., 224, 238. 244 Dixon, M., 208, 244 Dobie, D. L., 399, 411, 428, 451 Dobson, G. M. B., 106 Dodd, R. E., 131, 156 Doermann, A. H., 337n. Dognon, A., 286, 293, 320, 324 Dollinger, E. J., 405, 423 nc4 RADIATION HIOLOGY Dorcas, M. J.. 113, 156 Dorii. H. F.. 553, 55(1, 558 Dorno, C. Ill Dotv, P., 184, 196 DourIhs, C. a., 137. 156 Downos, A., 365, 383. 423 Down.s, J., 75, 91 Doyle. M. E.. 71. 93 Draisin. W.. 154 Dr.'oscMi. W. C. 7(). 91 Dvihnniilh. W.. 530, 558 DuHuv. II. G.. 7(), 93 Dutr^ar. B. M.. 324, 3 13, 311. 361, 366- 367. 383-385. 388, 3!)7, 399, 401, 423, 133. 434. 437, 441, 442, 449, 453, 55U, 558 Dulbccco, R., 304, 324, 335. 345, 349, 350-355, 360, 362, 394-395, 410, 423, 441, 457, 459, 461-463, 467- 470 Duncan, A. B. F.,'l47. 156 Diinkelman, L., 137, 156 Dunn, C. G., 368. 424, 447-449 Dunn. .J. E., 76, 91 Duntlov, S. Q., 503, 505, 524 Durand, E., 102, 103, 253, 281 E Easley, M. A., 157 Eddy, C. E., 367-368, 428 Edgerton, H. E.. 156 EdI6n, B., 156 Edmondson, M.. 282 Edsall, J. T., 235, 244 Edwards, E. A., 503, 505, 524 Ehrismann, O., 384-385. 423 Ehrlich, P., 215, 244 Eicher, M., 169, 511-512, 514, 524 Einstein, A., 1, 4 Eisenbrand, J., 144. 156 Elbe. G. von, 18, 36, 39 Elenbaas, W., 156 Elion, G. B., 191. 196, 197 Ellinger, F., 184, 196, 492, 516-517. 524 Ellis, C.. 41, 91, 120, 145, 156, 366. 383. 424 Ellsworth, L. D., 404. 424 Emelius, H. J., 36. 40 Emmett, J., 319, 324 Emmons, C. W., 84, 91, 276-277. 281- 282, 399, 406, 411, 424 425, 435. 437-439, 449, 450 Engelbreth-Holm, J., 551. 558 Engstrom. A., 211, 244 Engstrom, R. W., 136, 138. 139. 141. 166 Ephrati, E., 336, 361, 380, 417. 427 lOphrussi. B.. 432. 445. 448, 460, 451 Epstein, L. F., 13, 40 Euler, 11. von, 192. 196 Evans, F. R., 396, 423 Evans. .1. W.. 1 17, 156 Evans, T. C., 294 296, 300. 324 Ewell, A. W., 65. 91 Ewest. H., 83, 91 Ewing. D. T., l',)3, 196 Exner, F. M., 335, 337, 340, 362, 380. 427 Faliergd, A. C, 264, 281 Failla, G.. 324 Fano, U., 236. 244, 33S Farkas, L., 21, 35. 37, 38 Fellgett, P. B., 132, 156 Ferguson, L. N.. 165. 171, 196 Feulgen, R., 216, 217, 244 Fieser, L. F., 187, 196 Fieser, M., 187, 196 Findlay, G. M., 529, .532. 558 559 Finkelstein. H., 363 FinkeLstein, X. A.. 156 Finkelstein, P., 172, 185. 196 Finley, H. E., 320-322. 324, 330 Finsen, N. R., 488, 508. 524 Fish, F. F., 319, 324 Fitzpatrick. T. B.. 505-506. 524, 526 Fixl, J. O., 173, 199 Flax, M., 214, 216, 239, 244 Flint, J., 405, 414, 423 Flogler, E. A., 395. 426, 476 Flood, v., 323 Florv, L. E., 142. 156 Fluke, D. J., 343. 360 Folkers, K., 192, 195, 198 Fonda, G. R.. 129, 156 Foote, W. S., 154 Forbes, G. A., 26, 38 Forbes. G. S.. 133. 143, 147, 156, 159 Ford, J. .M., 399, 411. 424, 427-428, 435, 436, 440, 445, 450 452 Forro, F., Jr., 340. 342. 363 Forssberg, A., 293, 325 Forster, T., 7, 13, 16, 168. 197 Forsvthe, W. E.. 120. 145. 149. 167 Foucault, J. B. M., 487 Fowle, F. E., 102, 107 Fowler, R. H., 11, 38 Fraenkel-Conrat, H.. 208. 219. 244 Fragstein, K. von, 14(i, 157, 159 Fram. H., 368. 424, 449 Francis, D. S., 305, 327 Franck, J., 3, 6, 9, 11, 13, 38, 168. 197 Frankenburger, W., 133, 163, 506. 516, 625 Franklin, R. G., 163 Eraser, 11. T.. 76. 93 Eraser, R. D. B., 134, 167 NAME INDEX 565 Freed, S., 183, 197 Freeman, P. J., 400, 422 French, C. S., 152, 157 French, D., 235. 244 Frenkel, J., 13, 38 Fricke, H., 295 Friedewald, W. F., 335, 349, 358, 360, 380, 424 Friedman, H., 100 Friedrich-Freksa, H., 173, 195 Fries, L., 432, 450 Fries, N., 432, 450 Froelich, H. C, 129, 157 Fuerst, R., 413-414, 430, 453 Fuller, F. W., 330 Fulton, H. R., 386, 388, 423 Funding, G., 531-532, 558 G Gaffron, H., 10, 11, 13, 38 Gair, C. J. B., 493, 527 Gaither, N., 289, 300, 304, 309, 312, 321, 327, 328, 395, 410, 426, 479, 480 Galston, A. W., 37, 38 Gantz, H., 396, 427 Gard, S., 334, 361 Garen, A., 458 Garner, J. M., 320, 327 Gasvoda, B., 323 Gates, F. L., 42, 344, 361, 363, 371, 384, 386-389, 397, 414, 424 Gaw, H. Z., 291, 330 Gay, H.. 245 Gaydon, A. G., 146, 160 Gebbie, H. A., 115 Geckler, R. P., 309, 311-313, 325, 328, 359, 361 Gee, G., 29, 37 Geigy, R., 251, 282 Germeshausen, K. J., 156 Gersh, I., 211, 227, 235, 245 Gershon-Cohen, J., 76. 93 Gest, H., 361 Gettner, M,, 234, 245 Gibson, G. E., 143, 157 Gibson, K. S., 178^., 197 Giese, A. C., 286, 292, 294, 295, 297, 300, 302-308, 314, 316-318, 320-322, 325, 326, 329, 330, 366, 386, 395, 400, 424, 430, 433, 434, 450, 457, 479, 482, 514, 525, 550, 558 Gilder, H., 190, 197 GUes, G. M., 522, 525 Giles, N. H., Jr., 375, 404, 422, 439, 443, 446, 447, 450 GUI, W. A., Ill, 112 Gilles, A. R., 149, 157 Ginsberg, B., 506, 525 Ginther, R. J., 129, 155 Click, D., 210, 245 Glover, A. M., 137, 157 Goeppert-Mayer, M., 174n., 197 Golay, M. J. E., 131, 132, 157 Goldfarb, A. R., 185, 197 Goldschmidt, R., 216, 245 Goldstein, M., 230, 245 Goodgal, S. H., 410, 424, 441, 442, 450, 453, 479 Goodwin, T. W., 185, 197 Gottschevvski, G., 251, 282 Gotz, F. W. P., 97, 102, 106 Gould, B. S., 185, 198 Gowen, J. W., 343, 346, 361, 363, 368, 404, 427 Gracely, F. R., 521, 524 Grady, H. G., 493, 501, 526, 533-535, 538, 550, 558 Grady, L. D., 129, 154 Granick, S., 190, 197, 215, 246 Gray, C. H., 404, 424 Green, A. B., 367, 374, 424 Green, C. B., 154 Green, J. W., 326 Greenberg, D. M., 244 Greenstein, J. P., 411, 425 Grey, D. S., 155, 226, 229, 243, 246 Grier, H. E., 156 Griese, A., 192, 201 Grigg, G. W., 407, 424 Gross, P. R., 302, 326 Groth, W., 157 Guild, W. R., 185, 200 Guillaume, A. C., 509, 525 Gurney, R. W., 16, 39 Gutmann, A., 359, 362, 420. 427 Guyenot, E., 251, 255, 282 Gyorgy, E. M., 161 H Haam, E. von, 532, 535, 557 Haas, E., 143, 157 Haas, F, J., 331, 402, 413, 424, 429, 430 Haber, F., 35, 38 Haberman, S., 404, 424 Haddox, C. H., 413-414, 430, 453 Hadley, L. N., 145, 157 Hagen, J. P., 99 Haines, R. B., 367, 370-375. 380-381, 387-388, 392, 414-416, 427 Halban, H. von, 143, 144, 157 Halberstaedter, L., 286-288, 291, 293- 295, 297, 319, 320, 323, 326 Halford, R. S., 141, 161 Hamilton. J. B., 505, 525 566 RADIATION BIOLOGY Hammarsten, 10., 221. 245 Ilamnior. V.. 488. 525 Haniporl, II. L'.. I'.IU, 492, 503, 505, 525 Haiu-ock, M.. 131. 158 Hanson, D. F., 110. 161 Han.son, J., 409, 423 Harding, C. V., 300. 326 Hardv, A. C, 43, 123, 157 Harm, W., 394, 424 Harrington, N. J., 21(1. 245 Harris, D. G., 190, 197 Harris, L., 130, 133, 157 Harrison, G. R., 120. 125, 134, 135, 149, 150, 152. 158, U)9, 197 Hart, D., 71, 74, 91 Hartock, P.. 35. 38 Hartelius, H., 232. 245 Hartley, W. N., 105, 171, 197 Hartmann. H., 181, 197 Hartwig, S., 172, 173. 195, 200 Harvey, E. B., 315, 326, 329 Harvey, E. N., 286, 292. 321. 326 Harvey, G. G., 161 Harvey, R. A., 358. 363 Haskins, C. P., 453 Hauschka, T., 319, 326 Hausmann, W., 510, 525 Hausser, I., 492, 525 Hausser, K. W., 488, 494, 502, 525 Hawk, P. B., 182, 197 Havashi, K., 193. 197 Haynes, H., 85, 93 Heath, H. D., 294, 295, 303, 318, 325 Hedcn, C, 225, 246 Heidt, L. J., 133, 143, 155, 156, 158, 185, 197 Hcilbninn, L. V., 295, 315, 322, 326 Heinmcts, F., 388, 395, 397, 424 Heitler, W., 167, 170, 173, 174, 197 Hellstrom, H., 192. 196 Helmke, R., 502, 525 Henle, G., 349, 351, 361 Henle, W., 349, 351, 361 Henri, V., 384, 404, 424, 488, 525 Henriqvies, 0. M., 531-532, 558 Henschke, U., 490, 492, 503-500, 522, 525 Henshaw, C. T., 327 Henshaw, P. S., 298, 299, 302, 305, 326, 327, 368, 427, 443, 445. 450 Hercik, F., 368, 370, 386, 389, 425 Herlitz, C. W., 529, 658 Herold, W., 171, 201 Herschel, Sir William, 487 Herscher, L. W., 141, 163 Ilcrshov, A. D., 343, 345, 353, 361, 183 Herwerden, M. A. van, 213. 217. 245 Herzberg, G., 2. 4, 6. M, 38 Herzfeld, K. F., 23. 38, 173, 197 Heve.sy, G., 297, 327 Hewitt, H. B., 337, 361 Heyroth, F. F., 41, 91, 156, 366, 383, 424 Higginbottom, C, 10 1, 423 Higgins, G. C, 134, 158 Higinbothani, W. A., 140, 158 Hill. R., 244 Hill, W. R., Jr., 1 10, 158 Hillier, J., 234, 245 Hinics, M., 209, 216, 244, 246, 247 Hirshlield, H. I., 307, 308, 328 Hitchings, G. H., 191, 196, 197 Ilockcnhull, D., 432, 450 Hodge, E. S., 135, 162 Hodgins, J. W., 155 Hoorr, N. L., 211, 212, 245 Hoffman, R. M., 158 Hogeboom, G. H., 210, 217, 245 Hogne.ss, T. R., 30, 38 Hogue, J. i\I., 492-493, 502, 521, 524 Holden, H. F., 189, 197 Holla, W. A., 75, 93 Holladay, L. L., 63, 68, 70, 77, 92, 93, 158, 492, 526 Hollaender, A., 42, 45, 46, 76, 81, 84, 91- 93, 272, 276-277, 281-282, 284, 290, 314, 315, 326, 327, 329, 343. 344, 361, 365-421, 422-426, 429-430, 435, 437-439, 441, 442, 446-448, 449 453, 462, 550, 558 Holmes, B., 362 Holt, A. S., 157 Holtz, F., 529, 535, 559 Holweck, F., 287, 288, 307, 320. 327, 364, 367-368, 426, 443, 444, 447, 448, 451 Horecker, B. L., 192, 197 Hornig, D. F., 131, 158 Horning, E. S., 321, 327 Horowitz, N. H., 432. 451 Horsfall, F. L., Jr., 364 Horvath, J., 321, 327 Hotchkiss, R. D., 185, 197 Hottinguer, H., 432. 450 Hovdahan, M. B., 281 Howland, R. B., 321, 327 Hover, H., 215, 244 Hoyle, F., 95, 98 Hoyle, L., 349, 361 Hrenoff, M. K., 193, 201 Huber, W., 371, 426 Hubert, G., 505, 525 Hudson, W., 360 Hueper, W. C, 538, 558 Huggins, W., 105 Hulburt, E. O., 103, 111, 520, 554 Human, M. L., 347, 362 Hunt, R. E., 143, 158 Hutchings, B. T... 191, 199 NAME INDEX 567 Hatchings, L. M.. 327 Hutchins, A., 449 Hyde, J. X., 530, 558 Hyde, W. L., 131, 154 Hyden, H., 232. 245 Hyman, C, 321, 327 Illuminating Engineering Society, 58, 87, 92 Ingraham, H. S., 76, 93 International Commission un Illumina- tion, -14, 92 Iversen, S., 544, 551, 558 Ives, J. E., Ill, 112 Jablonski, A., 7, 9, 38 Jacob, F., 470 Jacobs, J., 174/i., 197 Jacobs, L., 42, 91, 92, 327 Jacobs, L. E., 181, 198 Jacobson, W., 191, 198 Jacoby, F., 211, 245 Jaeger, L., 213, 246 James, T. H., 134, 158 Janes, R. B., 137, 158 Jansen, M. T., 503, 525 Jansky, K. G., 99 Jarrett, E. T., 76, 92 Jencks, P. J., 160 Jenkins, F. A., 145, 158 Jennings, R. K., 320, 327 Jenrett, W. V., 411, 425 Jensen, K. A., 432, 451 Joffe, C. L., 133, 159 Johnson, B. K., 134, 158 Johnson, E. H., 395, 426 Johnson, F. H., 476 Johnson, F. S., 98, 102, 129, 158 Johnson, J. B., 136, 158 Jolit, M., 475 Jones, L. A., 131, 132, 134, 158 Jones, M. F., 42, 91, 92, 290, 327 Jones, R. N., 171, 188, 189, 198 Jones, T. T., 31, 39 Jucker, E., 189, 198 Jukes, T. H., 192, 195 Jundell, I., 529, 558 Jupnik, H., 243 K Kabat, E. A., 185, 196 Kaczka, E., 192, 195, 198 Kalmus, H. P., 142, 158 Kamen, M. D., 361 Kaminsky, J., 133, 157 Kamm, 0., 193, 196 Kandler, L., 173, 200 Kaplan, R. W.. 399, 412, 426 Kariakin, A., 17, 40 Karrer, P., 189, 191, 192, 198 Kasha, M., 7! 9, 12, 39, 143, 159, 168, 175/(., 198 Kaspers, J., 14, 37, 143, 155 Kassel, L. S., 20, 21, 39 Kaufman, W., 396, 427 Kaufmann, B. N., 284, 453 Kaufmann, B. P., 213, 245, 272, 282 Kausche, G. A., 363 Kavanagh, A. J.. 229, 245 Ke, C.-L., 12, 39 Keilin, D., 244 Keller, P., 490, 503, 509, 525-526 Kelley, E. G., 215, 245, 246 Kelner, A., 45, 50, 92, 366, 394-395, 410, 426, 437, 439, 441, 445, 446, 451, 456, 470, 471, 475-479 Kennedy, J. W., 361 Keresztery, J. C., 192, 201 Kerr, G. P., 78, 92, 130, 137, 159 Kihlman, B., 432, 450 Kimball, R. F., 289, 300, 302-304, 308, 309, 311-313, 321, 327, 328, 395, 410, 426, 479-481 King, A. S., 159 Kirbv-Smith, J. S., 493, 501, 509, 524, 526, 533-535, 538, 550, 558 Kirk, I., 451 Kiiwan, D. P., 399, 411, 424, 436, 440, 445, 450 Kistiakowsky, G. B., 159 Kjeldgaard, N., 362, 420, 427, 470 Klasens, H. A., 129, 159 Kleczkowski, A., 395, 422, 470, 483 Ivlein, A., 317, 324 Klevens, H. B., 170, 199 Kline, B. E., 531-532, 549, 559 Klingstedt, F. W., 195, 198 Knapp, E., 253, 274, 280, 282, 411, 426 Knoll, G.M., 552, 558 Knowles, T., 78, 92, 474 Koana, Z., 160 Koehring, V., 294, 328 Kohler, A., 220, 246 Kohn, H., 146, 159 Kolb, R. W., 76, 93 Koller, L. R., 41, 65, 71, 92, 93, 120, 125, 137,159,388,391,426,453 K0lmark, G., 451 Kolnitz, H., 517, 526 Koniuszy, F. R., 192, 195 Kornberg, A., 192, 197 568 RADIATION HIOLOGY Korsoii, R.. 242. 247 Koza, R. W.. 2 It.. 245 Kratky, O., 173, 199 Kroniers, H. C, 149, 159 Ivrotchnior, X.. 18o. 198 Kroner, F. A., \2\). 159 Krogh, A., 517, 526 Knipa, H. F., 5()ti, 527 • Kiuliler, L., ;VA. 39 Kiick, K., 3Gi», 430 Kiihn, H., \7An.. 198 Kunitz, M., 172, 173, 187, 198 Kuper, J. B. H., 137, 159 K\irnick. N. B.. 2l(i, 246 Kurtz, II. F., 151, 159 Lacassagne, A., 287, 288. 307. 320. 327, 328, 339. 364, 307-368, 426, 433, 434, 443, 444, 447, 448, 451, 551, 558 Ladonberg, R., 120, 159 Lagorstadt, S., 234, 246 Lagoni, H., 390. 427 Laidler, K. J., 17. 39 Lairn, G. I., 364 Lambert, R. H., 166, 200 Landen, E. W., 433-435, 437. 451 Lange, B., 135, 159 Langley, S. P., 99, 101 Langmuir, A. D., 76, 93 Larionov, L. T., 233, 243 Larson, D. A., 129, 162 Lash, J. P., 142, 159 Lafarjot, R.. 336, 343, 345, 346. 356, 357. 361 363, 366, 3()8, 380, 389. 404-405. 416-417, 419-420, 423, 426 427, 429, 445-448, 451, 459, 467, 470. 475. 477, 478 Lauffer, M. A., 334, 362 Launer, H. F., 131, 133, 137. 159 Laurens, H., 41, 92, 507. 526 Lawrie, N. R., 322. 328 Lea, D. E., 41. 277. 282, 287. 298, 299. 302, 328, 335-342, 362, 366-367. 370-375, 380-381, 387-388. 414-41(). 427, 444, 451 Loderberg, E. ^L. 402-403, 416-417, 427, 430 Lederberg, E. Z., 439, 450 Lederberg. J., 402-403, 416-417, 420, 427, 430, 432. 451 Lee, II., 312, 328 Leicher, A., 83. 91 Loighton, P. A.. 2(). 28. 31, 33. 35, 39, 135, 158, 286, 2i»2, 310, 320, 321, 325 Leigliton, W. C!., 133, 159 Leitgeb, H., 218, 246 Lembke, A., 396, 427 Loim.'ird-.Jones, J., 174. 198 Leppelnieier, E. T., 78, 92 Lerner, A. B., 505-506, 524, 526 Leslie. I., 238, 244 Leuchtenbergcr. (.. 216, 221, 234. 235, 238, 239, 246, 247 Leuchtenbergcr, !{., 246 Levaditi, J. C, 320, 328 Levene, P. A., 182. 198, 216, 217. 246 Leverenz, H. W., 129. 159 Levin, H.-S., 294, 328 Levinson, S. O., 343, 350. 362 Lewandowski, T., 76. 92 Lewis, G. X., 7, 12, Iti, 18. 32. 33, 36, 39, 165. 167, 171. 173, 198 Lewis. R.. 330 Lewis, T., 517-518, 526 Lewschin, W., 3, 13, 39 Ley, H.. 159 L'ileritier, P., 360. 362 Libby, R. L., 404, 429 Lichstein, H. C., 400. 422 Lifschitz, J., 133, 159 Lignac, G. 0. E., 505, 526 Lill. X. D., 76, 93 Lincoln, R. E., 368, 404. 427 Lindegren. C. C. 437, 448. 449 Lipkin, D. L., 32, 33, 39, 167. 183, 196, 198, 230. 244 Lippincott, S. W., 522, 531, 536, 558 Lison, L., 210. 212, 215. 218. 225, 235, 246 Liston, M. D.. 131, 142, 159 Little, E. P.. 324 Litwer, G.. 319, 328 Lively. E., 403, 416 417. 427 Livingston, R., 1-37. 38, 39, 10. 168, 197 LlcweJlyn, V. B., 136, 158 Lock, C., 137, 156 Loeb. .J., 314, 328 London, J., 522 Longuet-Higgins, H. C.. 174, 198 Loofhourow, J. R., 123, 146, 150. 158, 159, 169. 173, 183 185, 197, 198, 200, 226. 227, 247, 295, 328, 3()6. 383. 385. 387, 401, 412-413, 427, 433. 434, 451, 550, 558 Loos, G. M., 298, 301, 302, 304-307, 323, 395, 422, 481. 482. 489, 516 517, 524 Lord, R. C., 150. 158, 169, 197 Lorenz, K. P., 368, 427 Loring, H. S.. 185. 199 Lotz, C, 413, 423, 449 Lovisatti, X., 509. 526 Lucas, X. S.. 493. :)16. 526 Luck, J. M., 324 Ltick6, B.. 322. 328 NAME INDEX 569 Lucke, W. H., 437, 438, 443, 445, 448, 461, 452 Liu-kicsh, M., 45, 4(i, G3, M. ()8. 70, 77, 78, 92, 111. 130, 169, 385, 427, 492, 514, 526 Lucv, F. A., 33, 39 Lui, C. K., 129, 169, 160 Luntz, A., 293, 297, 326 Luria, S. E., 333-335. 337, 340, 345-347, 350-353, 356. 362, 364, 369, 371, 380, 419, 427, 459, 461, 467, 468 Liirie, M. B., 75, 92 Luscombe, H. A., 552, 558 Luther, R., 30, 39 Liitkemever, H., 23. 37 Lutz, W.', 503, 526 Luyet, B. J., 434, 437, 443-445, 461, 453 Lwoff, A., 359, 362, 420. 427, 470 M McAlistar, E. D., 146, 160, 290, 292, 330 McAulay, A. L., 287. 328, 399, 411, 427- 428, 435, 436, 440, 451, 452 McClintock, B., 262. 264. 282 McClure, D. S., 12, 39, 168, 198 Maccoll, A., 165. 173, 198 McDonald, M. R., 245 MacDougall, M. S., 310, 328 McEIroy, W. D., 432, 452, 453 McGinnies, R. T., 130, 157 Mclhvain, H., 419, 428 Mackenzie, K., 256-257, 273, 282 McKhann, C. F., 71, 91 McLaren, A. D., 14, 34, 38, 39, 143, 160, 172, 185, 196, 199, 343, 344, 363, 433, 452, 516, 526 Maclean, M. E., 143, 144, 160 McNicholas, H. J., 160 McQuate, J. T., 257, 267, 282 Magel, T. T., 32, 33, 39 Magill, M. A., 185, 199 Magnusson, A. N. W.. 552, 558 Mahdihassan, S., 244 Makishima, S., 151, 160 Mallette, M. F., 191, 195 Malmgren, B., 225, 246 Malter, L., 163 Marchbank, D., 369. 430 IVIarenzi, A. D., 185, 199 Markham, R., 362 Marks, H. F., 30, 39 Marshak, A., 301, 304-306, 328, 395, 427, 482 Martin, B. F., 211, 245 Martin, F. L., 366, 369, 372, 375-377, 425, 429, 444-446. 453 Massey, H. S. VV., 168, 199 Masson, P., 503, 526 Matplsky, h, 83, 92 Mathews, A. P., 213, 219, 246 ALathews, M. M., 395, 422 Mathieson, D. R., 330 Matsen, F. A., 174, 199 Matthews, M., 482 Matz, C. H., 160 Maiitner, L., 140, 160 ?kIaximov, A. A., 489. 526 Mazia, D., 213, 246, 307. 308, 328 Mazza, L., 160 Mefferd, R. B., Jr., 379, 430 Meirowskv, E., 505, 526 Mellon, M. G., 248 Mellors, R. C, 225, 226, 233, 234, 246 Melville, H. W., 31, 39 Menczel, S., 180, 199 Menkin, V., 518, 526 Menzel, D. H., 95, 98 Mercer, F. E., 368, 429 Merrill, D. P., 160 Merton, T. R., 134, 160 Meutzner, L, 366, 394, 419. 429 Meyer, A. E. H., 41, 88, 92 Meyer, H. U., 252, 275, 278, 281 282, 410, 428 Meyer, P. S., 509, 526 Michaelis, L., 215, 246 Miescher, G., 490, 505, 509-510, 519, 526-527 Mikeska, L. A., 246 Miller, E. G., Jr., 215, 246, 246 Miller, E. S., 184, 199 Miller, H., 432, 462, 453 Miller, W. E., 36, 40 Miller, W. R., 76, 92 Millikan, R. A., 160 Milovidov, P. F., 217, 246 Milzer, A., 350. 362 Minch, F., 367, 428 Minder, H., 505, 519. 527 Minkoff, G. J., 146, 160 Minsk, L. D., 71, 93 Mirsky, A. E., 216, 223. 231. 233, 234, 239, 246-248 Mitchell, J. S., 516-517, 527 Mitchell, P., 184, 199, 366, 428 Miwa, M., 298-300, 329, 331 Miyake, Y., 133, 160 Moelwyn-Hughes, E. A., 11, 39 Mohan, B. N., 330 MoUer, M., 488. 527 Monne, L., 219, 247 Monod, J., 475 Monsees, H., 409, 423 Montgomery, H., 506, 624 Moon, P., 102, 519, 527 570 RADIATION' inOLOOY Moon.'y, R. L., Ua, 160 Morgan. M. N., 55(1, 558 Mori, K., 208, 29»>, 329, 331 Mori, T.. 246 Monish, A. 11., liiT, 160 Morso. M. L., 375-376, 380, 380. 40 1 , 422, 428 Morton, G. A., 137, 130, 111, 160 Morton, R. A., 184, 185, 187-100, 102, 105, 197, 199 Moses, M. J., 225, 227, 247 Mosovitch, E., 185, 197 Mott, N. F., 16, 39 Mottram. J. C, 310, 329 Mouroin.soft, G., 387-380. 428 Mowat, J. H., 101, 199 Movfho, v., 488, 525 Muller, H. J., 255-257. 267, 270, 273, 281 282, 404. 428 Miillor, K., 172, 173, 200 Mulligan, H. W., 330 Mullikon, R. S., 31, 39, 173, 174. 199 Mullink, J. A. M., 401, 404, 501, 517, 527 Mund, W., 399, 422 Muntz, J. A., 418, 421 Murphy, J. B., 363 Murthv, S. N. K., 453 Myers, V., 132, 160 N Nachtrieb, N. H., 225, 247 Nadson, G. A., 433, 434, 443, 452 Nagy, R., 120, 160, 387-380. 428 Nakamoto, K., 173, 199 Nanavutty, S. H., 386, 421 Xaora, H., 236, 247 Nastiukova, O. K., 304, 323 Nathan, 517, 527 National Re.scarch (^oiincil, 7(1, 92 Nauman, R. V., 12, 39 Noal, J. L., 362 Neal, P. A., 76. 91, 93 Nebel, B. R., 314, 329 Neuberger, A., 172, 196 Newcombe, H. B., 402, 405. 407. 410, 428, 476, 478 Noethliiifi, W., 255, 258. 261, 278-270, 282 283, 384-385, 411, 423, 428 429 Norberg, li., 210, 247 Nordberg, M. E., 137, 160 Norman, A., 360, 372, 417, 421, 428, 437, 438, 443, 445, 446, 448, 449, 452 Norris, K. P., 226, 220, 247 Norri.sh. R. G. W., 15. 37, 160 Nottingham. \V. B.. 160 Novick, A., 395, 410, 428, 470-474, 476- 479 NovikolT, A., 211, 247 Noyes, W. A., 26, 28, 31. 35, 39 () Oakley, H. E. H., 367, 429 O'Hrien, B., 134, 160, 555 556, 559 (Aldio, T. H., 367 36K, 428 O'Keefe, B. J., 131. 158 Oliph.-mt, J. W., 81. 91, 344. 361 OLsou. A. R., 33, 39 Oppcnhoimer, F., 362 Opi)onh(>im(>r, J. R., 1(18, 170. 195 Ornstein, L., 200, 210, 231, 23(1. 247 Oser, B. L., 182, 197 Oshima, K., 160 Oster, G., 172, 1S4, 199, 201, 343, 344. 363 Oster, R. H., 434, 435, 440-443. 448, 452 Osterberg, H., 243 Oszy, A. J., 120, 160 Overholt, R. H., 71, 74, 92 Pack, G. T., 552, 559 Packard, C., 202, 329 Palade, G. E., 245 Panijel, J., 234, 247 Parpart, A. K., 142. 160, 328 Pasteels, J., 225, 235. 246 Patat, F., 33. 39 Patel, C, 319, 329 Pauling, L., 167, 173. 199 Peacock, A., 172. 185. 200 Pearson, A. R., 403. 527 Pearson, G. L., 154 Pearson, S., 143, 160 Peck, S. M., 503, 507, 527 Peller, S., 553, 559 Penney, M., 195 Percival, G. H., 517, 527 Perkins, J. E., 75, 93 Perrin, F. H., 43. 123. 157 Perry, J. W., 140, 160 Perthes, G., 500-510, 527 Pettit, E., 08, 102, 103, 113, 114, 521. 527 Petty, C. C., 115 Pfankuch, E., 346, 363 Pfund, A. H., 130. 131, 161 Phalen, J. M.. 522. 527 Phelps, A., 207. 329 Phelps, E. B., 50. 91 Phillipov, G. S., 433, 434, 443, 462 Pierce, J. V., 102, 195 Pierce, W. C., 225. 247 Piffault, C., 286, 203-205, 320, 324, 328, 329 Pinckard, J. H., 33, 40 NAME INDEX 571 Pioro, E. R.. 1.57, 161 Piatt, J. R.. 170. 174, ISl. 198, 199 Plocser, J. M., 185. 199 Plomley, N. J. B.. 452 Plus, N.. 360, 362 Plymale, W. S., Jr., 140. 161 Polanyi, M., 17, 23, 40 Policard, A., 211, 247 Pollard, E. C, 333, 340, 342, 343, 360, 363 Pollister, A. W., 205, 208, 212, 213, 215, 216, 218, 221-225. 227. 230. 231, 233-236, 238-241, 246, 247 Polster, H. D., 146, 161 Pomper, S., 431, 437-439, 441, 443, 445, 448 Pontecorvo, G., 404, 423 Porter, G., 160 Posner, I.. 76, 91, 399, 425 Post, J., 247 Powell, W. M., Jr., 149, 161 Powers, E. L., 303, 312-314. 329 Preer, J. R., 311, 329, 359, 363 Price, J. P., 298, 301, 302. 305. 323, 481. 550, 558 Price, W. C, 167, 199, 334, 343, 362, 363 Pringsheim, P., 7, 9, 12, 40, 129. 161 Prins, J. A., 10, 40 Proctor, E., 368, 424 Promptov, A. N., 255. 282 Pruckner, F., 189. 199 Prudhomme, R.-O., 320. 328 Puck, T. T., 458 Pugsley, A. T., 367-368. 428 Purcell, J. D., 98, 102 Putschar, W.. 529. 535, 559 Q Quantie, C., 159 Quinn, C. E., 159 R Rabideau, G. S., 157 Rabinowitch, E., 13, 37. 40 Rachele, J. R., 209. 243 Raff, F. A. v., 30. 39 Rahn, O., 48, 93, 366, 372, 428 Rajchman, J. A., 139, 161 Ralston, H. J., 287, 329 Raman, C. V., 146, 161 Ramasastry, C., 161 Ramberg, E. G., 137, 139, 142, 163 Ramsden, W., 159 Rao, S. K. S., 432. 453 Raper, C., 312, 313, 329 Paper, K. B., 406, 411, 425, 439, 451 Rapkino, S., 362, 420, 427 Ratcliffe, H. L.. 75, 93 Rayleigh (Lord), 106 Read, J., 337, 361, 375. 430 Reaume, S. E., 432. 452 Rechen, H. J. L., 133, 155 Recklinghausen, M. V., 79, 93 Reckncgel, R. O., 304. 322. 331 Rector, C. W., 174, 198 Reed, E. A., 302, 304, 315, 318, 322, 326, 329 Reed, G. B., 404, 429 Regener, V. H., 147, 161 Reinhard, M., 237, 248 Reisner, E. H., Jr., 242, 247 Rekling, E., 531-532, 558 Rentschler, H. C., 137, 161, 286, 320, 321, 329, 387-389, 391, 428 Reuss, A., 252, 256, 277, 282-283, 411, 426 Ricca, A., 328 Rice, C. E., 404, 429 Rice, F. O., 168, 170. 199 Rice, O. K., 149, 155 Richards, O. W., 243 Richardson, R. A.. 114 Rickes, E. L., 192. 195 Riehl, N., 343, 363 Rieke, C. A., 173, 174, 199 Rigdon, R. H., 319, 329 Ris, H., 208, 218, 227, 230, 234, 235, 237, 238, 247, 248 Risse, O., 282, 411, 426 Ritter, J. W., 487, 527 Rittner, E. S., 135, 161 Ritz, E., 362, 420, 427 Rivers, T. M., 344, 363, 368, 430 Robb, C. D., 131, 154 Roberts, R. B., 366. 371. 391, 393, 400, 415, 419-421, 429 Robertson, E. C., 71, 74 Robertson, M., 303, 322, 328, 329 Robinow, C. F., 403, 429 Robinson. J. C., 304-307, 395, 422, 481, 482 Roegner, F. R., 410, 429 Roepke, R. R., 368, 404, 429 Roffo, A. H., 529-533, 535, 550, 553, 559 Rollefson, G. K., 12, 15, 36, 40 Roman, H.. 262. 266, 268. 283 Romand, J., 157 Roothaan, C. C. J., 174n., 199 Rosenbhmi, M. B., 76, 93 Rosenstern, I., 71, 93 Roskin, G. R., 320, 322. 330 Ros.senbeck. H.. 216. 248 Roth, J. S., 326 Rothman, S., 506-507. 516. 527 572 K \I)I A'l'ION mOLOGY Kotnian. R., ."^5:1 361 Hotticr, P. B., 4<»1, 4!»4, 501, T)!?, 527 Ihibin, B. A., 314, 330 Huhin, J.. ."ilC). 527 Huch. v., 2M). 248 l{u(li.s('ll. II.. ;n'.t, 329 Rusch. H. P.. 531 532. 51',», 557, 559 Rviss, S., 367, 374, 3<»<.). 422 Russol, T. A., 134. 160 Russell. P. B., I'll, 196 Riissdl, P. F., 319. 330 S Sack, 517, 527 Sadun. E. 11., 319. 329 Sage, S.. 154 Saidel, L. J.. 185, 197 Salaman. M. II.. 340-342, 362 Sancier, K. M., 183, 197 Sanderson. J. A., 520, 554. 559 Sandvik; O., 134. 158 Sausome, E. R., 282, 30(i. 411. 425, 435. 437, 438, 446, 451, 452 Sarachek, A., 437, 438, 443, 445, 448, 462 Sargeant, W. E., 159 Sauer, L. W., 71, 74, 93 Savage, G. M., 437, 445, 452 Savitzky, A., 141, 161 Sawires, Z., 195, 199 Sawyer, R. A., 120, 150, 160, 161 Schaeffer, A. A., 311, 330 Schall, L., 491, 502, 509. 527 Schauenstein, E., 172, 173, 184. 199-201 Scheibe, G., 172, 173, 182, 195, 200 Scheuing, G., 238, 248 Schlafer, H. L., 181, 197 Schlenk, F., 185, 200 Schlesinger, R. W., 349, 351, 363 Schlesman, C. H., 131, 161 Schmidt, C. L. A., 244 Schneider, E. G., 120, 149, 161 Schneider, W. C., 245 Schneiter, R. A., 76, 93 Schoen, A. L., 135, 162 Schoenborn, H. W., 288. 330 Schonmann, E., 102 Schormuller, J., 186, 200 Schou. S. A., 193, 194, 200 Schrader, F., 219, 234, 246 Schramm, G., 184, 200 Sfhreiber, H., 274, 282, 411, 426 Schrek, R., 553, 559 Schulman, J. H., 129. 162 Schultz, J., 217, 243, 262-263. 272. 283 Schultz, R., 490, 492. .503 .506. 522. 525 Schwarz, E., 131, 162 Schwarzenback, G.. 192. 198 Schwegler, R., 129, 166 Scott, C. M., 286, 330, 517, .527 Scott, G. G., 159 Scott, G. H., 211, 248 Scott, G. W., 405, 428, 476 Scott, J. F., 169, 181, 183, 200, 231, 232, 248 Seeds, W. E., 172. 201, 247 Seifriz, W., 320. 321. 330 Seitz, E. O., 41, 88, 92 Seitz, F., 129, 169 Sell-Beleites, I., 277, 283 Senib. J., 191, 199 Serra, J. A., 218, 248 Setlow, R. B., 185, 200 Shalimov, L. G. 292, 330 Sharlit, H., 505- .506. .527 Sharp, D. G., 363, 387, 429 Shaughnessy, H. J., 362 Sheets, G., 324 Shefner, D., 303, 312, 313, 329 Sheild, A. M., 530, 559 Sheppard, S. E., 166, 180, 182, 200 Sheremet'ev, G., 7, 9, 40 Sherman, A., 173, 201 Shettles, L. B., 287, 330 Shimkin, M. B., 551, 559 Shin-Piaw, C., 104 Shirley^ E. S., 320-322, 324, 330 Shishliaeva, Z., 320, 322, 330 Shpolskil, E., 7, 9, 40 Shiigar, D., 456 Siendentopf, K., 143, 157 Silverman, H. F., 75, 93 Simard, R. G., 157 Siminovitch, L., 362, 420, 427, 470 Simon, S., 322, 330 Simpson, D. M., 191. 198 Simpson, W. L., 211. 248 Simpson, W. T., 174/(.. 200 Singer, M., 218, 244 Singer, T. P., 418, 421 Singleton, W. R., 274, 283 Sinsheimer, R. L., 146, 162, 169, 173, 183, 187, 200 Sizer, I. W., 172, 185, 198, 200 Skarzynski, B., 194, 200 Sklar, A. L., 170, 173, 174h., 197, 200 Skovsted, A., 432, 452 Slater, N. B., 11, 38 Slaughter, J. C., 324 Slautterback, D. B., 219, 247 Slizynski, B. M., 257, 267, 283 Small, M. IL, 404. 429 Smetana, II., 319. 323 Smiljanic, H. M.. 506. 627 Smith, C. E., 75, 93 NAME INDEX 573 Smith, E. C, 431, 453 Smith, F. C, 185. 200 Smith, K. M., 362 Smith, K. O., 161 Smith, L. I., 193, 200 Snvder, R. L., 139, 161 Sollmann, T., 514, 526 Solmssen, U., 192, 198 Solowey, M., 50, 91 Sommer, A., 136, 137, 140, 162 Sonneborn, T. M., 288, 309, 312, 313, 330, 359, 363 Souder, C. G., 553, 559 Spear, F. G., 370-373, 429 Spencer, R. R., 310, 330, 371, 429 Spiegel-Adolf, M., 510, 525 Spikes, J. D., 315, 330 Spindler, L. A., 330 Sponer, H., 6, 38 Spooner, L. W., 156 Sprague, G. F., 258-260, 283 Stacey, M., 217, 248 Stadler, L. J., 249, 254, 258-262, 266, 268, 274-276, 279-280, 283, 411, 429 Stahmann, M. A., 432, 453 Stair, R., 103, 109-111, 130, 137, 155, 492-493, 502, 521, 524 Stamm, R. F., 152, 162 Stapleton, G. E., 366, 368-369, 372, 374- 380, 382-383, 391, 400, 418, 421, 425, 429, 444-448, 453 Staude, H., 146, 162 Stauffer, J. F., 432, 453 Steacie, E. W. R., 17, 27, 40 Stearns, E. I., 33, 40, 146, 155, 208, 248 Steiger, R. E., 185, 199 Stein, W., 366, 394, 419, 424, 429 Steinberg, R. A., 432, 453 Steiner, R. F., 184, 196 Stenstrom, W., 237, 248 Stent, G. S., 360 Stephenson, C. G., 553, 559 Stern, A., 189, 199 Stevens, J. R., 192, 201 Stiller, E. T., 192, 201 Stimson, M. M., 186, 201 Stockbarger, D. C., 149, 162 Stokes, A. R., 172, 201 Stokstad, E. L. R., 191, 192, 195, 199 Stoll, A. M., 319, 330 Stone, F. M., 185, 196 Stone, R. S., 76, 92 Stone, W. S., 296, 331, 402, 412-414, 424, 429-430, 453 Stoughton, R. W., 12, 40 Strait, L. A., 193, 201 Straub, J., 265, 283 Streim, H. G., 246 Strickland, A. G. R., 330 Striker, G. O., 142, 158 Strong, L. C, 542, 559 Stubbe, H., 255, 258, 261, 278-279, 283, 363, 411, 428-429 Studer, F. J., 129, 162 Sturm, E., 344, 363 Style, D. W. G., 29, 40 Subbarow, Y., 191, 199 Subramaniam, M. K., 432, 453 Summers, D., 29, 40 Summerson, W. H., 182, 197, 506, 524 Sunkes, E. J., 75, 93 Suntzeff, v., 493, 523 Swanson, C. P., 254, 268-272, 274, 276, 278, 283-284, 399, 425, 441, 442, 446, 447, 451, 453 Swanson, W. H., 434, 450 Sweet, M. H., 140, 162 Swenson, P. A., 400, 422, 441, 453, 457, 479 Swift, H. H., 207, 227, 228, 230, 233, 235, 236, 239, 240, 247, 248 Syverton, J. T., 340, 358, 363 Szilard, L., 395, 410, 428, 470-474, 476- 479 Tang, P. S., 291, 330 Tatum, E. L., 402, 404, 424, 430, 432, 462, 453 Taussig, J., 552, 559 Taylor, A. H., 45, 64, 78, 85, 92, 130, 137, 159, 162, 474, 492, 514, 526 Taylor, A. R., 343, 363 Taylor, C. V., 295, 316, 324, 330 Taylor, E. C., Jr., 191, 195 Taylor, H. S., 36, 40 Taylor, M. C., 286, 328 Taylor, R., 185, 198 Taylor, W. W., Jr., 388, 395, 397, 424 Tchakotine, S., 315, 321, 331 Teece, E. G., 248 Teller, E., 168, 170, 199 Tennent, D. H., 297, 321, 331 Terenin, A., 17, 40 Terent'ev, A. P., 16, 40 Terrien, J., 149, 162 Terus, W. S., 491-492, 495-497, 499-500, 502, 510-512, 514, 517, 523, 524 Thayer, R. N., 129, 162 Theorell, H., 189, 201 Thoday, J. M., 375, 430 Thom, C., 432, 453 Thomas, J. O., 330 Thomas, L. E., 218, 248 Thomas, L. J., 306, 326 574 RADIATION BIOLOGY TlK.mpson, T. L., 379, 430 Tliorcll, B.. 208. 218, 220. 221, 223, 22.'). 227-230. 232, 234, 23(), 237, 243, 244, 248 Tiiu()r."('lY-H(>sso\v.skv, N. W ., 363 Tisdall, F. F., 71. 93 Tobias, C. A., 417, 430, 1 14, 445, 448, 453 Tomkins, F. S., 193, 196 Torriani, A., 475 Totter, J. R., 191, 201 Touscy, R., 98, 103, 158 Troiber, E., 172, 184, 200, 201 Ts'ai, L.-S., 30, 38 Tsi-Ze, N., 104 Tsuboi, K. K., 173, 201 Turk, W. E., 136, 140, 162 Turkowitz, H., 443, 445, 447, 448, 449, 450 U Ubor, F. M.. 134, 162, 2.54, 273-27(1, 279- 280, 283-284, 344, 363, 411, 429 U.S. Pvitilic Health Service, 79, 93 Unna, P. G., 530, 559 Ureck, C, 35, 37 Vahle, W., 488, 502, 525 Vaiulenbelt, J. M., 193, 196 Van der Lingen, J. S., 388-389, 422 Vandiviere, H. M., 75, 93 Van Vleck, J. H., 173, 201 Van Voorhis, C. C, 159 Vassy, A., 106 Vavilov, S. I., 13, 40 Vendrely, C, 246 Vendrely, R., 246 Vigroux, AI. E., 105 Vilallonga, F., 185, 199 Vincent, H. B., 120, 161 Vint, F. W., 553, 559 Vodar, B., 157 Vogels, H., 7, 40 W Wagner, R. P., 413-41 1, 430, 432. 436, 453 Wahl, R., 343, 345, 361, 363 Walilgren, F., 529. 558 Wald, G.. 490, 527 Walker, R. D., 166, 200 Waller, C. W.. 191, 199 Wallis, R. F.. 35, 38 Wanza, J. W.. 324 Warburg, E., 21, 40 Warburg, O., 190-192, 198, 201 Ward, F. S.. 71, 93 Ward, P. A.. 330 Warren. S. L.. 340, 358, 363 Warshaw, S. D., 438. 453 Wataiiabe, K., 158 Watens, W. A., 167, 201 Wat.son, J. D., 336. 337, 310. 341, 346, 348. 350, 352, 355. 360, 363, 409 Watson, W. F., 13. 40 Waxier, S. H., 319, 330 Webster, L. T., 350, 363 Wedding, M., 508, 527 Weidel, W., 360 Weigert, F., 30, 39, 40, 146. 162 W(-igle, J. J., 360, 120, 430, 170 Wei.ssnian, S. J., 13, 40 Wells, A. A., 41, 91, 156, 366, 383, 424 Wells, J. M., 511, 525 Wells, M. W., 75, 93, 391, 430 Wells, P. H., 304, 305, 330, 395, 430, 482 Wells, W. F., 63, 75, 93, 391, 430 WeLs, P., 321, 330 West, W., 36, 40, 143, 162 Westergaard. M., 451 Weyde, E., 133, 163 Whalen, J. J., 152. 162 Wheeler, S. M., 76, 93 Wheland, G. W., 174. 180. 201 Whitaker, D. M., 394. 430, 456 White, H. E., 145, 158 Whitehead, H. A.. 407. 410. 428, 478 Wichterman, R., 286. 292. 295, 316, 320, 330 Widniark, E. J.. 487-488. 527 Wieland, H., 238, 248 Wiggins. L. F., 248 Wilbur, K. M.. 304. 315, 322. 326, 330 Wilder, T. S., 75, 93 Wilkins, M. H. F., 172, 201, 247 Wille, B., 33, 40 Williams, A. H.. 12. 28. 36, 37 Williams, G. D.. 552. 559 \Mlliams, R. C., 461 Willmon, T. L., 76. 92, 93 Wilson, E. B.. 213, 248 Wingchen, H., 159 Wislocki, G. B., 215. 248 With. C.. 508-509, 528 Witkin, E. M., 366. 368, 371. 387, 389, 391-392, 405, 416, 418-419, 421, 430, 470 Wokes. F.. 190. 191, 196 Wolf, A., 331 Wolf, 1). i:., 192, 195, 198 Wolf. K. L.. 171. 201 WoUenton, R. W., 160 Wollman, E., 337, 339, 340, 364 NAME INDEX 575 Wollman, E. L., 360 \yood, R. E., 35. 38 Wood, R. W., 135, 163 Wood, T. R., 192, 195 Woodruff, C. E., 522, 528 Wottgo, K., 321, 322, 330 Wright, N., 141, 163 Wright, W. H., 290, 292, 330 Wulf, O. R., 166. 201 Wyckoff, H., 141, 163 Wyckoff, R. \\. G., 48, 93, 368, 373, 384- 387, 430, 434, 437, 443, 445, 453 Wyss, O., 296, 330, 379, 402, 412-413, 424, 429-430 Yamashita, H., 2<)8, 299, 329, 331 Young, R. A., 295, 326 Zahl, H. A., 131, 163 Zahl, P. A., 433-435. 437, 442, 453 Zain, H., 319, 331 Zechmeister, L., 33, 40, 188, 189, 201 Zelle, M. R., 76, 345, 365-429, 430, 463 Zeuthen, E., 322, 326 Zhalkovsky. B. G., 297, 331 Ziegler, J. E., Jr., 351, 364 Zimmer, E., 282, 406, 411, 425, 435, 439, 446, 451 Zimmer, K. G., 363 Zimmerman, G., 16, 40 Zimmerman, W.. 163 Zinder, N. D., 403, 416-417, 427 Zirkle, R. E., 304, 331, 444, 453 Zotterman, Y., 517, 526 Zscheile, F. P., 190, 197, 208, 248 Zworykin, V. K, 135, 137, 139, 142, 163 SUBJECT INDEX A Abdomen exposure to ultraviolet in Drosophila, 251-253, 256, 277 Abnormal form in protozoa, radiation- induced, 310 Absorption {see Ultraviolet radiation) Absorption coefficient, 104 Absorption cross section, 179 Absorption laws, Bouget-Lambert, 492 in cytochemistry, 204-210 Acceleration of cell division by radia- tion, 297 Acenaphthene, 432 Acid-base equilibria of excited mole- cules, 16-17 Acidophilia, 219 Acriflavine, 432 Actinometer (see Detectors, photo- chemical) Action spectrum {see Ultraviolet radia- tion) Activation of eggs by radiation, 314- 315 Adaptive enzyme synthesis, inhibition by ultraviolet, photoreactivation, 479 Adenosinetriphosphate, 401 Air mass, 101 Algae, unicellular, 293 Alpha radiation, 335, 340, 369, 370 division delay produced by, 307 effect of, on cell motility, 286, 288, 320 on centrosomes, 288 on fungi, growth of, 443 lethal, 443, 444 mutagenic, 444 on kinetosomes, 288 lethal effects of, 287, 288 polonium, 368, 370, 372 Amblystoma larvae, 395 Amino acids, 414 dopa, 506 tyrosine, 507 ultraviolet absorption, 184, 185 Arnoeba, 294, 307, 308, 320-322 Angiosperms, 253-254 Anomalous dispersion in cytochemistry. 231 Anthocyanin pigments, ultraviolet ab- sorption, 193, 194 Anthracene, photochemical dimeriza- tion of, 30 Antibodies, 510 Antirrhinum, 249, 253-255, 258, 261, 273, 278-279 majus, 411 Arbacia, 295, 296, 298-301, 304-307, 314, 315, 322 gametes of sea urchins, 395 punctulata, 395 Strongylocentrotus purpuratus, 395 Arginine, cytochemical test for, 218 Ascaris, 290, 292, 299 Ascorbic acid, ultraviolet absorption, 192, 193, 208 Aspergillus, 276 melleus, 437 nidulans, 432 niger, 433-435, 437, 442, 444 terreus, 435, 437, 438, 441, 442, 444- 448 Associated volume method, 339-342 Attenuation coefficients, 494, 498, 511 Autooxidation. 11-12, 28-29 Azure, metachromasia, 215 Azure A, absorption curve, 239 B Bacteria, Achromobacter fischeri, 368, 400 Aerobacter aerogenes, 405 Azotobader, 380 Bacillus anthracis, 367, 387, 390, 404 megatherium, 386, 388-390 mesentericus, 370-372, 387, 388 pyocyaneus, 367, 385 subtilis, 389-390, 404 Corynebacterium diphtheriae. 390 Eberthella typhosa, 390 Escherichia coli {see Escherichia coli) lysogenic {see Lysogenic bacteria) Micrococcus candicans, 385 candidus, 390 piltonensis, 390 sphaeroides, 390 Moraxella Iwoffi, 404 Mycobacterium tuberculosis, 404 577 578 RADTATIOX mOLOGY Ractoria, Xeisfirrin cainrrhalin, iiOO nonlysoKcnic, !WG photorcactivjition {src Photorcactiva- tion) Phiitoinonas slctrartii, 404 tuincfaricus, 'MO Proteus vulgaris, 390 Fseudoiiionas aeruginoHa. 390 fluorcscens, 390 "pyocyaniquo S," 367 radiation-induced mutations in, 3()5 Sahnonclln ttiphiinurium, 308 Sarcina lutea, 3G8, 390 Serrnlin iHarcesans, 3()8, 37 1 . 385, 387, 390, 399, 404, 412 Shigella paradt/senteriae. 390 Spirillum ruhrutn, 390 Staph i/loeoccus albus, 387, 390 aureus, 307, 370-371, 384-385, 387, 390, 404. 412 Streptococcus hemoh/ticus, 390 lactis, 390 viridans, 390 survival curve, 472 Bactericidal action (see Germicidal action) Bacteriophage, 333-364 genetic recombination, 353 inhibition of development l)y lisht, 470 interaction with host, 346 intracellular irradiation. 356-358 mixed infection, 350 mutual exclusion, 350 photoreactivation (see Photoreactiva- tion) plaques, 352 type-hybrid, 345 viruses (see \'iruses) BAL, 337/1. Band width, definition of, 148-149 Basal cell, 533, 551 Basic staining (ba.sophilia), 213-216, 218 \ Ba.sophilia, 213-216. 218 Beer's law, 4 in cytochemistry, 206, 221 Beta radiation, cleavage delay produced by, 298 effects on protozoa, 292. 312. 313 mutagenic action of. 312. 313 radium, 367 radon disintegration, 370 Bimolecular steps. 17-19 Blank in photometric analysis of tis- sues, 222. 230. 231 Blepharisma, 297, 302, 316. 318, 322 Bodenstein stcidy-state approximation, 21 26 Bouger-iiauilxTt alisorj)! ioii law, 492 Breakage-fusion-hridge cycle, 261, 277 c Cadmium photocell, 42 Caffeine, 432 Cage effect, 15 Camphor, 432 Cancer, breast, 535 "buccal cavity," 554 crude-tar induced, 529 cutaneous (see Skin cancer) face, 530, 552 feet, 552 genitals, 552 hands, 530 kangri, 552 in Kenya natives, 553 latitude in relation to, 553-556 in light-complexioned persons, 530 lung, 535, 551 north-south distribution of, 553 skin (see Skin cancer) tropical ulcers of leg, 553 in ury)an areas, 553 (See also Carcinomas; Sarcomas) Carbon arc, 487, 513 Carbon dioxide, 375 Carcinogenic agent, 533 in chimney sweeps, 552 crude petroleum, 552 Carcinog(>iiic wave lengths, 531 Carcinogens, effects on protozoa, 310 Carcinomas, 533, 534 • growth period, 542 induction period. 542 (See also Cancer) Cell death, causes of, 286-290 delayed. 287-290 differences in sensitivity, 291 fractionated dose in relation to, 293 immediate, 286, 287, 290. 293 relation to division. 287, 289, 290, 293. 307 role of chromosonie al)erration and mutations in, 287-290, 293 Cell-division retardation, photoreactiva- tion, 479, 481 (See also Cleavage delay : Division delay) Cell growth. 288, 290, 303. 307. 311 Cell membrane, damage to. by radia- tion, 2S7. 290. 315 Cell morphology. 203 204 Sf^JECT INDEX 579 Cell multiplication, 287, 288. 290. 303, 311 Cells, (k'udiitic, 503 diploid. 416. 417 frrowiiis, 418 haploid. 416, 417 localized irradiation of. 315 multinucleate, 416. 418 poison, 415 prickle, 503, 509 relative sensitivity of, 389 resting. 391, 418 suboptimal-temperature incubation of, 382 tissue culture, 367 uninucleate, 416, 418 vegetative. 372, 374, 389. 390 wild-type, 407 Centrosome, effect of alpha radiation, 288. 307 role in cleavage delay, 305 Chaetomium cochlioides, 443, 446. 447 glohosum, 411, 434, 440, 445 Chaetopterus, 302, 305, 314, 322 Chain reactions, 23-25, 28-29 polymerization, 30-31 Chaos, 294, 311 Chemical equilibria of excited molecules, 16-17 Chemical protection, 377 additivitv, 379 BAL, 375. 378-379 j8-propiolactone, 409 British anti-Lewisite, 375 carboxylic acids, 378 chloroform, 396 cj'^steine, 337n., 377-379 2,3-dimercaptopropanol, 377 ethanol, 377-378 formate, 377 furmarate. 377 glucose broth, 375 glycerol, 377 glycine, 396 glycols, 377-378 hydrogen donation, 379 hydrogen svUfide, 396 hydrogenase system, 379 hydrosulfite, 378 isopropanol, 377 lactate, 377 2- (2-mercaptoethoxy )-ethanoI, 377 mercaptosuccinate, 377 methanol, 377 phenol, 396 plateau concentrations, 379 propanediol. 377 propylene glycol, 377 Chemical jirotection, pyruvate, 377 .sodium formate, 378 sodium hydrosulfite (Na2S204), 375, 377-379 succinate. 377 sulfhydryl compounds, 377-379 triethylene glycol, 377 Chimeras, chromosomal, 265 C'hitin, absorption of ultraviolet by, 252- 253 Chloramine T, 432 Chlorophyll, 208, 210, 233 Cholesterol, 550 Chondroitin sulfate, metachromasia, 216 Chromatinic bodies, 403 Chromidial hypothesis, 213, 216 Chromonucleic acid, 213 Chromosome aberrations, achromatic lesions, 270 breakage-fusion-bridge cycle, 264, 277 and cell death, 287-290, 293 chromatid, 268-273 deficiencies. 257-258. 261-273. 275, 277. 279-280 interstitial, 257-258, 265-266, 268 terminal, 257, 263-272 deficiency translocations, 265-266 deletions, 257, 268-273 terminal, 265 effect of idtraviolet on X-ray in- duced, 272-273 half-chromatid, 269-270 induced by ultraviolet, 257-258, 268- 273 Gasteria, 265 inversions, 256, 264 isochromatid, 268, 271 in pollen tubes. 268-273 potential, 263 rearrangements, gross, 267 minute, 257 relation of, to dosage, 269, 278 to stage of division, 269-271 to wave length, 274 in salivary glands, 257 translocations, 256-257. 259-260. 264-265, 268, 270-272 Chromosome breaks, behavior of, 262, 272-273 restitution of (healing), 258. 263, 271-273 reunion of, 271 Chromosomes, absorption curve, 220 iso-. 265, 268, 270. 272 matrix, 271-273 ring, 262-265, 273 580 HADIATION JUOLOGY rhroinosoiiu's, riiiK, loss of. 2()2 2(V.\ X. 252- 25:^. 2(17, 277 XX. 252 V, 258 Chromospljoro, '.)7 Chronic exposure to radiation, W,], 310, 311 Ciliata, 286-289, 291-292, 297, 302, 305, 310, 316, 320, 321 Cis-trans isomerization, 31-33 Classical rosonanoo, 13 Cleavage delay, 295, 29() by beta radiation, 298 role in, of eentrosoine, 305 of nucleus and cytoplasm, 304-307 time course of, 298-302 by ultraviolet radiation, 298, 300 302, 304, 305, 307 by X rays, 298-302, 304, 305 Coagulation of protoplasm by radiation, 286, 287 Colchicine in pollen tube cultures, 254 Collisions, intermolecular, 11 multiple impact with solvent mole- cules, 14 Colloid osmotic pressure, 411 Colorimetry, 207, 239 Colpidium, 292, 295, 310, 316 colpoda, 367 Comparison eyepiece, 224 Complexion, 552 Coprinus Jimetarius, 432 Corneum, 489-494, 497, 501, 509-510, 516, 551 sterol in, 494 Corona, 97, 99 Cyanide, 432 Cyclotron, 368-370 Cysteine, 33 7n. cytochemical test for, 218 Cystine, cytochemical test for, 218 Cytochemistry, absorption laws, 204-210 anomalous dispersion in, 231 Beer's law, 206, 221 fixation in, 234, 235 Lambert's law in, 206, 230 photometric analysis in. errors, 229- 236 technique, 225-229 Cytochrome c, specific absorption, 208 Cj'tological studies, 370 Cytoi)lasmic fac^tors, 359-360 Cytoplasmic particles, effect of radiation on. 311. 312 D Decomposition, photochemicil. of for- maldehyde, 26-27 Decomposition, photochcmic.-il, nf HI. 21 23 of H,S, 26 Degradation of excitiition energy, (i, 8 Denaturation of proteins. 33-.34 Densitometer, 236 DeoxyiH'ntose nucleic acid (D.\A), amount per nucleus, 2!i7 239 Beer's law, 221 nucleal reaction for, 217 specific absorption, 208 Deoxyribomiclease, 214, 216 Deoxyribonucleic acid (DXA), 384, 389, 400, 411 (See also Deoxypentose nucleic acid) Deoxyribose nucleic acid (see Deoxy- pentose nucleic acid) Dennatological lore, 552 Dermatologist, 557 Dermis, 488, 490 corium, 489 dilation of minute vessels, 488 Detectors, fluorescent, 129 converter, use with phototube, 129 magnesium tungstate, (luantum efficiency for, 129 sulfides, 129 zinc silicate, binders for, 129 maximum excitation of, 129 quantum efficiency of, 129 photochemical, 132 triphenylmethane dyes, ([uantum yield. 133 temperature coefficient, 133 uranyl oxalate, quantum yield. 133 temperature coefficient, 133 photoelectric, 135 image orthicon, 142 ultraviolet microscope, 142 use in idtraviolet spectroscopy, 142 photoemissive cell, 136 current-voltage characteristic, 138 precision, principal limitation of, 136 spectral response, 138 threshold wave length, 136 photomultiplier cells, 139 amplification in, 139, 140 construction of, 139 power supply for, 140 response of, 140 time resolution, 140 variation in characteristics of, 141 "venetian-blind," 140 photovoltaic cell, electric circuits for, 135 SUBJECT INDEX 581 Dectectors, photoelectric, photovoltaic cell, envelope materials, 136 quantum efficiency of, 137 quartz envelopes for, 136 photographic, accuracy of, 133 fluorescent sensitizer for, 134 sensitivity of, 134 f thermal, bolometer, 130 response time of, 131 sensitivity of, 131 Golay cell, 131 sensitivity of, 132 sensitivity, ultimate limit of, 131 thermocouple, 130 thermoelectric power, 131 sensitivity of, 131, 132 Deuterons. 335, 340, 342 Development, effects of radiation on, 290, 300, 303 Diazomethane, 432 Dichroism, 172, 173, 177, 183, 230 DitTusion-controUed processes, 19-20, 36 Dimerization, photochemical, of an- thracene, 30 Direct effect of ionizing radiation, 337 Disinfection by ultraviolet, absorptive liquids, 78-80 blood plasma, 78-85 exposures of films, 82 by film spreaders, 80, 81 controls, 82, 85 laboratory methods, 81 milk, 78-81 penetration into, 78-80 sugar syrups, 78, 79 vaccines, 78-81 air, 67 effective exposures, 67 health value, 73 conservative appraisal, 76, 77 hospitals, 74, 75 institutions, 76 Navy barracks, 76 schools, for colds, influenza. 75 rooms, 69 ceiling height, 71, 72 circulation of air, 70 hospital, 71-73 hospital barriers, ultraviolet, 71, 74 intensity of ultraviolet in upper air, 71 louvered ultraviolet fixtures, 73 occupied, 69 open ultraviolet fixtures, 72 protection of occupants, 52-70 unoccupied, 69 upper-air method, 70, 71 Disinfection by ultraviolet, air, rooms, and ventilation, 70, 71 standard, tentative, 73 air duct, 52, 53, 67, 68 effect of size, 68 intensity of ultraviolet, 67 reflective walls, 68 relation to make-up air, 69, 70 . turbulent flow, 68 surface of solids, 82, 83 fruit, smooth skins, 83 grain, 83 sugar, 83 water, 77 absorption of ultraviolet, 77, 78 devices for, 79 exposures, 77-79 Dispersive power, definition of, 148 Dissociation, and cage effect, 15 direct optical, 3-4 internal conversion, 6-7, 15 predissociation, 5-6 Dissociative changes. 404 Distribution curves, photometric data, 240-241 Distributional error, 235 Division delay, 285, 289, 293, 297, 302- 304 by alpha radiation, 307 role of nucleus and cytoplasm in, 307- 309 time course of, 302, 303 by ultraviolet radiation, 285, 289, 293, 297, 300, 302-304, 307-309 by visible light, 297 by X rays, 297, 303 DNA (see Deoxypentose nucleic acid; Deoxyribonucleic acid) Dosage curves, flattening of, 275-278 Drosophila, 249, 251-258, 267-268, 272- 273, 277-278, 404, 410 "genoide" of, 360 E Echinoderm photoreactivation, 481-482 Efficiency or quantum yield, 497 Einstein photochemical equivalence law, 1 Electric arcs, 487 Electrical stimulation, effects of, on radiation injury, 304 Electron transfer processes. 15-17 Embrj'O, abortion, induction of, 261 chimeras, 265 deficiencies, 261-263 physiological damage by ultraviolet, 252 582 RADIATION niOLOGY ICncountors. intermolfcular. in coiKicnscd JlllMSO. 1 1 Kmlospfiiii. (U'licifiifics. 25'.) '2{h'>. 270- 271. 27:{-28U ontiic. 2()2. 204. 270 fractional. 259, 2()2-2()4, 270 relation of, to doso, 27:^ 278 to wave lonpith. 2.5t) 2(11. 27(i- 280 marker K<'nes, 253. 259-204. 2t)0 mosaics. 204 iMicrf^v, absorbed, 300 erythema] eff(>ctiv(\ 490. 499, 500 excitation, deK'":>, 380 inactivation of, photochemical, 33-34 Epidermis, 490, 500, 503 hmiian, 500. .501 hyperplasia, 489 malpifihian layer, 489-494. 497-498. 501, 509. 514 melanin pigment, 488, 503. 505 mouse, 501 Equivalence law, Einstein i)liotochcini- cal, 1 Erythema, 488-490, 500 by antidromic impulses, 517 inhibition of, 499-500 after superficial burn from heat, 501 intracellular edema. 490 migration of leukocytes, 490 shari)ly limited, 517 Erythemal action, ultraviolet, 42 44, 51- 55 exposures, 51-53 AMA tolerances, 53, 54 face and eye injury, 54 first aid, 55 protection, 54, 55 wave-length function. 42. 51 factors in, 44 2907 A and 2537 A, 42. 44 Erythemal effective energy. 49(). 499, .500 Erythemal radiation, factors affecting, fog. 521 "glacier burn," 521 I'.rylhcmai radiation, of human forearm. untaniHMl volar surface. 501, .")07 rcllcction from sky, 521 "snow burn," .521 transmission spectra, 50(J Erythemal tiucshold, 191, 490, 510 dilator substance, 499 individual, 513 palm of hand. 491 scoto])ic \ision. 490 skin of torso, 491 sole of foot, 191 Escherichia roli. 347. 300-308. 370,372, 384, 380-390, 394, 399-401. 410 purineless reverse mutations, 412 radiation-resistant mutant. 300 strains, adenine-reciuiring. 413 B. 300, 374, 387, 390-391 B/r, 374, .391, 395-390. 407. 410, 412 Ti bacteriophage in, 403 Crook. 383 80G. 374 Gratia, 374 H-52, 374 histidineless, 399 K12, 390, 416 lysogenic. 420 purineless. 407—408 SD-4, 407, 410 streptomvcin-dejM'udent. 407-408. 412 streptomycin-nondependent. 408 Tennessee, 374 Texas, 383, 400 streptomycin-resistant mutations, 405 Excitations, 339 Excystment of protozoa, effects of radia- tion on, 315-317 Exponential survival curve, 368 Extinction, 200 Extinction coefficient, 4 Eyes, protection and first aid to, 54, 55 F Fast green, protein stain, 208 Feulgen-positive bodies, 403 Feulgen reaction, 208, 222, 233, 240 {See also Nucleal reaction) Filters, absorption, 144 Christiansen, spectral selectivity of, 146 transmission of, 146 focal isolation, 14t) spectral selectivity of, 147 interference, spectral .selectivity of, 145 transmission of, 145 SUBJECT INDEX 583 Filters, miscellaneous, 147 polarization intcM-fcrencc, 147 rotary disp(M\sion, 147 total internal reflection, 147 Fixation in cytochemistry, 234, 235 Fluorescence, 209n. depolarization of, 13 of diatomic molecules, 3 long-lived, 7 of molecules in condensed phase, 3, 7-9 quenching; of, 12-13, 28 self-, 13 sensitized, 13 yield of, 5 Foot and mouth virus, 340 Formation, photochemical, of HBr, 23-25 Fractionated doses, 292, 293, 313 Franck-Condon principle, 2 Free radicals, 330 Freeze-dry method, 211, 212 Frequency factor of bimolecular reac- tion, 11 Fuchsin, Schiff's reagent, 239 Fucus eggs, 394 Fungi, Aspergillus niyer, 372 terreus, 369, 391, 399, 406, 411 Chaetomium globosum, 399 Neurospora, 406, 410-411, 413, 417 conidia, 407, 413, 417 Penicillium chrysogenum. 410 notatum, 395, 399, 432, 435, 439, 441, 446 survival curves in, 438, 445, 446 Trichophyton mentagrophytes, 406, 411 yeast, Saccharomyces cerevisiae, 395, 400 Streptomyces griseus, 394 yeast cells, 382,' 394 G Gamma radiation, 355, 340 Co"", 370 effect of, on invertebrate eggs, 298 lethal, on fungi, 444 mutagenic, on fungi, 444 on protozoa, 303, 310, 320, 321 radium, 370 Gasleria, 249, 253-255, 265 induced chromosomal aberrations, 265 Genes, 374, 391, 411, 415, 416, 520 mutation (see Mutations) Germicidal action, 42 bacteria, 43 Escherichia coli, 43—53 humidity, 43, 49, 53 susceptibility to kill, 43, 48 tubercle bacilli, 75 Germicidal action, exposures, 51 conversion factors, 59 depreciation related to germicidal effectiveness, 66-67 injury, 47 intensity, and time reciprocal, 51-53 unit of, 51, 59 kill units of, 49 killing, logarithmic function, 48 lethal, lethe, 63.2 per cent, 43-49 mutation, 48 molds, new species, 83-85 susceptibility, 43-47 and ventilation, 49 fimgi, 43, 47 growth prevention, 47 mutation, 83-85 spore killing, 43, 47 logarithmic kill, 48, 49 svuilight, 50 wave-length function, 42 of longer wave length, 45, 46, 50 reactivation, 45, 50 of optimum wave length, 42 of shorter wave length, 44 tentative factors for mercury lines, 44 2537 A mercury line unique, 46, 47 Germless seeds, 272 Growth cell, 288, 290, 303, 307, 311 H Half life, 4 of phosphorescence, 7-9 Heat, sensitization to, by radiation, 317-318 Helium, 375 Hemagglutination, 349 Hemangiomas, 534 Hemoglobin, 210, 218 Herpes virus, 340 Heterokaryon method, 440 Heterokaryotic complex, 432 Higher plants, photoreactivation, 483 Histidine, 218, 336 Histone, 218, 223, 237 Hit theory, 337-343 Hormones, 505 "Hot" molecule, 6 Humidity affecting X-ray sensitivity, 447, 448 Hydrogen, 375, 380, 381 Hydrogen peroxide, 380, 381, 412-414 Inactivation, photochemical, of en- zymes, 33-34 Inactivation dose, 335 584 RADIATION BIOLOGY Tndir.'ct .'ITccts. 3:^5 337 Iii(iu('('(l polyploidy, 443 Iiuluccd prcdissociat ion. ti. 11 Infectivity, cffei-t^; of r;idi:it ion on, '2HH. 291, 319 Influenza virus, 349, 351 separation of properties of, 349 Infrared intensity, 114-117 Infrared speetrum, 115, 116 Inheritable effects of radiation on i)ro- tozoa, miscellaneous, iilO 312 mutation, 289, 309, 310, 312-314 Inheritance of differences in sensitivity to radiation, 291 Inhibition of chain reactions. 30-31 Injury substances, 295 Intensities. 5 Intercombination rule, 2 Interference ph(>nomena, 348. 350, 351 Intermittent ilhnnination. 31 Internal conversion, 6-9, 27 and dissociation, 15 and isomerization, 33-34 Invertebrates, marine, 292, 298-300, 303-305, 315. 321, 322 Iodine, 432 Ionization, 339 densities, 372 direct effect of, 416 indirect effect of, 374 quantum absorption, 415 residual effect. 415 Ionizing radiations, 335 direct effect of, 337 indirect effects of, 335-337 Isomerization, photochemical, 31-34 K K saltation, 434-436 Kanpri cancers, 552 Kappa inactivation by radiation, 311, 312 Kinetics, first-order, 372 Kinetosomes, effect of alpha radiation on, 288 Lagrange's law, 123 Lambert's law in cytochemistry, 206, 230 Latent viruses, 358-360 Lethal action (see Ciermicidal action) Lethal effects of radiation {see Cell death) Lethe, unit of kill, 43. 45, 49 l.cMikotaxine, c!i|)illary i)erme!d)ility, 518 increase in, 518 Life time of excited state, actual. 5 natural, 5 Light, inhibition of bacteriojjhage devel- oi)ment by, 470 reactivation of injured organisms by, 50 visible (see Visible light) Light sources, energy output. 12()-128 life time of, 126-128 means of excitation, arc. 121 discharge, \2i\ spark, 121 thermal, 126 power re(juirem(>nts, 126-128 source size, 12()-128 spectral distribution of emitted light, 126-128 Liverworts (Sphaerocarpus) , 249, 253, 274, 280-281 Localization, intracellular, alkaline phosphatase, 211 fixation for, 210-212 iron, 211 nucleic acid, 212-217 protein, 217-219 by ultraviolet absorption, 219-224 Localized irradiation of cells, 315 Long-lived em^rgetic states, 9-12 Luminescence, 400 Lycopersicum esculentutn (tomato), 265- 266 Lysogenic bacteria, 351, 396 induction of, 359 photoreactivation, 470 M Macronucleus, effect of radiation on, 288, 313 xMaize (Zea mai/s), 249, 253-255, 258- 268, 270, 273-277, 279-280 Maize pollen grains, 411 Malpighian layer, 535 Mastigophora, 287-289, 293, 297, 303, 316, 320-322 Mating type, effects of radiation on, 312, 316, 317 Mean lethal doses (MLD), 370, 372 37 per cent survival dose, 372 Mean life of excited state, 5 Mechanism of reaction, 1, 20-21, 25-26 photosensitized reaction. 10 and steady-state approximation, 21- 26 SUBJECT INDKX 585 Medium, effects of irradiated, 292, 294- 297, 299 Melanin, dopa, 506 reduced Icuko form, 505 Melanin formation, 505 water-extractable sulfhydryl com- pounds, 506 Melanization, 504, 506, 519 melanotactic, 518 pigment migration, 502 Mercury arc, corex D filter of, 495 intermediate-pressure, 495 low-pressure, 495, 496, 531 pyrex filter of, 495 radiant flux, 495 Mercury-arc radiation, 529, 532 Mercury lamps, amalgam, 88-90 depreciation of killing effectiveness, 66, 67 affected by starting, 66 high- and low-pressure, 55 high-pressure, 87 characteristics, 86 conversion factors, output, in- tensity, exposures, 59 depreciation, 66, 86 solarization, 88 intensity variation with distance, 61-63, 87 line intensities, 47, 86, 87 specification of ultraviolet intensity in research, 88 starting and restarting, 87 life of, affected by length of operat- ing period, 66 low-pressure, 55-57 characteristics, 56, 57 efficiency of 2537 A production, 58 line intensities, relative, 60 low intensity, inherent, 55, 58 output increase by forced operation, 58 ozone by 1849 A, 60, 65 operation of, temperature and venti- lation, 65 radiation use, 60 absorption by mercury vapor, 63, 64 air absorption negligible, 67 average intensity in space, 63 calcvdation of intensity, 58, 62 conversion factors, output, inten- sity, exposures, 59 reflector materials, 63, 64 relative line intensities, 47, 60 specification of ultraviolet intensity in research, 60, 61 variation with distance, 61, 62 Metachromasia, 215, 216 Metastable state, 7-9 Metastasis, 535 Methyl green, 216, 239 Methylcholanthrene, mutagenic effect, 432 sensitization to light bj-, 442 Mice, albino, 530-532, 535 body, well-furred parts, 536 ears, 533-534 hairless, 536 epidermis, 501 paws, 536 snout, 536 strain A, 534, 551 tail, 536 Microincineration, 211 Millon reaction, 208, 218, 219, 239-240 Mineral content of Paramecium, effect of X rays on, 293 Mitosis, inhibition of, 367 Modification of radiation effects, by electrical stimulation, 304 by starvation, 304, 308, 309, 318 by temperature, 299, 300, 304, 317, 318 by various substances, 293-296, 304, 315 by visible light, 294, 304, 306 Moisture content, 391 Monochromator, 226 grating, 152 angular dispersion of, 152 diffraction order of, 152 prism, 149 constant deviation, 150-151 construction of, 150 double monochromators, 150 fluoride, characteristics of, 149 liquid-filled quartz, 149 quartz, characteristics of, 149 transmission of, 149 Morphology, cellular, 404 colony, 404 Motility and behavior of protozoa, 286, 288, 316, 317, 320 Mucor dispersus, 437 genevensis, 433, 443 guilliermondi, 433 Multiplication, cell, 287, 288, 290, 303, 311 Multitarget theory, 369 Mutagenic effects of transmutation, 314 Mutagenic substrates, 413 Mutagens, chemical, 414 Mutants, biochemical, 404 K, 411 resistance to antibiotics, 412 586 RADIATION BIOLOGY Mutants, somatic, 542 St ropt oiny cin-resistant, 407 Mutations, 543 analysis of, 250 hioclicniical, 407 rhl(in)i)hvll, 2(U>-2C7 coincitionce of, 252, 258-259, 277 coI()r-r(\^ponsp, 410 coini)l('\ity of, 250 dominant! 258-259 cnil-|)()int, 405, 406 endosperm, 259 200, 203-264, 273- 280 function of time after exposure, 257 gene, comparison of ultraviolet and X-ray induced, 259-200, 202- 204, 266-208, 272 lethal, 253, 256, 268, 277, 280-281 dominant, 259 recessi\-e, 252, 255-250, 259-200 sex-linked, 253, 255-257, 207- 268 and nucleic acids, 281 relation of, to chromosome aberra- tions, 250, 255-268 to dose, 253, 274-278 to ultraviolet wave length, 250, 253, 260-201, 273-281 visible, 252, 255, 277 haplo-viable, 258, 260 in higher organisms, 249-281 induced, 405 by photoreactivatiou, 476-479 in invertebrates, 309 lethal, 415, 416 lethal-mutation hypothesis, 416 nature of, 250 in protozoa, 289, 309, 310, 312-314 recessive lethal, 416-418 replication of, following ultraviolet radiation, 252, 255-256 reverse, 407 somatic, 544 somatic-mutation theory, 542 spontaneous, 278-279, 402, 405 by ultraviolet, 83, 84 molds, new species, 85 sunlight, correlation, 84 zero-point, 405, 406 N Nadsonia fulvescens, 433, 443 Necrosin, 518 Nematode eggs, 290, 292 Neurospora, 251, 277 crassa, 432, 435, 437-439, 441-443 Neutrons, 335 effect on Euglena, 320 Nicoiiana (jlulinosa, 395 Ninhvdrin, 432 Nitrite, 432 Nitrogen, 375, 408 Nitrogen mustard, 432 cfTect on protozoa, 311-313 Xonspecilic light lo.ss in microscopical prejiarations, 221, 222, 230-232 Nucleal reaction, 216, 217 Nucleases as cytochemical reagents, 213, 215 Nucleic acid, 344, 516 Nuclein, 216 Nucleinic acid, 217 Nucleolus, ultraviolet ab.sorption, 221, 234, 240 Nucleoproteins, 516-517 ultraviolet absorption, 222-224 O "One-hit" processes, 34 Ophiostoina niuUiannidntuni, 432 Optical density, 206 Optical dissociation, 3-4 Organic peroxides, 413, 414 Osteochondrosarcoma, 535 Oxygen, 407, 408, 416 photochemical reactions of, 27-29 Oxygen concentration, 360, 408, 412 Oxygen tension affecting X-ray sensi- tivity in fungi, 443, 447, 448 OxyhemoglolMu, specific absorption, 208 Ozone, absorption of, 105 atmospheric, 100 Ozone formation by 1849 A, half life, 05 permissible concentration, 05 P32, virus inactivation by, 343, 345 Pa})illonia virus, 335, 340 intracellular irradiation, 358 Fandorina, 280, 287, 291, 293, 297, 320 Paramecin, 359 Paramecium, 280-289, 291-297, 300, 302-304, 307-314, 310-318, 320- 322 mineral content, effect of X rays on, 293 mutations producetl hy radioactive isotopes, 312, 314 Paramecium aurelia, 395, 410 kappa factor of, 359 Pathogenicity, 404 SUBJECT INDEX 587 Penicillium chrysogenum, 410 notatum, 395, 399, 432, 435, 439, 441, 446 Pentose nucleic acid. Beer's law, 221 intracellular distribution, 213, 217 specific ultraviolet alxsorption, 208 Permea})ility, effect of radiation on, 294, 322 Peroxides, 336 hydrogen, 380, 381, 412-414 mutagenic effects of, in fungi, 432, 437 photochemical formation of, 11-12, 28-29 role in radiation damage, 295, 296 in ultraviolet-irradiated culture me- dium, 437 Phenotypic expression, 405 Phenylalanine, 218 Phosphorescence, 7 Photocells (see Detectors, photoelectric) Photochemical effects, catalysis of oxi- dation, 65 odorous substances formed and destroyed, 65 of 2537 A and 1849 A, 65 Photochemical process, primary, 389, 492, 502 Photochemical reaction, 385, 415 effect of stirring on, 20 quantum yield, 10-11, 14, 30 in solutions, 14-15 Photochemical sensitizers (see Sensi- tizers) Photodecomposition, 414 Photodvnamic action, 37, 297, 302, 315, 317. 318, 320, 321, 399 Photoionization, 15-16 Photometer, 225, 226 Photometric analysis, errors in cyto- chemistry, 229-236 photograph}' in, 225, 226 quantitative, tissues, 236-243 technique in cytochemistry, 225-229 Photometry, heterochromatic, 134 homochromatic, 134 Photomultiplier tubes, 225 Photooxidation reactions, 27-29 Photoreactivation, 45, 50, 345, 351, 355, 394, 410, 412, 417, 420, 455-486 action spectrum, 468-469, 474-475, 481, 482 adaptive enzyme synthesis, iahibition by ultraviolet, 479 bacteria, 470-478 action spectrum, 474-475 kinetics, 473-474 survival curves, 472 Photoreactivation, chemical actions, 469, 475, 482 constant dose reduction principle, 459, 471 bacteriophages, 457-470 action spectrum, 468-469 adsorption and, 458 chemical actions, 469 kinetics, interrupted light, 465 light intensity, 464 multiple infection, 468 single infection, 461-468 temperature, 465, 466 in liquid, 458 lysogenic complexes, induction, 470 photoreactivable sector, 461 photoreactivation curve, 462 on plate, 458 survival curves, 457, 459 temperature coefficient, 465 cell-division retardation, 479, 481 dark reaction, 466 echinoderm, 481-482 cell-division retardation, 481 in fungi, 441, 478-479 higher plants, 483 hit theory, 473 kinetics, 461-468, 473-474 light intensity, 464, 473 light reaction, 466, 473 model, 466 multicomplexes, 468 mutation induction, 476-478, 479 plant viruses, 470 poison theorj', 473 postirradiation treatment, 366 protozoa, 479-481 cell-division retardation, 479 reduced vigor, 480 structure of macronucieus, 481 salamander larvae, 482 temperature, 465-466, 471 ultraviolet inactivation, 455-486 ultraviolet wave length, 479, 482 of viruses, 345, 351, 355 visible light, 304, 306 X-ray inactivation, 469, 470, 481 yeast, 479 Photoreactivation rate, 463 Photorecovery, protozoa and inverte- brate eggs, 304, 306 Photoreversal, 397 Photosensitive mutagen poison, 410 Photosensitization, 508 eosin, 508 lipstick, .508 sulfanilamide, 508 Photosphere, 95, 96 588 RADIATION BIOLOGY Photosynthesis, 37 rhysar'iuin. 320. 321 Pi(;inonted ncvi, 552 IManck constant, 497 Phmt viruses, photoreactivation, 470 Phisnionucleic acid, 213 Polar cap te( liiii(nie, 251, 258, 273, 277 Pole cells, irradiation with ultraviolet, 251-252 multiplication of, 252 mutations induced in, 252 techniques of exposure to ultra\ iolrt, 251, 255, 258 Pollen, 253, 259, 200. 204, 273-274 defective, 259-200, 204, 200 transmission of ultraviolet throuffli, 254. 270, 273-274, 279 tube chromosomes, 208-273 tube culture, 254 255 Pollen tube, limitations of culture tecli- nique, 254-255, 208 Polymerization reactions, photoclienii- cal, 30-31 Polytoma, 287, 288, 307, 320 Postirradiation treatment, increasinfi; temperature of incubation, 300, 394 photoreactivation, 300 Potential energy diagrams, 3, 7—8 Predissociation, 5 induced, 6, 14 Primarv photochemical process, 389, 492, 502 Primary steps, 1 Probability of transition, 2, 4 Proliferation, 543 Prophage, 359 Protamine, 218 Protection, of products, antibiotics, molds, 84 meat storage, aging or curing, 85 syrups, juices, 85 wine storage, 85 sunburn, 508 (See also Modification of radiation effects) Protective agents, 335 Protein synthesis, 400 Proteins, denaturation of, 33-34 staining and tests, 217-219 ultraviolet absorption, 208, 219, 222-224 Proton transfer reactions, 10-17 Protoplasmic constituents, 414 Protozoa, 285if. motility and l)ehavior, 280, 288, 316, 317, 320 mutations, 289, 309, 310, 312-314 nitrogen mustard effects, 311-313 Protozoa, parasitic, 288, 291, 319 photoreactivation, 479-481 radiation effects, inheritable, 289, 309-314 radium effects, 292, 297, 303, 310, 322 Q Quantum efficiency, 385 Quantum-hit interpretation, 387 (Quantum yield, 342 of fluorescence, 5, 10-11 of photochemical reactions, 10-11, 14, 30 (Quenching of fluorescence, 12-13, 20 R Uadiant flux, definition of, 122 Uadiant intensity, definition of, 122 Radiation, alpha (sec Alpha radiation) bactericidal effects, lethal, killing, inactivation, 30() beta (see Beta radiation) coagidation of protoplasm l)v, 286, 287 effect of, on cytoplasmic particles, 311, 312 on development, 290, 300, 303 on excystment of protozoa, 315-317 on infectivity, 288. 291. 319 inheritable, on protozoa, 289, 309- 314 lethal {see Cell death) on macronucleus, 288, 313 on mating type. 312, 310, 317 recovery from, 292, 293, 297-303 {See also Modification of radia- tion effects) on respiration, 290, 322 erythemal {see Erythemal radiation) exposure to, chronic, 303, 310, 311 gamma (see Gamma radiation) infrared, 502 ionizing, 335-337 ionizing protons, 369 mercury-arc, 529. 532 oxygen-dependent effects, 420 oxygen-independent effects, 420 sensitivity to {see Sensitivity to ra- diation) toxic substances produced by, 290 ultraviolet (see Ultraviolet radiation) X rays, 367/. copper-K, 368 40-kvp, 368 soft, 367 "Y" and "Z" ray.s, 522 SUBJECT INDEX 589 Radical reactions, 18-20 Radioactive isotopes, mutations in Pnra- tnecium produced by, 312, 314 Radium, effect of, on invertebrate eggs, 322 on protozoa, 292, 297, 303, 310. 322 Radon, effect of, on Amoeba, 294 on Pseudocentrotus eggs, 298 Rats, 530, 532 Reactivation, by light {see Photoreac- ti vat ion) of viruses, 335, 351-356 Reciprocity law, 386 Recombination of atoms and radicals, 18-19, 22 and cage effect, 15 Recovery from radiation effects. 292, 293,' 297-303, 306-308, 317 (See also Modification of radiation effects : Photoreactivation) Reflecting objectives, 226, 228 Reflectors to increase ultraviolet in- tensity, 63 Reproducibility of photometric data, tissues, 240-242 Respiration, effect of radiation on, 296, 322 Reversing layer, 97 Rhizopus nigricans, 437, 444, 445 suinus, 433, 434, 437, 442 Riboflavin, specific absorption, 208 Ribonuclease, 214, 221, 222 Ribonucleic acid (RNA), 400 {See also Pentose nucleic acid) Ribose nucleic acid (see Pentose nucleic acid) RNA (see Pentose nucleic acid) Rodents, 553 Rotating sectors, 134 Saccharomyces cerevisiae, 432-435, 437, 439, 441, 442, 444, 445, 447, 448 ellipsoideus, 443, 446, 448 Salamander larvae, photoreactivation, 482 Sarcomas, 533-534 probable cause, connective tissue, 534 muscular elements, 534 spindle-cell, 534 (See also Cancer) Scattering, ultraviolet, in tissues, 221, 222, 231 Sea urchin, 294-296, 298-305, 307 (See also genus names) Sebaceous gland tumors, 534, 535 Secondary' steps, 1, 17-20 Seeds, germless, 272 Selection rule, 2 Self-(iuenching of fluorescence, 13 Self-reproducing elements, 358 Semiquinones, photochemical formation of, 16 Sensitive cross section, 338 Sensitive volume, 338 Sensitivity to radiation, differences in, during division cycle, 289, 299-302 inheritance of individual, 291 life cycle, 291, 304, 319 species, 292, 304, 319, 320 Sensitization to heat by radiation, 317- 318 Sensitized fluorescence, 13 Sensitized reaction, 9-12, 29, 31, 34-37 Sensitizers, photochemical, atomic, 34-35 molecidar, 35-37 pigments and dyes, 9-12, 37 Serum albumen, ultraviolet absorption, 208, 223 "Single-hit" processes, 34 Skin, cadaver, 505-506 desquamation of, 488, 500 Negro, 508, 510 template, 491 vitiliginous, 508 Skin cancer, 530, 533, 535, 551 "farmer's skin," 553 Indians, 553 keratoses, 557 Negroes, 553 "peasant's skin," 553 precancerous changes, 553 "seaman's skin," 553 in vineyard workers, 530 white races, 553 Sky brightness, 114 Sodimn sulfathiazole, 392 Solutions, photochemical reactions in, 14-15 Somatic mutants, 542 Spectral quality of sunlight, 488 Spectrum, antirachitic, 555 action, 555 Balmer, 98 factors influencing, cloudiness, 555- 556 dust, 555-556 ozone, 555 smoke, 556 infrared, 114-117 maximum absorption, 494 mercury arc, 495-496 provitamin D, 551, 555 solar, 97, 98, 102, 103 standard erythemal, 499 ultraviolet, 97, 98. 103 590 KADlA'i'lON HlOLOfiY Spciiii.itozoa. 251 25:^. 2")t> 'l'^~ . 277 SpcniiMtozoids, swiiiiniiiin. 2"):^ 2") I. 274. 280-281 Sphan-orfiri>iis (livciwoits i. 2 I'.t. 2"):{, 274, 2S() 2S1 Spirostomum, 316, 320-322 Sporofioiiia, sphaor<)eari>us, 2S0 S([U;Ulioiis cell, r>X\. ryiW Steady-state approximation, 21-26 Stop wcdfics. 134 Stcradiaiicy, ilolinitioii of. 122 Sterility, ultraviolet induced, 2y>i\. 258, 277 Stirring, effect on photochemical reac- tions, 20 Stokes's law, 3 Stream birefringence. 411 Streptomyces fiaveolus, 437, 43*), 445. 446 (jriseus, 437, 441, 445 StrongylocentrolHS. 2<)!), 300, 301, 3()(i, 314, 318. 322 Sti/Ionychia. 292. 316. 321 Substrate, chemical trc-itnicnt of. 412 irradiation of, 412 Sun. chromo.sphero. 97, 98 corona, 97, 99 infrared spectrum. 114-117 photosphere. 95, 96 radio emission. 99 reversing layer. 97 spectral intensity. 102, 103. 108 ultraviolet spectrum. 97, 98. 103 X-rav emission. 100 Sunburn. 487. 508 of eyes. 488. 520 inflammation substances. 518 inHammatory processes, 489 inflammatory responses. 518 protection, 508 Sunlamps, 90-91 Sunlight, 488. 492. 522, 553, 557 absorption of ozone, 520 active hyperplasia, 550 optics of skin, 488 jjrophylactic measures, 557 Survival curves, bacteria, 472 bacteriophages, 457, 459 exponential, 368 in fungi, 437, 438, 445, 446 Target theory, 287, 288. 338-343, 370, 372. 374. 415 TCA (see Trichloroacetic acid) Temperature, effects of, on radiation injtiry, 299. 300. 304. 317. 318 on ultraviolet killing. 442 Termolecular steps. 19, 22 Thermocou|)le (see Detectors, tlicrnial) Thiamine, speeilic ab.sorption, 208 'I'hiourea, 33() Tobacco niosiac virus, 336, 340, 344, 346, 380 Tobacco necrosis virus. 340. 395 Toluidine blue, metachromasia, 215 Tomato (Lycopersicum esculentutu), 265- 2()6 Tomato bushy st\int virus, 340 Torulopsis cremoris, 445. 447, 448 Toxic substances produced l)y radiation, 290 Tradescantin, 249, 254-255, 268-274, 278. 381. 387 ultraviolet-induced aberrations in, 249. 254-255, 2()8-274. 278 Transfer of excitation. 13-14 Transmi.ssion. 205. 236 carbon dioxide. 1 16 coefficient of, 96 ozone, 116 water, 115 water vapor, 115-117 Transmutation, mutagenic effects of, 314 Tricliloroacetic acid (TCA) as cyto- chemical reagent, 214, 221, 222 Trichophyton. 276 nientnyrophytcs, 435, 437, 438, 446, 447 Triosephosphate deliydrogenase, reacti- vation, 456-457 Triplet states of complex molecules, 7, 9-12 Trypanosoma, 286, 288, 319, 320 Tryptophane, 336 specific absorption, 208, 218 Tumors, 530, 533-535, 542, 551, 552 development, 537, 548 development time of discontinuation of dose, 548 growth of, 543 progressive acceleration of, 548 induced by spectacles, 552 sebaceous gland, 534, 535 (See also Cancer) Two-hit killing curves, 369, 370 Tyrosine, specific absorption, 208, 218, 237 U Ultraviolet-irradiated culture medium, 436, 437 peroxide in, 437 Ultraviolet killing, temperature effect on, 442 SUBJECT INDEX 591 Ultraviolet potentiation, bj' cyanide, 442 by dinitrophenol, 442 by nitrogen mustard, 442 Ultraviolet radiation, absorption, 251, 253, 258, 273-274 amino acids, 184, 185 anthocyanin pigments, 193, 194 ascorbic acid [see vitamin C, below) carotenoids, 188, 189 catechins, 194-195 by cells, 250, 253, 273-274 by chitin, 252-253 coefficient of, 104 coenzymes I and II, 191, 192 cross section, 179 cytosine, 181 desoxyriljonucleotides, 187 dichroism, 172, 173, 177, 183, 230 "end", 171 b.y extranuclear components, 251, "253 "fairly clear" air, 105 flavins, 190-191 flavone pigments, 193, 194 "forbidden" transition, 170 Franck-Condon principle, 170 intracellular localization by, 219- 224 Kundt's rule, 180 molecular orbital, 173-177 nucleolus, 221, 234, 240 oxygen, 105 ozone, 105 Planck's relation, 166 in pollen grains, 258, 273 by pollen tubes, 254-255 porphyrins, 189-190 pterins, 190, 191 steroids, 186-189 tissues, apparatus, 226-229 living cells, 233 nucleic acid in, 185-187, 344, 434-436, 516 proteins, 172, 184-186, 208. 219, 222-224, 435, 436 reproducibility, liver nuclei, 240 triplet state, 168 vitamin A, specific, 208 vitamin Be (pyridoxine), 192 vitamin B12, 192 vitamin C (ascorbic acid), 192, 193, 208 vitamin E, 193 vitamin K, 193 actinic rays, 522 action spectrum, 286, 287, 290. 307- 309, 314-317, 384. 38() Ultraviolet radiation, action spectrum, for K saltants, 43() for killing of fungi, 434-436 for mutation in fungi, 434-437 activation of eggs by, 314, 315 chemical rays, 487 chromosome aberrations, 257-258, 268-273 cleavage delav by, 298, 300-302, 304, 305, 307 in combination with X rays, 272-273 cosmetic filter, 511 development time, 546-547 disinfection by (see Disinfection by ultraviolet) division delav by, 285, 289, 293, 297, 300, 302-304, 307-309 dose D, 546, 547 dose-effect curves for mutation in fungi, 438-440 dose fractionation, effects of, 257 dose-reduction ratio, 394, 395 doses genetically effective, 256, 269, 275-281 effects of, on development, 290 on fermentation, 434 genetic, 249-281 in comparison with X rays, 250, 256-260, 262-264, 266-268, 271-272 on growth of fungi, 433 inheritable, 310-312 lethal, 286, 287, 289, 292, 294, 319 microscopical preparations, 232, 233 miscellaneous, on protozoa, 316-321 on respiration in fimgi, 433, 434 experimental procedure, biological, 251-255 extreme, 396 gross chromosomal changes, induction of, 264 inactivation by, definition, 456 of enzymes, 516 of viruses, 343-345 intense flashes of, 286, 292, 320, 321 intensity of, 109-114 effect on, of haze, 108-109 of smoke, 111 reflectors to increase, 63 internal filtration, 254, 259, 274-275, 277-279 lethals, induction of, 255-257 long, 366 physiological damage l)y, 251-253, 256-257, 277 (See also Mutations) quartz transmission of, 488 i02 BADIATIOX IJTOT.OOY Ultraviolet radiation, sliort-vi.siV)le, 3GG spoctnim, 97, 98, 102, 103 .st(Mility, induction of, 25()-258. 277 stimulation by. 433 suppression of enzymatic .•ulai>t:it ion by, 441 translocations, induction of, 2')ti 257, 259-2()0, 2ti4 2()(), 2()8. 270 272 variations in sensitivity to, 292, 304 wave-length dependence for muta- tions, 250, 256-258, 2(1()-2()1, 273-281 window-glass filter, 488 Ultraviolet scattering in tissues, 221, 222, 231 Ultraviolet sources, 55-57, 86-90 mercury, 55 high-pressure, 86-90 characteristics, 86 low-iiressure, 55-58 characteristics, 56-57 metals other than mercury, 88, 89 simlamps, characteristics, 89 fluorescent, mercury, 90 Ultraviolet survival curves (see Sur- vival curves) Unimolecular steps, 19 Unto, 321, 322 Ustilago zeae, 433, 434, 437 Vacuolization, radiation-produced, 286, 321 Vascular dilation, 502 effects of histamine, 517-518 Vasodilation, 517 Viruses. 333-364 bacteriophage, 380, 405 active, 420 synthesis, 400 bacteriophage-resistant, 402 composition, 333 foot and mouth, 340 inactivation, 334-345 by deuterons. 340, 342 by ionizing radiations, 335-343 by P^^ 343, 345 quantum yield of, 342, 344, 345 rate of, 335 by ultraviolet, 343-345 by visible light, 345 inactivation dose, 335, 340 interference, 348, 350, 351 intracellular irradiation, 356-360 latent, 358-360 mutations of, 346 Viruses, nonlethal cfTects on. 340 papilloma, 335, 340, 3.58 phage svnthesis, 400 phage Tl. 103 photoreactivation of, 315, 351, 3.55 plant, crystals of, 349 ral)l)it iia))illoma, 380 reactivation, 335. 351-356 hy multiplicity, 351-356 reprothictive delay, 346 sensitive cros.s-section, 338 sensitive volume, 338 separation of properties of, 346-350 Tl l)acteriophage, 405 in E. colt B/r, 403 titration, 334 tobacco mosaic, 336, 340, 344, 346, 380 tobacco necrosis, 340, 395 tomato bushy stunt, 340 vaccinia, 340. 341 (See also Bacteriophages) Viscosity, protoplasmic. efTect of radia- tion on. 304, 322 Visible light, biochemical changes in protozoa induced by, 322 division delay by, 297 modification of radiation effects by, 294, 304. 306 photodynamic action, 297, 302, 315, 317, 318, 320, 321 photoreactivation. 304, 306 virus inactivation by. 345 w Wall effects. 20-22 Water, irradiated, effect of, 295. 296 ^ Water vapor, transmission of, 1 15-117 X X-ray survival curves in fimgi, 445 X rays, 335, 340-342, 355 absorption analvsis, 211 cleavage delav by, 298-302, 304, 305 division delay by, 297. 303 effect of intensity, 299, 300 fractionated dose, 292, 293 genetic effects of, on cell motility, 286 on cell permeability, 294 in combination with idtraviolet, 272-273 in comparison with ultraviolet. 259-260. 262-264. 266-268. 271- 272 SUBJECT INDEX 593 X rays, genetic effects of, factors alter- ing, 447 on growth of fungi, 443 indirect, 292, 294-296 inheritable, 311-313 lethal, 286-290, 292-294. 319 in fungi, 444 miscellaneous, on protozoa. 316-322 on mutation in fungi, 444 X rays, variation in sensitivity to, 291, 292, 304. 305 Zea mays (maize), 249, 253-255, 258- 268, 270, 273-277, 279-280 Zi/yorhynchus molleri, 443 prior anus, 433