Marine Biological Laboratory Received. July 30, 1952 A M SS^73 Accession No. ^. n '^otm Wiley and Sons, Inc. Liiven By ^ y Pic ■ m ! -D ; nj I m czi i r-R a nn Symposium on Radiobiology The Basic Aspects o£ Radiation Effects on Living Systems 7} S3 Symposium on Radiobiology The Basic Aspects of Radiation Effects on Living Systems OBERLIN COLLEGE June 14-18, 1950 Edited by JAMES J. NICKSON Editorial Committee P. MORRISON M. BURTON E. S. GUZMAN BARRON G. FAILLA H. M. PATT Sponsored by The NATIONAL RESEARCH COUNCIL of the NATIONAL ACADEMY OF SCIENCES JOHN WILEY & SONS, INC., NEW YORK CHAPMAN & HALL, LTD., LONDON Copyright, 1952 BY John Wiley & Sons, Inc. All Rights Reserved This book must not be reproduced in whole or in part in any form without the written permission of the publisher, except for any purpose of the United States Government. Library of Congress Catalog Card Number: 52-5046 PRINTED IN THE UNITED STATES OF AMERICA Foreword The Subcommittee on Radiobiology of the Committee on Nuclear Science of the National Research Council had, for some time, considered the need for a symposium on radiobiology. At the first meeting of the special committee, appointed to consider the nature and scope of the symposium, the need and desirability of such a symposium were very thoroughly reviewed. It was readily apparent that radiological and other societies had been conducting and were continuing to conduct meetings in which various aspects of radiobiology had definite representation. In addition to this, the Atomic Energy Commission was holding meetings on a reg-ular schedule at its various facihties in which current work and progress in radiobiology were presented. It was the consensus of the committee that additional meetings of the character outlined above would serve little purpose and could hardly augment these meetings in any substantial fashion. In this regard it was felt that the Subcommittee on Radiobiology might properly recommend to the Atomic Energy Commission, to the Radiological Societies, and to the Federated Societies for Experimental Biology a continuation of their usual conferences and programs at their regularly established meetings. The committee turned its attention in another direction and after long dehberation and examination of the various facets of the problem concluded that a symposium, if it were to be held, should concern itself with the basic aspects of the radiation effects on living cells. It was decided that the objective would be a thorough examination of the fundamental concepts that e.xist in radiobiology. Since the interaction of ionizing radiation in living matter should be considered in as orderly a manner as possible, it was further decided that the subdivisions and their order would be essentially as follows: First, a survey of the physical interaction of ionizing radiation and matter. Second, a discussion and elucidation of the chemical vi FOREWORD changes arising from the transfer of this physical energy. Third, an examination of the biochemical effects; and, finally, a discus- sion of the changes occurring in living tissue. Much attention was centered upon the manner in which radiation effects in living tissue should be surveyed. It appeared desirable to avoid numer- ous subdivisions in order to facilitate the handhng of this problem. The committee concluded, therefore, that it should first center on the simplest living unit — the cell — and then transfer directly to the complex living system. The mammalian organism, which would in almost all circumstances be our eventual target, was therefore chosen. The agenda followed closely upon these deliberations. It was proposed that the essayists would present the informa- tion available on each of these subjects in its proper background and perspective, giving full development to the subject from its basic aspects through to the most complex. The presentation of original data was to be minimized, such data being utilized only to develop and expound the main thesis. With the material pre- sented in this fashion it was felt that avenues to the solution of existing problems might be pointed up and hiatuses in our present knowledge would be more certainly delineated. Our objectives were fundamental concepts and ideas rather than isolated informa- tion which had not yet found its proper position in radiobiology. Although the symposium may not have achieved this ambitious goal, the participants established a very sound basis for further developments and, in a large measure, aided in defining the field of radiobiology. Whatever success the symposium may have achieved is due to the essayists, but special mention of their efforts is hardly neces- sary since their approbation will come from the readers who will have an opportunity to examine their collective work. It appears appropriate, however, again to extend our thanks to the foreign scientists. Dr. Walter M. Dale, Dr. George Hevesy, and Dr. Ray- mond Latarjet, who came long distances at the expenditure of considerable time and effort. The labors and unflagging zeal of the members of the special symposium committee, Drs. R. E. Zirkle, A. K. Solomon, J. J. Nickson, M. D. Kamen, H. J. Curtis, and A. M. Brues, were particularly important and contributed greatly to the organization of the symposium. The symposium program was arranged under the five subdivisions already men- FOREWORD vii tioned. The presiding chairmen for these sessions were, respec- tively, G. Failla, S. C. Lind, A. M. Brues, Karl Sax, and A. H. Dowdy, whose stimulating guidance in moderating the various phases of the program was greatly appreciated. The committee is especially indebted to its executive secretary, Dr. Harvey M. Patt, who implemented the arrangements for the symposium to the smallest detail and devoted the greater part of a year to its preparation. The hospitality of Oberlin College, made available through the good offices of Mr. Donald Love, secretary of the college, and President William Stevenson, contributed immeasurably to the success of the meeting. The symposium committee is also deeply appreciative of the efforts of Dr. Joseph G. Hamilton, Chairman of the Subcommittee on Radiobiology, of Dr. L. F. Curtiss, Chairman of the Committee on Nuclear Science, and of Dr. R. C. Gibbs, Chairman of the Division of Physical Sciences, of the National Research Council, who gave constant encouragement and smoothed the road to the final culmination at Oberlin. The symposium was honored by the attendance of Dr. Detlev W. Bronk, Chairman of the National Research Council, who addressed the assembly on the nature and scope of the Council's activities and called attention to the opportunities and responsibilities of scientists, as set forth in its charter, to serve in an advisory capacity to agencies of the government in matters pertaining to science. The subcommittee acknowledges with deep appreciation the support of the symposium by the Atomic Energy Commission and the Office of Naval Research through contracts with the National Academy of Sciences. H. L. Friedell, Chairman Special Symposium Committee January, 1952 Preface This volume reports the papers and discussions presented at the Oberhn Symposium on Radiobiology in June of 1950. As Dr. Friedell has said in the Foreword, the purpose of the meeting was to present in orderly fashion the state of knowledge of the field at that time, and further to indicate the hiatuses that exist in our knowledge, particularly in areas that seemed susceptible of in- vestigative attack. It is believed that the volume to a considerable degree fulfills the purpose set forth above. It is unfortunate that publication was delayed, but the vagaries of authors, publication committees, and the international situation were such that our deadlines were largely honored in the breach. The chairman of the publication committee wishes here to record his appreciation for the wholehearted cooperation of Drs. P. Mor- rison, M. Burton, E. S. G. Barron, G. Failla, and H. M. Patt, who were responsible for the editing of the material of the five divisions of the meeting. In addition the efforts of Dr. I. Rachwalsky and the Misses E. Tyree and E. Sobel in my office were invaluable in preparing the material for publication. J. J. NiCKSON January, 1952 Contents 1 Radiation in Living Matter: The Physical Proc- esses 1 P. Morrison 2 Secondary Electrons: Average Energy Loss per Ionization 13 U. Fano 3 Beams of High-Energy Particles 25 Robert R. Wilson 4 Neutrons and Their Special Effects: Recoil Effects 40 A. K. Solomon 5 General Statements about Chemical Reactions Induced by Ionizing Radiation 56 Robert Livingston 6 Chemical Reactions in the Gas Phase Connected WITH Ionization 70 Merrill Wallenstein, Austin L. Wahrhaftig, Henry Rosenstock, and Henry Eyring 7 On the Primary Processes in Radiation Chemistry and Biology 97 Robert L. Platzman 8 Elementary Chemical Processes in Radiobio- logical Reactions 117 Milton Burton 9 Influences of Details of Electronic Binding on Penetration Phenomena, and the Penetration OF Energetic Charged Particles through Liquid Water 139 Robert L. Platzman 10 Some Aspects of the Biochemical Effects of Ioniz- ing Radiations 177 W. M. Dale ii^tiV' xu CONTENTS 11 Ionizing Radiation and Cellular Metabolism 189 George Hevesy 12 The Effect of Ionizing Radiations on Some Systems OF Biological Importance 216 E. S. Guzman Barron 13 Some Factors Influencing Cell Radiosensitivity BY Acting at the Level of the Primary Bio- chemical Action 241 Raymond Latarjet 14 On the Localization of Radiation Effects in Molecules of Biological Importance 259 Martin D. Kamen 15 Recent Evidence on the Mechanism of Chromo- some Aberration Production by Ionizing Ra- diations 267 Norman H. Giles, Jr. 16 Physical and Chemical Factors Modifying the Sensitivity of Cells to High-Energy and Ultraviolet Radiation 285 Alexander Hollaender 17 Gene Mutations Caused by Radiation 296 H. J. Muller 18 Speculations on Cellular Actions of Radiations 333 Raymond E. Zirkle 19 The Dependence of Some Biological Effects of Radiation on the Rate of Energy Loss 357 Cornelius A. Tobias 20 The Influence of Quantity and Quality of Radia- tion on the Biologic Effect 393 Titus C. Evans 21 Some Physiological Factors Related to the Ef- fects OF Radiation in Mammals 414 Hardin B. Jones 22 Mammalian Radiation Genetics 427 W. L. Russell 23 Analysis of Mammalian Radiation Injury and Lethality 441 Austin M. Brues and George A. Sacher I LlI / . I LIBRAkY T • • T • • NT/- \. i^Ass. yj^/ Radiation in Living Matter:\^^ — ^^ The Physical Processes p. MORRISON Cornell University Ithaca, New York Radiation as a Localized Reagent The beam of radiation, whatever its type, is a source of energy in highly available form. One can estimate roughly the free energy of such a beam, taking into account both the energy it contains and its entropy, or high-degree order. For a typical x-ray beam, say 50 r per min at 1 mev, it is easy to show that one is dealing with a sample of radiation at a temperature of about 10* degrees Kelvin. A few minutes of exposure to such a beam corresponds to the introduction into the irradiated volume of free energy fully comparable to that made available by the injection of a strong reagent like nitric acid, up to a concentration of, say, 10~^ molar. It is no wonder that rather small total energies have widespread biological effects. It is likewise evident that the history of this free energy, between its introduction in such potency and its final expression in the reaction of an organism, is bound to be a long and complex story, which the 5 days of our symposium will by no means be able to detail. Examination of a typical cell, say an individual of E. coli, on the atomic scale will help fix the space-time picture of the radiation inter- action in recognizable terms. Such a cell is a wonderfully organized collection of some 10^^ atoms, mostly in the molecules H2O, of course, with many others. About 10"^ ion pairs within the cell produced by x- radiation are enough to lower by a good factor its chances of indefinitely multiplying. (See Fig. 1.) Those ions are formed not at a constant rate, but in short bursts of a hundred at a time, bunched in 10~^" sec or less, and spread out along a tortuous and branching path crossing the cell volume. With alpha particles the same effect on multiplication requires a few dozen alphas crossing the cell one by one, leaving in their roughly straight wakes similar short bursts of ionic produce, arranged in columns with nearly every molecule on the path seriously disturbed. 1 2 PHYSICAL PROCESSES IN LIVING MATTER With ultraviolet a similar effect would be produced by a steady rain of quanta, with a few million photons independently absorbed more or less uniformly in the protoplasmic molecules, excluding the water but some- what concentrated in the side chains of the nucleoproteins. It is no wonder that these very different first steps in the distribution of the available energy lead to differing mechanisms of the consequent effects; it is perhaps more remarkable that the final effects can be so similar. Doubtless this simplicity is in large part an artifact of our gross means of observation. 1000 rep 100 rep ofPoa's of Coy's 1 micron Fig. 1. The distribution of disturbed atoms immediately after irradiation of an E. coli cell with dosages indicated. Note the great difference in spatial distribution and the various delta rays, scattered electron paths, etc. The history of radiobiology has led to the emphasis on ionization as the measure of absorbed energy, since, in the x-ray domain especially, the means of measurement depended on the easy collection of ions in air. The roentgen and its definition in terms of ion pairs produced have fixed this point of view; but we shall use the roentgen, or perhaps better the so-called rep, as a measure of the density of energy absorbed. The generality of the definition is evidently helpful; to say that when 93 ergs per gram of ordinary tissue is the energy density absorbed we have 1 rep is to connect this physical factor with all the biological experience. But it must be evident that the description of the complex class of radia- tion reagents by the absorbed energy density alone is inadequate, even if we have already far extended the ion-pair definition of the radiologists. We know that qualitative differences can exist among radiations and their effects, even for identical gross energy transfer. The examples above for E. coli colony growth corresponded in energy units to about 5000 rep for the x-rays, 20,000 rep for the alphas, and roughly half a million rep if we stretch the concept to include the non-ionizing ultra- violet. The last figure is equivalent to a temperature change of about 1°. With this general picture in mind, we shall here attempt to outline the principal mechanisms for the transfer of energy from the hot beam of IONIZING RADIATIONS 3 radiation to the atomic and molecular structures of the tissue. It is hard, of course, to separate cleanly the job of this panel from that of our colleagues the chemists. Very roughly, we quit when we have given the energy to an atom or even an electron whose mean velocity is not too much greater than that of the thermal motion, and which has settled down to a recognizable state which may persist through at least a few atomic collisions. Our story is much longer to tell than to watch, I am afraid, by a factor of some 10^*. Ionizing Radiations The characteristic physical tool of the radiobiologist is actually an ionizing particle. Excluding the ultraviolet region, which from its high molecular specificity is the proper province of the photochemist, most of the effects of radiations of every kind, from x-rays to neutrons, are due to ionizing particles. This does not mean that most of the effects are due to ionization itself; quite the contrary. But the initial transfer of energy comes to most atoms through the more or less close approach of a charged particle, whose electrostatic repulsion or attraction for the nucleus itself and for the electrons of the atomic shells is the mechanism of energy transfer. The disturbed atoms are very frequently ionized, and because of its adaptability to electrical measurement it is the ionization which is for the physicist the most conspicuous effect of the passage of the particle. In the materials of interest, the electrons of the atomic shells move with rather low velocities. Even the fastest electrons in the main atoms of biological materials have energies of only a few thousand electron volts; by and large they are much slower. If the velocity of the incoming fast particle is considered (not its energy, but its velocity matters), calculations on classical mechanical lines are adequate. One simply considers the hyperbolic orbit of the motion of the incident particle in the inverse-square electrostatic force field of the atomic electron; the whole collision, which is an intimate one, more or less head- on, will be over and done before the atomic electron has had a chance to move in its slow orbital motion about the nucleus. As long as the energy transfer in collision is large compared to the energy by which the electron is bound to its atom, the effect of the atomic binding forces can be neglected safely. The incident particle may be appreciably deflected. The diagram of Fig. 1 sketches the mechanical relations in such a collision (1). Such close collisions are often called knock-on collisions. They may be handled with high accuracy. The spectrum of secondary electrons 4 PHYSICAL PROCESSES IN LIVING MATTER falls off with energy as 1/E^. The secondaries can ionize in their turn, and the total energy removed from particle motion converted by such collisions can be computed. For such encounters, one electron is like another, and the energy removed is independent of the kind of atom traversed, except in so far as the composition determines the total electron density of the medium. The space rate of energy loss depends, for knock-ons, only on the velocity — not the mass — of the incoming particle and increases as the square of its charge. If the incident particle is an alpha, or an even heavier ion, like the recoil C^* nucleus of slow neutron capture in nitrogen, the charge will not remain constant. Passage of the heavy ion through the atoms of the material can be regarded in a different frame of reference as a kind of bombardment of a stationary ion by the electrons of the matter; sometimes the not completely stripped ion will lose an electron by ionization; sometimes the ion will pick up an electron moving just in its direction, and the charge will be reduced. The process reaches a kind of slowly shifting equilibrium as the particle slows down. It is most important for slow and heavy ions, for which the simple considerations will no longer be adequate, and ionizations may proceed strongly even if the ion is on the average nearly neutral. Further to complicate the problem there are the very important collisions, often called glancing collisions, in which the ionizing particle does not approach any electron very closely. It may pass through the atom, or even many angstroms away. The distinguishing feature of these collisions is a small energy transfer, not overriding the atomic binding forces, and corresponding to a very slight deflection of the incident particle. The effect of such an undeflected and more or less remote charge sweeping by can to a good approximation be replaced by a strong, rapid, electric pulse, very like a burst of light uniformly dis- tributed in frequency. The equivalent electromagnetic radiation may excite or even ionize the atom as a whole, just as a beam of real photons would do. For this type of collision the simple mechanics of electro- static forces is entirely inadequate; the effect of light of all colors on the atom must be known. Evidently the problem is more complex and demands a full knowledge of the quantum mechanics of atomic struc- ture (2). In particular, we must recall the following: 1. The atomic electrons now move during these relatively slow and weak encounters. The accurate treatment of the whole process requires a knowledge of the electron orbits so complete that the effect has been worked out in detail only for hydrogen atoms. We have to depend upon this calculation for a guide and supplement it with observed semi- empirical regularities (3). IONIZING RADIATIONS 5 2. It is by no means obvious that collisions like the glancing ones, which are sometimes quite distant, can be treated as the direct inter- action of only two systems: the moving particle and the struck atom. Surrounding or intervening atoms may be polarized by the varying electric fields. Their distorted charge distributions will in turn affect the net force felt by the atom under consideration. Thus the energy loss will be dependent on the chemical composition, and not simply on the electron density. This effect seems rather small for overall energy loss, with a notable exception in the case of very fast particles, where the difference between gases and solids is very marked (4). 3. When quantum effects play a role, it becomes clear that the simple picture in which an electron is either removed from its bond entirely, forming an ion pair, or is simply shaken up and allowed to return to its original state without any energy gain, is not correct. In fact, the energy lost by the incident particle must eventual^ appear in one of three forms; excitation of the atoms or molecules of the material to discrete excited states; ionization into the continuum; or kinetic energy of secondaries too small to excite any more atoms. Clearly the last form will be negligible in complex material like protoplasm or even water; whereas the first form may be of great importance and may lead to irreversible chemical change, not merely to thermal motion of the stopping material. Radiation of light quanta may intervene, and is in fact to be expected for fast-moving particles. Of the relative importance of ions and excited atoms Fano will have more to say ; it is enough to observe here that for every ion pair formed one \xi\\ expect two or three excited atoms. The precise kind and number of excitations will in general depend on the stopping material, but not much on the velocity of the particle, so long as it still moves fairly fast compared to the atomic electrons. In spite of these difficulties we can give a pretty fair account of the space rate at which energy is lost from a particle in tissue. The accom- panying graph (Fig. 2) is a fair sample of the dependence on velocity and charge; we summarize (Fig. 3) [after L. H. Gray (5)] the energy loss per micron of path for typical radiation types. The energy set free on ionization may, of course, include considerable kinetic energy given to the newly freed electrons. Most of this energy appears in electrons of rather low energy, which are capable in their subsequent motion of exciting and ionizing a few more atoms them- selves. Thus very frequently the ions are produced not in single pairs but in little clusters of some two to four or five pairs with the corre- sponding excited atoms. Once such a secondary electron has received, as it occasionally will, sufficient energy- to produce more than these few PHYSICAL PROCESSES IN LIVING MATTER Incident proton M 5 = 2e^lmv^ e = 2/mv^ • e^b Fig. 2. Approximate mechanics of simple knock-on collision. The distance the struck electron is displaced during the encounter, perpendicular to the path of the incident proton, is called 5; the angle of deflection of the incident proton (considered small) is 6; the energy transfer to the initially stationary electron which moves finally with velocity Vf is AT, and E'mc = Mv'^/2 is the initial energy of the incoming proton. The distance b, often called the impact parameter, is the distance at which the inci- dent particle would pass if there were no force of attraction. Small b means good aim. Note especially how large energy transfers are associated with large angles of deflection, small impact parameters, and big displacements, 5. The faster the inci- dent particle, the greater the energy transfer for a definite deflection, but the smaller must be the impact parameter, and therefore the better the "aim." High energy transfers are evidently less likely. 200 o 100 a. 0 Electron s; Proton 1 — " Energy- for ch transfer density arged particles 1 1-10^ 1 2-10^ cm/sec 1 1 0.5 1.0 kev 1 1 0.5 1 1.0 1 1.5 2.0 mev 1 1 8 mev Fig. 3. Space rate of energy transfer, measured in kev per micron of tissue, for various charged particles as a function of energy. IONIZING RADIATIONS 7 pairs, it will form a secondary track, far from straight, tortuously branching off from the main primary track. Such recognizable secondary electron tracks are called delta rays, distinguished from the secondary ion pairs made close to the primary path by low-energy secondaries only by their length. Delta rays are responsible for about half of the energy transfer for all ionizing particles, except the slowest electrons, and extend considerably the volume affected by the concentrated ionizing forces of a heavy alpha particle. Lea [(6), esp. pp. 26 ff.] has estimated that delta rays extend the length of the alpha-ionized column by a factor of 2 or 3, that of recoil protons by 20 per cent or so, that of fast electrons very little. This means that the ions and excited atoms lie in a narrow column a few atom-diameters wide along the actual path of an ionizing particle, but out of this main more or less linear spine there come numerous short branches, producing a feathery structure, especially for the heavier particles. (See Fig. 4.) Energy-Transfer Density, kev/ju tissue Minimum ionization particles 0.22 kev/n 20-mev betatron gamma rays 0 . 28 Cobalt gamma rays 0 . 42 1-mev supervoltage x-rays 0.5 200-kev deep-therapy rays 2 . 8 X-ray region 3 . 5 12-mev protons 10.0 Cyclotron neutrons: Be + D 23.0 Thermal-neutron capture recoil ions -^100.0 Polonium alphas 150.0 U fission fragments '^4.0 mev Fig. 4. Space rate of energy transfer for a variety of types of beam. Note the wide variation possible. In the time that any such projectile crosses the cell, some 10"^"* sec, the main energy transfer takes place. Out of the atoms in the wake of the particle there then come secondaries, while molecular transitions and "free-radical" formation take place in the excited atoms. This stage takes perhaps even less time, say about the time of a few electron orbit passages. Some molecular disassociation, now already sure to occur, will not be complete for a considerably longer period, since the nuclei must move. Meanwhile the secondaries are moving out, most of them slowly, and ionizing and exciting as they go, again for such a period of time. After not more than 10~^^ sec, then, there is a feathery arrangement of excited molecules and atoms with free electrons and positive ions, none of them possessing enough energy to ionize further, 8 PHYSICAL PROCESSES IN LIVING MATTER though they may still excite complex molecules and transfer their kinetic energy in elastic collision into simple heat motion. We are almost at the point of chemistry. Loss by elastic collision would require perhaps 10~^° sec, but much molecular excitation might take place earlier. Now the free electrons will become captured by a molecule of the medium. In water one will most frequently find this capture leading to a free negative hydroxy 1 radical, setting free a neutral H atom as well Density (arbitrary units) Initial effect Angstroms from track Lateral distributions Fig. 5. Qualitative representation of the lateral distribution of disturbed atoms and molecules across an ionizing track. To the left is shown the distribution immediately- after passage of a fast electron; to the right, the distribution of diffusing positive and negative ions after an alpha particle has traversed water, about 10"^** sec earlier. Note the charge separation due to the faster motion of the secondary electrons which are captured some distance from the track. [After Lea (6).] in the reaction. What the spatial distribution of these initial products will be is a little obscure. The feathery track will be smeared out by the diffusion processes, but the caging effect of the surrounding medium and the frequently high density of ions along the track itself will combat this free diffusion by making recombination easy, and even by the overall electric field which attracts negative ions toward the generally more concentrated positive-ion core. (Figure 5 suggests the situation.) It is most doubtful that any of the quantitative treatments yet given [see (6), p. 59, and (7)] are adequate for the complex problem involved here. It can be said that the diffusion from a lightly ionizing track may spread the radicals over sizable distances, even a tenth micron or two, whereas in the alpha case it seems likely that most radicals will recom- bine before some 10~^ sec, have passed, in a distance of only a couple of ELECTROMAGNETIC RADIATION 9 hundred angstroms. Of course, the proximity of any molecular system more complex than the expected water polymer — such as a long nucleo- protein cylinder — will locally much modify this picture. But these problems belong to the chemists. Even after the initial particle or its electron secondaries have lost so much energy that they are unable to ionize at all, and their passage is invisible to the cloud chamber or the ionization-sensitive device, they will have a possibly important effect in the stopping material. Heavy particles, however, can ionize appreciably, even after they have become neutral, by capture, though eventually they slow down so far that they cannot transfer to a single electron enough energy to ionize or, finally, even to excite an atom. This limiting energy is not so small, since the maximum transferred energy depends on the velocity of the heavy particle. A carbon recoil from neutron capture in nitrogen might possibly spend a large part of its energy without making a single ion pair. This effect may be of some importance in special cases; it is apparently observed in the effects of bombardment on solid materials. The theory here is far from complete (8) . It is plain that the transfer of momentum to the stopping atoms does not always take place wholly along the direction of motion. The primary particles are deflected by these collisions. The many small- energy-transfer collisions impose on a heavy particle a successive series of angular deflections to all sides of the path. For fairly fast, heavy particles the mean square displacement angle will grow proportionally with path traversed; this will give rise to a statistical distribution of energies after a fixed straight-line segment of path. Such struggling can be important at the end of a high-energy track, as Wilson will show. Electrons and very slow, heavier particles will be scattered more drasti- cally; their paths may, in general, differ widely from a straight line. Sufficiently slow electrons will appear to diffuse from collision to collision. In tissue all electrons below some hundreds of kev will be very greatly deviated from a straight-line path. Electromagnetic Radiation The effect of electromagnetic radiation cannot, of course, be described for the whole spectrum at once. For the usual range of interest it is, however, fairly satisfactory to observe that every quantum absorbed can affect at most one primary absorbing atom. The secondary product of the absorption, an electron ordinarily, will then be set free to repeat the history of an ionizing particle in living matter. For the typical x-ray at, say, 100 kev, the ionizing events due to these electron secondaries 10 PHYSICAL PROCESSES IN LIVING MATTER are about 600 times as numerous as the atoms which directly interact with radiation. The effect of radiation here, then, is just the effect of randomlj^ originating ionizing recoil electrons. We can distinguish a few important quantum energies in the whole spectrum as marking different regimes of radiation : 1. Near 4-5 ev. This ultraviolet region is marked by strong selective absorption in specific atomic and molecular structures. Only the direct action of the quantum is important here, as indeed for lower-frequency visible light, where the biological effects of radiation are the most important of all — photosynthesis. 2. Up to 50 or 60 kev. In this region of soft and medium x-rays, the principal process of energy transfer is through the photoelectric absorp- tion of the quantum by an inner electron of some atom. All the quantum energy appears in the ejected electron, mostly as its kinetic energy, but partly in the potential energy gain of ionizing the inner atomic shell. Here specific heavy atoms may somewhat affect the probability of the process, though, of course, the photoelectrons are responsible for the great bulk of all energy transferred to the tissue. 3. Up to about 20 mev. In this domain of gamma rays, high-energy therapy machines, and the betatron, the principal transfer process is the Compton process, in which the gamma ray is absorbed by an essentially free electron, and both a recoil electron and a secondary gamma emerge. The recoil electrons have a wide range in energy but are invariably fast from the point of view of their stopping effects. Only electron density counts in this region; no specific atomic effects are to be expected. 4. Above 20 mev, up to about 100 mev. Here the formation of positron- electron pairs is more important than the Compton effect. This essen- tially means that the quantum energy is converted to that of two elec- trons, of widely distributed, very roughly equal energies. 5. Beyond 100 mev. Here the cascade region is reached. A single quantum absorbed will lead to a whole chain of new quanta and electrons, dividing the energy up among many fast electrons in the end. The spatial distribution of the energy transferred will be markedly different from that in the other regions; in general the depth dose will exceed the entry dose. On the cellular, fundamental level, of course, all these radiations above the ultraviolet should have qualitatively similar effects : those of fast secondary electrons. But much fundamental dosimetric work still remains in the high-energy field, especially with multicellular organisms. NEUTRONS AND NUCLEAR COLLISIONS 11 Neutrons and Nuclear Collisions The irradiation of tissue with neutrons provides one more example of the deviousness of the path by which energy transfer finally is made by ionizing particles. Neutrons, having no electric charge, interact only negligibly with the atomic shell electrons. They collide, then, only with the nuclei; their mean free paths between collisions are measured in centimeters rather than in angstroms, because of the great difference in size between nuclear and atomic structure. A fast neutron collides almost entirely with the H atoms of tissue, setting them into rapid motion, with any energy between the incident Eq and 0. These secondary protons — for they will be generally stripped by the shock of collision — then move through the material, ionizing as they go. This makes protons of low range important agents of biological irradiations. Inelastic collisions of neutrons with nuclei are quite frequent as well, with very fast neutrons and heavier atoms. The energy lost to the motion is con- verted to a gamma ray or even two or three. When the neutron is moving so slowly that it can no longer cause strongly ionizing recoils, it still sets free neutral atoms to make more collisions, disturbing chemical bonds. Even after it has dropped below the excitation region for molecular transitions it will still persist until it reaches thermal equi- librium. Finally it will be captured by a nucleus, in tissue generally by N^*, to yield two recoil ions, a proton with less than 1 mev and a slow, heavy recoil C^* atom. These projectiles are especially important for thermal neutron effects, where only capture gammas and capture recoil ions can transfer appreciable energy. High-energy protons or mesons also yield nuclear reactions. Some of these will be sources of multiple highly ionizing tracks. Such star events, while not major contributors to overall energy transfer, may possibly turn out to be sources of specially observable effects, because a single cell may with one event be heavily damaged. Even the products of neutron or charged-particle nuclear reactions, which in general will be radioactive nuclei, may have a role. There is some evidence that the effect of the recoil energy and chemical change subsequent to a radio- active disintegration of a bound atom of P^^ may have a biological consequence considerably more important than that of the same ioni- zation energy delivered external to the binding molecule. The possi- bilities here are notable, and suggest caution in the use of energy- absorption data alone as a predictor of biological effect for specific tracer activities. Enough has been said in this very cursory survey to demonstrate that even within the first hundredth of a microsecond or less, the physicist's 12 PHYSICAL PROCESSES IN LIVING MATTER domain, the actual energy transfer from radiation to tissue matter is not simple in nature. The discussion of such a complex physicochemical system in detail is beyond us; add the evident subtlety of the biological problem, and you will see why facile all-embracing explanations find little favor with physicists. But it is equally clear that the construction of models, like the very important and general target model, and the elaboration of such models both in concept and by experimental changes, are the only means of progress. By the steady growth and test of our oversimple ideas we will weed out of the whole picture those features which are decisive in each of the many problems which interest the radio- biologist. We know already the importance of energy density for many processes; we know the importance of diffusing products for others. It is the hope of the physics panel, as the other essayists continue to fill in the details of the physical picture I have sketched, that from this snap- shot of the events within an atomic collection workers in the other fields will be able to create a colorful and penetrating set of artistic and convincing portraits. REFERENCES 1. See, for example, the discussion of N. Bohr in the monograph on stopping prob- lems, Kgl. Danske Videnskab. Selskab, Mat.-fys. Medd., 18: 8, 1948. 2. The standard treatment is that due to H. A. Bethe, Handbuch der Physik, vol. 24/1, pp. 491 ff., Berlin, 1933. 3. Compare H. A. Bethe, Revs. Modem Phys., 22: 213, 1950, and 9: 261, 1937. 4. Bohr, A., Kgl. Danske Videnskab. Selskab, Mat.-fys. Mcdd., 24: 19, 1948. Halpern, O., and H. Hall, Phys. Rev., 73: 477, 1948. 5. Gray, L. H., Brit. J. Radiol., Supplement 1, p. 7, 1947. 6. Lea, D. E., Actions of Radiations on Living Cells, Cambridge, 1946. 7. Jaffe, G., Ann. Physik, xlii: 303, 1913. Read, J., Brit. J. Radiol, 23: 504, 1950. 8. See the discussion and paper by R. Platzman, p. 158 in this volume. 9 Secondary Electrons: Average Energy Loss per Ionization U. FANO Radiation Physics Laboratory National Bureau of Standards Washington, D.C. Following Morrison's picture of the physical action of ionizing radiations on matter, I should like to elaborate a little on some details. As a part of our general program, I shall deal with two particular topics, namely: (1) the distribution of radiation energy by secondary electrons, and (2) the factors that control the amount of ionization produced in matter, but particularly in gases, per unit amount of energy distributed by radiation. Spectrum of Secondary Electrons Secondary electrons are ejected from atoms under the impact of other fast charged particles. They are ejected with a kinetic energy that may be anywhere between zero and a certain upper limit. The upper limit is set by the conservation of momentum and energy in the collision [(1), p. 494]. If the incident particle is a heavy one (proton, alpha particle, etc.), it cannot impart to a secondary electron more than twice its own speed. Thus, for example, if the incident proton has an energy of 1 mev, the maximum energy of secondary electrons equals 2200 ev. On the contrary, if the incident particle is itself an electron, it may share any fraction of its energy with an atomic electron in the course of a collision. WTien the two energies are comparable after the collision, one usually calls "primary" the faster of the two electrons and "secondary" the slower one; this convention amounts to fixing the upper limit to the energy of the secondary electrons at one-half the energy of the primary. Even though the upper limit to the energy of the secondary electrons depends on the nature and energy of the primary particle, the energy distribution of the secondaries depends but little, on the whole, on these conditions. The reason is that the energy distribution is completely 13 14 SECONDARY ELECTRONS skew; for example, many more secondary electrons have an energy between 10 and 20 ev than between 210 and 220 ev, and still fewer have an energy between 410 and 420 ev. Therefore the location of the upper limit determines only the cut-off point of the far tail of the energy distribution. Compare Fig. 1. The shape of the energy distribution can be discussed qualitatively on the basis of the classification of the collisions of the primary particle into two classes, namely, "glancing" and "knock-on" collisions. This (electrons) Fig. 1. Number of secondary electrons per unit energy, N{E), receiving total energy E from an incident ionizing particle, plotted against E. Note that every secondary must get at least the ionization energy, /, if it is to leave the atom. The great bulk of the energy transfers occur at low energy, and thus the position of the maximum energy transfer, iS'inax, which may vary greatly with type of incoming particle, has in spite of the great variation no large effect on the distribution of secondary energies. The shaded region locates the region important for total energy loss. classification has already been explained to you by Morrison. Glancing collisions are much (about 8-10 times) more frequent than knock-on collisions in typical cases. One may wish to characterize the shape of the energy distribution by its slope n on a logarithmic plot, that is by assuming a distribution law of the type N(E) dE = dE/E'^. Now, if there were only glancing col- lisions, the slope n would be roughly 4.5. If there were only knock-on collisions, n would be equal to 2. Therefore the glancing collisions, even though by far the most frequent, are much more unlikely to produce high-energy secondaries than the knock-on collisions [(1), pp. 515^.]. Low-energy secondaries, say up to 50-100 ev, are due overwhelmingly to glancing collisions. In this energy range the slope n of the logarithmic plot of the spectrum should be of the order of 4. High-energy secondaries are due to knock-on collisions. Above 100-200 ev the slope n should approximate 2. (Figure 2 graphs these relations.) These qualitative ENERGY DISSIPATION BY SECONDARY ELECTRONS 15 theoretical predictions are confirmed by the available experimental evidence. However, this evidence is not very abundant. [ \ ^^^^ Total from all collisions V^-"Glancing" collisions ^IJE^ [ N \ \v \ ^"Knock- on" collisions ^IJE -^ 100-200 W- %s^ [ 1 1 Sv 1 log/ log£ Fig. 2. The energy distribution among secondary electrons, plotted now on a log-log scale. Note that the shaded region, which again represents the bulk of the second- aries, comes mainly from the glancing collisions. The knock-on collisions become dominant only for the infrequent but relatively energetic encounters. The slope of the skew distribution is much steeper for the glancing than for the knock-on col- lisions, as explained in the text. It would be helpful to have reliable and detailed tables of the energy distribution of secondary electrons, but such tables do not seem to be available at this time. Energy Dissipation by Secondary Electrons As we have seen, the great majority of the secondary electrons have a rather low energy, even though their aggregate energy amounts to about two-thirds of the energy lost by a fast particle. Electrons whose energy amounts to no more than 100 or 200 ev can transfer energy only to the external electrons of atoms, and this only when passing right through or very close to an atom. On the other hand, every passage in the proximity of an atom has a fair chance of leading to a collision with energy transfer. Also, low-energy electrons experience frequent, re- peated, large-angle deflections. Low-energy secondaries dissipate most of their energy within a short distance from their point of origin. This distance is of the order of 10 A in solid or liquid materials and about 1000 times as large in gases at atmospheric pressure. This energy is dealt out in the form of activations (excitations or ionizations) at points irregularly scattered in the proximity 16 SECONDARY ELECTRONS of the atoms from which each electron was ejected. These activations are said to form a "cluster" (2). Some of the activations in a cluster are produced by the secondary electron which originates the cluster, but some are produced by other electrons ejected with sufficient energy as a result of ionizing collisions within the same cluster. Electrons that have spent nearly all their energy usually wander around in a kind of diffusion path, undergoing numerous elastic col- lisions, until finally they are captured by some neutral atom or molecule to form a negative ion. Cloud-chamber pictures, which provide much of the scanty observational evidence on clusters, show the position of negative as well as of positive ions. A large proportion of the clusters appear to consist of a single pair of ions, corresponding to energy-poor secondary electrons. The larger the initial energy of a secondaiy electron, the longer and the more nearly straight is its path. The transition from cluster forma- tion to an arrangement of activations along a clear track takes place gradually, of course, as the energy increases from about 100 to about 500-1000 ev. Secondary electrons whose energy amounts to at least several hundred volts are loosely called delta rays. We can now form the following picture of the action of primary fast charged particles. The tracks of fast electrons are marked by a series of variously spaced clusters of various sizes and by a few delta rays. Occasional delta-ray tracks of unusually high energy may fork out from the main track. Heavy particles of energy up to about 10 mev undergo collisions so frequently that the clusters of activations produced by their secondary electrons merge and blend to form a sort of "column." Thus the mapping of the energy distribution by secondary electrons appears to be qualitatively understood. Nevertheless a detailed quantitative picture of this process is still missing. To produce a detailed mapping would constitute a fairly laborious task. [This spatial distribution of ions represents a quantity which is under rough control. By varying type and energy of incident particles the mean spacing can be varied over very wide limits. If the effects of individual ionizations, and accompanying excitations, turn out to be independent, the spatial size of the structures involved must be large compared to the mean ion spacing. If the biological structures are not large compared to the ion spacing, correlations will be found. The high-densit}^ columns of ionization will almost always be expected to show correlation effects. This type of analysis is, of course, greatly oversimplified. It is in particu- IONIZATION YIELD 17 lar not clear that different biological effects could not originate from ion clusters of different size, even with fixed mean spacing. In such a case, the whole correlation analysis would be washed out by the ion-cluster- size distribution. Morrison] Ionization Yield Ionization constitutes a particularly drastic form of molecular acti- vation. When an electron is ejected from an atom, the resulting separa- tion of electric charges lasts for a much longer time than the minor dis- locations of atomic electrons which accompany simple excitations. It is uncertain whether the somewhat larger energy involved in ionization processes than in excitations and the greater permanency of charge separation have a particularly great significance in relation to biological effectiveness. The separation of charges which results from ionization processes in gases affords a convenient and sensitive method for the physical measure- ment of radiation effects. It is frequently assumed, on somewhat uncertain grounds, that essentially equal amounts of ionization are pro- duced in a given amount of matter whether the matter is in gaseous, liquid, or solid state. Our information on the subject of ionization con- cerns mostly the occurrence of this phenomenon in gases. The amount of ionization produced in a gas generally serves as an index of the total energy dissipation. The main reason for this stems from the following considerations. I shall be speaking primarily about the effect of glancing collisions, but the smaller number of knock-on collisions does not modify the qualitative conclusions. Some of the glancing collisions merely raise the external electrons of an atom or molecule to an excited state ; others transfer more energy and lead to an ionization. The relative frequency of occurrence of transitions to different levels of excitation and ionization can be inferred from theoretical or experimental data on the absorption spectrum of the particular atom or molecule, since the glancing collision has the same effect as an electromagnetic radiation with a continuous spectrum of uniform intensity. Loosely attached external electrons, that is electrons with a low ionization potential, are generally apt to oscillate with comparatively low frequency and with great intensity while being raised to low excited states, whereas the opposite is true for electrons that are stiffly held. Therefore, excitations are relatively more probable than ionizations 18 SECONDARY ELECTRONS just in those atoms and molecules which require least energy to be ionized. In other words, just in those substances where an ionization can he produced cheaply, in terms of energy, a large amount of energy has to he spent in excitations. (In substances whose ionization potential has a typical average value, around 10 ev, the relative frequency of excitations and ionizations is of the order of 2 to 1.) As a result the ratio of the energy delivered to a material to the number of ionizations produced varies within remarkably narrow limits from one substance to another. This ratio is also, of course, essentially the same for different ionizing radiations, since most excitations and ionizations are produced through glancing collisions affecting the surface layers of atoms. Its numerical value for most gases is in the neighbor- hood of 30-35 ev per ionization. Because of this circumstance, the number of ionizations produced in a gas is very frequently taken as a measure of the energy spent by a radiation within it. All these considerations pertain to the action of very fast charged particles. As a particle begins to slow down, the excess of glancing collisions with respect to knock-on collisions is no longer very large. Accordingly, the ionization yield increases a little, because every knock- on collision produces an ionization, whereas glancing collisions produce excitations as well. The whole picture changes substantially when a particle slows down to and below the velocity of atomic electrons. (A heavy particle can move more slowly than an atomic electron and still have a substantial kinetic energy. This is the case, for example, for a proton of 20 kev or a nitrogen atom of 300 kev.) Then the particle becomes unable first to ionize and then to excite at all. The residual energy is presumably dissipated through bodily impacts against atoms which can no longer be easily penetrated. The energy dissipated in this manner probably escapes detection by ordinary radiation-measuring devices. Neverthe- less, it may well cause a very substantial dislocation in the structure of matter and thereby acquire a particular biological significance. The final products of ionization in a substance like water will include negative ions, like the hydrated 0H~ formed by electron capture. The subsequent chemical action of such molecules may be of high importance in biological material. REFERENCES 1. Bethe, H. A., Handbuch der Physik, vol. 24/1, Berlin, 1933. 2. Lea, D. E., Actions of Radiations on Living Cells, p. 27, New York, 1947. 3. See, for example, C. T. R. Wilson, Proc. Roy. Sac, A192: 1923. 4. Fano, U., Phys. Rev., 70: 44, 1946. DISCUSSION OF MORRISON'S AND FANO'S PAPERS 19 DISCUSSION OF MORRISON'S AND FANO'S PAPERS ZiRKLE : Is the probability of capture of electrons by the oxygen atom markedly greater than the probability of capture of the electron by water? Fano : The oxygen atom in the water molecule is the capturing agent. Latarjet : Fano stated that atoms with low ionization potential, such as hthium and cesium, are more easily excited, rather than ionized, as compared to atoms with a higher potential. Would this remark be of general value and be applicable to light atoms in the liquid or solid state, that is to living tissues? Fano : Yes. It is pointed out, however, that the elements in biological systems are all in the same bracket of ionization potential, except for the few metallic atoms. Latarjet : The ionization potential in this range varies from 9 to 20 volts. Fang : Only xenon and hehum have ionization potentials of about 9 volts. If negative ions, the probabihty of excitation is greater if excitation is to occur at all. Burton: I wish to comment on the previous two questions (by Zirkle and Latarjet) and on the rephes. It is undoubtedly true that the O2 molecule itself can capture thermal elec- trons to give negative ions and that this is an important process in the gaseous state. Threshold energy for the capture of electrons by water in the gaseous state makes the cross section of this process so high that it does not compete effectively with capture by positive ions. In the liquid, however, solvation of the 0H~ contributes much energy. The potential-energy curve is so displaced that H2O now captures an electron in a dissociative process to yield 0H~ (aq) without threshold. The cross section for such a process is, of course, lower than the cross section for capture by positive ions, but the concentration of water molecules is so overwhelmingly higher than the concentration of positive-ion species present that the process e + H20- aq H + 0H~- aq becomes very important. In regard to the question of the relative probabihty of formation of two species of positive ions with distinctly different ionization potentials, it is im- portant to remember that, although both will be formed initially, the ion of higher ionization potential transfers its charge to the other species. There are 20 SECONDARY ELECTRONS two effects. The ion species of lower ionization potential predominates, and the difference in energy becomes available for excitation. Fano: I should like to ask Burton whether he would expect the cross section to change markedly. Burton: There is competition between the positive and negative ions after they are thermaUzed. The probability of capture increases as the electron gets nearer and nearer thermal energies, but we must consider also the probability of positive-ion capture against the probabiUty of negative-ion capture. Platzman: I think that at the present time the effect of the efficiency of ionization by slowly moving positive ions cannot be predicted adequately by existing theory. It may be that such ions are ionized with greater efficiency than heretofore reaUzed. Our knowledge of the efficiency of ionization by ions whose velocity is in the region of orbital velocity or Ko of that value is most inadequate. Fano : I have discussed this matter with London, who doesn't see how this could be. Platzman : The efficiency of ionization of a gas by a heavy particle penetrating with velocity comparable to or lower than the velocities of valence electrons in atoms of the gas is a question of very considerable importance which is often dismissed inadequately or erroneously and which merits brief mention here. To say that a heavy particle of velocity Vq {vq = c/137 = velocity of the electron in the lowest orbit of the hydrogen atom), which has a most appreciable energy (25 kev for a proton, 99 kev for an alpha particle), does not ionize with good efficiency is not justified by any established knowledge and is probably also incorrect. The basis for this frequently encountered statement lies in the fact that, as the velocity of the particle decreases, for values below vq, the particle spends an increasing proportion of its time as a neutral atom; effects on atoms of the gas therefore tend to be Umited to direct encounters, in which colliding and struck atoms interpenetrate, and such collisions take on an increasingly adiabatic character as the coUision velocity falls. (Some ionization, but with extremely small yield, is known to occur even at the lowest velocities.) Thus, at very low particle velocities, the probability of excitation or ionization of the gas atom will be small, whereas the probabiUty of scattering— deflection of the particle, with resultant energy less as direct momentum transfer to the atom as a whole— will be great. At sufficiently low particle velocity, therefore, the simple "nuclear coUision" is the dominant mode of energy loss. Just how low this velocity must be is an important question. Unfortunately, the theory of pene- tration phenomena has thus far been unable to cope with the problem, at least DISCUSSION OF MORRISON'S AND FANO'S PAPERS 21 quantitatively. And, although some relevant experimental data are available, they are meager indeed and not entirely concordant. However, it does appear that W, the mean energy required to produce an ion pair, starts to rise for a particle velocity somewhat lower than vq, and thereafter increases steadily as the velocity declines (cf. the work of Madsen*). The functional dependence of W on the particle velocity, in this velocity domain, is still largely unknown. The critical velocity, namely that for which W starts to rise appreciably, appears to be considerably smaller than vq. That the overall ionization by slower ions is not as inefficient as might be anticipated if the energy loss were simply a competition between nuclear col- lisions, which lead to virtually no ionization, and familiar excitation and ion- ization, which are often assumed to correspond to the same value of W as for higher velocities (an unjustified extrapolation), might possibly arise from the extremely important contribution to the energy loss, in the velocity domain under consideration, of capture and loss of electrons by the positive ion — a proc- ess often erroneously ignored in discussions of this problem. This process might quite possibly prove to have a value of W smaller than that for excitation and ionization by high-speed particles, and thus tend to compensate to some extent for the energy lost in nuclear collisions and therefore wasted as far as ionization is concerned. These matters are also discussed briefly in a later contribution to this volume ("On the Primary Processes in Radiation Chemistry and Biology," p. 97), and a detailed study of the problem by the writer is now in progress. Morgan : My question is directed to Fano. In the case of a fast neutron (with an energy of, say, 2 mev) colhding with an oxygen atom, the most probable energy given to the oxygen atom is approximately 0.2 mev. This corresponds to the energy of an electron of about 8 ev if the electron has the same velocity as the 0.2-mev oxygen atom. In other words, a 0.2-mev oxygen atom would not be expected to produce much, if any, ionization in tissue. It is my understanding from Fano's discussion that he advises including this energy loss of such heaiy ions (to energy exchanges other than ionization) in the calculation of the maximum permissible flux for fast neutrons. Failla: It depends on the energy of the neutrons whether or not this effect is going to be an appreciable fraction of the total. I would say that for permissible limits the figures currently at hand have such a large factor of uncertainty that, in general, this would not be a very important matter. Morgan: It is true that most (or greater than 95 per cent) of the fast neutron energy is lost to hydrogen in the proton production, and from this point of ^^ew the energy *B. Madsen, Kgl. Danske Videnskab. Selskab, Mat.-fys. Medd., 23: No. 8, 1945. 22 SECONDARY ELECTRONS loss of the recoil oxygen, nitrogen, and carbon atoms of tissue is negligible. How- ever, in some cases, such as with epithermal neutrons, this may not be true. Fang : I think that in the epithermal region the secondary reactions are the most important. Failla : There may be a very narrow region in which the effect discussed by Morgan would be an important factor. I think that, in general, for the very high-energy neutrons most of the energy is transferred to the protons. For very low energies, nuclear reactions which give off radiation probably will set the limit. However, perhaps there is some narrow region in which the effect under discussion might be an important factor. Loevinger: Morrison has pointed out that ionization may well not be the main mechanism by which radiation produces a biological effect. Yet all dosage computations are based on ionization measurements in air. One uses the average energy per ion pair in air and the relative stopping power to compute energy absorbed by the tissue or organism. Thus, there is the implicit assumption that the biolog- ically important events in tissue are proportional to the ionization in air. Is, then, ionization in air to be considered a satisfactory physical quantity to meas- ure for dosage purposes, or is there hope of finding a better physical quantity to measure for these purposes? MORRISGN : As I understand it, that was a point of Fano's discussions several years ago which was re-emphasized here; it was just the special property of ionization by glancing collisions in gases that gives a good proportionality between energy lost, the rep, and its measure in the gaseous ionization chamber. I think that it does mean, however, that if we use the very useful parameter of the rep to represent the energy-density distribution for various kinds of radiation, we must expect that 1000 rep may produce very different effects in different biological systems. However, I think that the rep is a very convenient physical unit, primarily because of the excitation-ionization relationship which Fano just showed. Failla: The way in which the absolute energy is calculated by ionization measure- ments involves the total energy. Since the number of ion pairs is divided by the total energy of the particle, the average value per ion pair is for the total energy lost and not the energy to produce the ion pair alone. Thus the excitation energy and energy lost by other means are averaged into the energy associated with the production of each ion pair. DISCUSSION OF MORRISON'S AND FANO'S PAPERS 23 Burton: Ionization potentials are really different in the gas phase and in the liquid phase. It is very likely that they are lower in the latter merely because of the effect of dielectric constant — a very substantial matter in aqueous systems. Thus, statements of jdeld per ion pair based on the assumption of, for example, 32.5 ev required per ion pair are definitely wrong. I think that we may object to the usage even if we recall that calculated ion-pair yields are merely a con- vention, for the numbers we thus derive are definitely prejudicial to the theory. During the war years on the Atomic Energy Project we adopted, instead, the convention of 100-ev yield, the number of molecules converted per electron volt absorbed. The convention has the merit that ordinary 100-ev yields in simple cases without important chain and without important back reactions turn out to be of the order of unity (that is, up to 6 or some such figure). Another merit is that no one is tempted to place any inherent theoretical emphasis on a number so calculated. Aebersold: Morrison has given us a very excellent summary of the state of knowledge of the physical processes resulting in matter from ionizing radiations. I was par- ticularly interested in his remarks concerning the time scale of the sequences that follow the passage of an ionizing particle. Before World War II those of us who were interested in comparing the results of different types of ionizing radiation kept in mind the immediate physical picture, as developed by Lea and others, of the ion clusters produced by the particles. This, I gather from Morrison, is the picture at 10~^* to 10"^^ sec after passage of the particle, and that actually the more important picture for comparative purposes is the position of the affected atoms and molecules at later times, say 10~'^ to 10~^ sec. Would Morrison care to review for us the comparative picture of the state and position of the affected molecules and atoms resulting from passage of a 1-mev beta particle and a 1-mev proton at these later times? Morrison : This is a difficult subject, since, in the subsequent motion, caging, recombina- tion, and other factors are involved. Our knowledge of what occurs in the col- umns of ionization from different particles with varjang ionization densities is not adequate. We know the events up to 10"^- sec in gases. The question of the conversion of electron energy and excitation in complex molecules is not clear at the present time. Tobias: I should like to make a comment and ask a question. It was implied in the foregoing discussion that ionization of the less abundant cell constituents may be neglected on account of the small quantity of these elements. In this con- nection one should mention that an atom of an element with high atomic number will, on the average, more frequently become ionized than an atom mth low 24 SECONDARY ELECTRONS atomic number, since the electronic stopping power does not vary rapidly with Z. For example, in an atom of zinc there are 30 electrons; if all these electrons are regarded as free, the Zn atom would have about 30 times higher chance of becoming ionized than a hydrogen atom. On the other hand, one should also remember that some of the essential trace elements are located in very important spots in the cells: zinc, for example, is an essential component of some enzjones. The question I want to ask is : Are reliable estimates available for the chance of an atom becoming doubly ionized when a charged particle flies by, particularly if the latter has a high rate of energy loss (for example, a low-energy alpha particle) ? Morrison : Under these circumstances, internal conversion may be appreciable. Fang: If one considers the situation electron by electron, all take the same amount of energy, but strongly tied electrons take more energy and are less likely to be ionized. That is to say, zinc, with atomic number 31, does not have 30 times the probability of ionization as compared with the hydrogen atom. 3 Beams of High-Energy Particles ROBERT R. WILSON Cornell University Ithaca, New York Introduction Nuclear physicists, in their quest for the ultimate elementary particles of nature, have constructed larger and larger accelerating machines. Of the fruits of this research, perhaps the most important is the availability of radiologically usable beams of nearly all the known particles. Thus, by means of a cyclotron, a betatron, a synchrotron, or a linear accelerator, one can get a well-collimated beam of high-energy protons, electrons, photons, neutrons, alpha particles, and, indeed, even of nuclei such as Be or C. The use of beams of mesons, the new particles intermediate in mass between electrons and protons, may soon be practical. This sudden wealth of unexploited radiological tools should be of considerable usefulness in the research of the radiobiologist, and in this paper will be described the general characteristics of such beams. A beam of nuclear particles is characterized by its range, ionization density, and homogeneity. The range of a beam is here given in terms of the distance in centimeters that it can penetrate tissue. The ioniza- tion density is the number of ions per cubic centimeter produced on the average at a given point along the beam. To be distinguished from this is the perhaps more important concept of specific ionization, namely the number of ions per centimeter along the track of a single particle. The statistical nature of the loss of the energy of the beam introduces inhomo- geneities into the beam : inhomogeneities of energy, of direction, and of penetration. This straggling, as it is called, causes a spreading of the beam and a corresponding decrease in ionization density. Nuclear interactions between the particle and then stopping medium can also become important at high energies and also contribute to the inhomo- geneity of the beam. These effects will be discussed in more detail as each particle is taken up in turn. 25 26 BEAMS OF HIGH-ENERGY PARTICLES Photons Let me begin with photon beams. You are probably more famihar than I with the low-energj^ x-rays, that is, a few hundred kev. Such radiations can be well collimated using lead slits but are rapidly ab- sorbed in tissue. The x-rays, of course, act in the tissue when they are absorbed to form low-energy electrons, the electrons having a range of 0123456789 10 1 Water, cm Fig. 1. Isodose contours in phantom, using 16-mev electrons from betatron. much less than 1 mm. As the energy of the x-rays is raised to the order of 1 mev, the character of the absorption process begins to change. The penetration becomes greater and the electrons formed now begin to have a range in tissue of several millimeters. Additionally, these electrons can now radiate part of their energy into secondary x-rays which in turn can be absorbed farther on in the tissue, and we now begin to see an increase in the dosage with depth. As the energy is increased further, this maximum in the depth-dosage curve becomes more pronounced and its position occurs at a depth below the surface which has been found to increase almost linearly with energy: the maximum dosage comes at ELECTRONS 27 3-cm depth when 20-mev electrons are used (1). (See Fig. 1.) The peaks are quite broad, however, and the intensity falls off very slowly thereafter. At energies higher than about 20 mev the exit dose will be almost as large as that received at the maximum position. Betatrons and synchrotrons now give energies in excess of 300 mev, but there seems little advantage from a radiological point of view in using such high- energy x-rays; the penetration is far too great. At all such high energies the biological effects of the x-rays should be roughly the same, inasmuch as the specific ionization density is nearly independent of the energj^, for energies higher than 1 mev. At very high photon energies, nuclear explosions or stars are induced by the photons. In such stars, several nuclear particles are emitted, thus offering a mechanism for producing high specific ionization density in the tissue. The phenomenon is not well studied as yet, but it does not seem to be large enough to be biologically significant. Electrons A real step forward was made by Kerst and his group at Illinois when the electrons were brought directly out of the betatron. An exposure to homogeneous high-energy electrons can now be made directly, without the usual transition of the electrons inside the betatron to a degraded x-ray or bremsstrahlung spectrum and then the additional transition and further degrading of this spectrum back to electrons in the tissue. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Depth, cm Fig. 2. Depth-dose measurements with electron beam from betatron, in phantom. 28 BEAMS OF HIGH-ENERGY PARTICLES Ideally the homogeneous electron beam from the betatron travels straight into the tissue a distance equal to the electron range and then stops. Since the ionization density along an electron track is nearly constant above 1 mev, we might expect nearly constant dosage out to the end of the range and then zero dosage beyond, possibly a slight increase just at the end. This simple picture must be modified somewhat. In passing through the tissue, the electrons can radiate secondary photons having a large fraction or all of the energy of the electron. This causes a large straggling in the range of the electrons which tends to give a decreasing ionization density at increasing depths. Fur- thermore, the radiated secondary x-raj^s now pass beyond the range of the electrons and, upon absorp- tion, contribute to the dosage there. Fortunately, the second- ary photons are so penetrating that the residual dosage is very small beyond the end of the elec- tron beam. The largest modifi- cation comes about because of multiple scattering caused by many very small deflections suf- fered by the electrons as they pass through the atoms of the tissue. The many scatterings add up to very large angular de- viations, and near the end of the range the electron motion is more or less a random diffusion. There are a disadvantage and an advantage to this diffusion. It adds to the straggling in range caused by radiation and so brings the total average straggling to about 30 per cent of the mean range. This tends to reduce the ionization density deep in the tissue compared to that at the surface. The scattering also causes the beam to spread out laterally by several centimeters. Thus, if one directs a needle-like beam into the tissue, the ionization density will be very low near the end of the J L 0 1 2 3 4 5 6 7 Depth in phantom, cm Fig. 3. Depth-dose measurements with x-ray beam from betatron, at various elec- tron energies. The target-to-surface dis- tance was 45 cm in each case. PROTONS 29 range of the electrons. On the other hand, and this is the advantage, if one uses a beam of large cross section, the lateral motion will tend to increase the ionization density near the end of the range, for the scatter- ing gives the electron an oblique and hence longer path in a given incre- ment of depth. The measurements of Skaggs (2), using an 1 1-cm-diameter beam of 16-mev electrons, show that the latter effect predominates, so that a slight maximum is observed (Figs. 2 and 3) . Skaggs finds also that the extrapolated range in centimeters in H2O is about one-half the energy in mev minus 0.5 cm. A 40-mev betatron would be about right for radiological work. Nuclear disintegrations are induced by such electrons, but the number of disintegrations is far too small to be biologically significant. Protons High-energy proton beams obtainable from synchro-cyclotrons offer considerably higher precision in delivering a large dose to a small volume without overexposing neighboring tissue. Whereas the electron-specific 1400 1200 o 1000 sz S" 800 ■o ^ 600 400 200 \ jl 1 \ 1 beam *" ^k \ '1 i / / ^ s — ngie -1 \ otc "' 0 6 8 10 12 14 16 18 Depth, cm Fig. 4. Calculated depth dose due to protons. The dotted curve shows the effect of a single 140-mev proton in tissue; the full line, the estimated depth dose for a well- coUimated beam. The difference shows the effect of straggling and scatter. Repro- duced by permission from Radiology, 47: 487, 1946. ionization is nearly constant for the energies we are considering here, the proton-specific ionization decreases nearly inversely with energy. (See Fig. 4.) The reason is that the electron motion is completely relativistic, that is energy large compared to that of the rest mass (0.5 mev), whereas the proton energy (150 mev) is small compared to 30 BEAMS OF HIGH-ENERGY PARTICLES its rest mass (934 mev). Also, because of its large mass, the proton does not radiate secondary x-rays; consequently the straggling in range is much less than for an electron. Similarly, multiple scattering is small for protons. Thus a 150-mev proton has a range of 16 cm in tissue, the mean range straggling is about 0.3 cm, and the mean lateral spreading is about 0.6 cm^. Accordingly, it should be possible with 150-mev protons to give a spherical volume of 1-cm diameter located 16 cm deep in tissue several times the dosage of any of the neighboring tissue. It is a radio- logical problem whether the much higher specific ionization at the end of the proton tracks is advantageous or not. It should be emphasized that at the peak of the ionization curve the protons have a broad energy dis- tribution, the mean energy being about 20 mev. The specific ionization of such protons is only several times that of fast electrons. Thus one should not compare the radiological effects to those of recoil protons produced by neutrons, for such protons have very much smaller energies, less than 1 mev. Nuclear effects become pronounced at these energies. The proton has a considerable chance of impinging on a nucleus of one of the atoms of the tissue before coming to rest (about 30 per cent chance in going 15 cm). In that case it may go right on through, for nuclei are partially transparent at these energies; it may exchange its charge with a neutron and so become a neutron of the same energy and direction as the proton before the collision; it may be scattered; or it may be absorbed. The proton, in going past a nucleus, may also be diffracted or scattered through a small angle. Quantum mechanically, the motion of a proton is described by an associated wave, and the diffraction of this wave is is exactly like that of sound or light around an obstacle. At these energies the w^ave length of the proton is small compared to the size of the nucleus and the scattering is predominantly forward. The nuclear effects all tend to flatten the sharp maximum in ionization density that would obtain if only atomic straggling w^ere effective. Thus the protons absorbed along the way excite the nuclei to such a high state of energy that several short-ranged particles may come off, and these will add to the ionization density at the point of disintegration. If the proton exchanges into a neutron, that neutron may exchange back into a proton farther on in the tissue and so contribute ionization beyond the sharp cut-off — a small effect at energies near 150 mev. Large-angle scattering of the proton is not too important; an occasional proton leaving the beam cannot contribute much ionization elsewhere; but its absence at the end of the range decreases the Bragg peak there. Dif- fraction scattering can be the most important of the nuclear effects if one is interested in confining a dose to the smallest volume possible. PROTONS 31 With beams of large cross section, on the other hand, diffraction scat- tering produces no effect, for it is predominantly forward and is elastic so that the protons penetrate just as far, ending up with a slight lateral displacement. Even with beams of small cross section diffraction scattering is not too serious. About 10 per cent of the initial protons of a 15-cm beam are scattered out of the beam, and these are spread more or less uniforml}^ over an area of about 10 cm^, so that the density of pro- FiG. 5. Photographic plate irradiated under water by a beam of 190-mev deuterons. Note spreading at end of beam and increased ionization. tons outside the beam drops to about 1 per cent of its value in the beam. Tobias and Auger (6) have made experimental studies using 190-mev deuterons, which are similar to protons. Figure 5 shows a direct picture of the beam taken by allowing the deuteron to pass through a photo- graphic film which had been immersed in water. One can observe the spreading at the end of the beam and also the increased ionization density. Figure 6 shows quantitatively the differences in ionization density or dose characteristics among x-rays, electrons, and protons. Figure 7 is a typical isodose curve for a beam of 190- mev deuterons. There are a few things to emphasize in the use of protons. Higher energies than necessary should not be employed. It is true that the protons can be slowed down in some other material outside the tissue, 32 BEAMS OF HIGH-ENERGY PARTICLES but the additional multiple scattering introduced can become serious very rapidly. Furthermore, the products of nuclear reactions occurring in the initial stopping material can enter the tissue, thus destroying the initial homogeneity of the beam. Another necessary precaution is to make sure that the protons enter the tissue immediately on leaving the vacuum chamber. Even a thin foil can cause an appreciable multiple scattering that will diverge the beam rapidly in air. 100 80 60 40 20 \^ 16.4-mev ^ electrons / \ \ J ^ kv x-rays copper filter \^190-mev deuterons vl; X^ 10 15 Depth, cm Fig. 6. Comparison of depth-dose curves in water for various kinds of beam, all adjusted to same maximum dosage density. It is interesting to consider just how the precision of a proton beam depends upon the initial energy. Precision of the beam here means the percentage spreading and straggling. To a first approximation, there is no variation of the precision with energy or range. More accurately, nuclear scattering causes the beam to spread out a bit more at high energies — an effect already discussed — but this is offset, in part at least, by the fact that the percentage spreading and straggling decrease very slowly with initial proton energy. Thus a 150-mev proton beam has a root-mean-square straggling of 0.94 per cent, while for 10 mev straggling is 1.2 per cent. The percentage spreading varies similarly. Figure 1, showing the specific ionization of a single proton, would indicate that the ratio of the ionization density at the Bragg peak to that at the beginning of the range would be much greater for high energies. It is true that the ion density decreases just as indicated, but the ioniza- HEAVIER PARTICLES 33 tion density at the peak also decreases, for the increase in stragghng of the beam spreads the region of high specific ionization over a great volume. Actually nuclear absorption and scattering of the protons reduce the ratio obtainable at high energies. Because the protons are charged, it should be easy to pass a divergent or parallel beam through a magnetic lens and so produce a convergent beam whose point of convergence comes in the tissue at a depth equal to the proton range. This is equivalent to cross fire and should produce I I I I 5 10 15 Centimeters Fig. 7. Isodose curves for 190-mev deuterons in water. 0 E 20 similar spectacular results in reducing the dosage in the surrounding tissue. The same idea could be used with any charged particles, such as electrons. Heavier Particles Heavier particles such as deuterons and alpha particles, or even nuclei of atoms, have some advantages over protons. Multiple scattering and struggling decrease as the square root of the mass. Hence the extremes of ionization density will more closely resemble the ideal specific ioniza- tion. These extremes are even more emphasized because of the well- known specific ionization dependence on the square of the charge of the particles. The dependence is not quite as good as it first sounds, for, as the particles slow down to velocities approaching those of the atomic electrons, electrons become attached to the particle and so reduce the effective nuclear charge. Eventually the principal loss of energy comes about because of nuclear collisions, and such collisions increase the straggling. 34 BEAMS OF HIGH-ENERGY PARTICLES Cyclotrons that accelerate deuterons can also accelerate without read- justment heavier particles such as the nuclei of carbon, the energy being greater in proportion to the increase in mass. Thus, in a 200-mev deuteron cyclotron, one could readily get small beams of 12-bev carbon nuclei. Apart from the factors just mentioned, the specific ionization would be increased by more than 36 times that of the deuterons. The range, however, would be reduced from 16 cm to about 2.5 cm — still usable — and the precision better by a factor of 3. Li'^ nuclei would have an energy of 700 mev, a range of about 6 cm, and a specific ioniza- tion greater than protons by a factor of 9. Such beams should be par- ticularly valuable in precision research work on small organisms. The larger accelerators now under construction at Brookhaven National Laboratory and at Berkeley will give greater penetration for these heavy nuclei and make it possible to use even heavier atoms. The nuclear effects will be somewhat enhanced, but will probably not be large enough to make the use of such beams impractical. Mesons Figure 8 shows the track of a negative meson in a photographic plate. At the end of its path, the meson comes to rest and is absorbed by a nucleus. An energy equivalent to the meson rest mass (140 mev) is then liberated, some of which appears in the charged fragments of the nucleus seen in the typical "star" at the end of the track. Thus we have a mechanism for depositing a large amount of ionization at the end of the range of these particles. However, since their mass is nearly one- tenth that of the proton, multiple scattering is very large and may vitiate any concentration of ionization by the star. Our understanding of mesons is still developing. It seems now that, if mesons become plentiful, they may seriously compete with neutrons, but do not seem to have any advantages over, say, a high-energy alpha-particle beam. Neutrons Solomon will discuss low-energy neutrons. It is possible to make reasonably homogeneous beams of high-energy neutrons by a process called stripping, but there seems to be little radiological interest in them, for the neutrons become effective in tissue by the production of recoil protons which are now more easily and more homogeneously available directly. NEUTRONS 35 ^-->.<,^^ ^^---.^^^^ ^ r ■ i . 1 5- ■■:' ' oc < t- i' - i-- o Ui s •'i : ■ V 1 >. ^ -Q 03 w 73 CJ fl rt O ^-^ OJ CJ a o X o3 C 3 n 03 j3 T3 03 bll a < c 2 L 5 10 15 20 1 25 30 1 En, ev (21.5) (32.4) Fig. 1. Recoil spectra for varying angular correlations of electron and neutrino, for hypothetical nucleus of 100 atomic mass units with maximum beta-ray energy of 2.0 mev. [From Edwards and Davies (17).] energies for a hypothetical nucleus of mass 100, giving off a 2-mev beta particle, have been calculated by Edwards and Davies (17) and are shown in Fig. 1. The maximum recoil energy for C^^ is 6.9 ev; for P^" it reaches the sizable amount of 76.6 ev. The amount of this recoil energy {Er in electron volts) available for bond strain or rupture is given by Ei = m/{M + m)Er, where Ei is the bond strain energy in electron volts and M and m are the masses of the recoiling atom and the rest of the molecule, respectively. Thus, the energy available for bond rupture following beta decay is large except when the rest of the mole- cule has a mass small compared with that of the recoiling atom. It is not entirely clear whether orbital electrons are lost after beta emission. If they are not, the increase in nuclear charge should corre- spond to a chemical oxidation of one unit. Gest, Edwards, and Davies REFERENCES 51 (19) report that, when trivalent La^^^ decays to Ce^'*^, 60 per cent of the Ce is found in the tetravalent state; hkewise when Se^^ in Se^^Oa^ decays to Br^^, 35 per cent of the Br^^ is found as BrOs". In view of the electronic excitation of the recoil atom that is caused by the beta- decay process, these orderly oxidations seem surprising and may very well be the final state of a complex molecular reorganization. The recoil energy that follows the gamma emission accompanying isomeric transition can be calculated from the expression used for obtaining the in, y) recoil energy, and the fraction available for breaking bonds can be obtained from the expression above. In some cases the gamma-ray energy is sufficient to break chemical bonds directly, causing Szilard-Chalmers reactions as already discussed. In others, as for example Br^°, the 48.9-kev gamma ray imparts a recoil energy of no more than 0.016 ev. The percentage of the recoil energy available for bond rupture is only 1.2 per cent in the case of Br^^ in HBr, compared with 26.7 per cent for Br*° in C2H5Br. The subsequent rupture of the chemical bond must therefore be due to internal conversion of the gamma ray, with the consequent emission of electrons in the K or L shell. This process coupled with the Auger effect leads to the loss of many electrons ; for Br^°, calculations predict that 60 per cent of the recoil atoms will lose four or more electrons. Seaborg, Friedlander, and Kennedy (20) have shown experimentally that, in the case of zinc and tellurium, internal conversion is necessary for bond rupture. The gamma ray emitted in the isomeric transition of Zn*^^ is unconverted and more energetic than the gamma rays emitted in the isomeric transition of Te^^^ and Te^"^. However, the tellurium gamma rays are largely converted, and Seaborg et al. were able to observe bond rupture in radioactive tellurium diethyl, whereas they could find none in zinc diethyl observed under the same conditions. In sum, we can see that recoil effects, whether from beta decay or gamma emission, almost inevitably disrupt chemical bonds and increase the disorder of the system. In the whole spectrum of such effects re- sistance to rupture or recombination must be viewed as a rare and occasional process. REFERENCES 1. Melkonian, E., Phys. Rev., 76: 1750, 1949. 2. Engelkemeier, A. G., W. H. Hamill, M. G. Inghram, and W. F. Libby, Phijs. Rev., 75:1825, 1949. 3. Kruger, P. G., Phys. Rev., 51: 250, 1940; Proc. Natl. Acad. Sci., 26: 181, 1940. 4. Zahl, P. A., F. S. Cooper, and J. R. Dunning, Proc. Natl. Acad. Sci., 26: 589, 1940. Zahl, P. A., and F. S. Cooper, Science, 93: 64, 1941. Zahl, P. A., and F. 52 NEUTRONS AND THEIR SPECIAL EFFECTS S. Cooper, Radiology, 37: 673, 1941. Zahl, P. A., and L. L. Waters, Proc. Soc. Expt. Biol. Med., 48: 304, 1941. 5. Szilard, L., and T. A. Chalmers, Nature, 134: 462, 1934. 6. Libby, W. I., J. Am. Chem. Soc, 69: 2523, 1947. Miller, J. M., J. W. Gryder, and R. W. Dobson, /. Chem. Phijs., 18: 579, 1950. 7. Miller, J. M., and R. W. Dodson, J. Chem. Phys., 18: 865, 1950. 8. Williams, R. R., Jr., and W. H. Hamill, J. Chem. Phys., 18: 783, 1950. 9. Sue, P., and G. Kayas, /. chim. phys., 45: 188, 1948. 10. Edwards, R. R., Frances Nesbett, and A. K. Solomon, J. Am. Chem. Soc, 70: 1670, 1948. 11. Ball, E. G., Octavia Cooper, and A. K. Solomon, /. Biol. Chem., 177: 81, 1949. Ball, E. G., F. L. Rodkey, C. Davison, and A. K. Solomon, unpublished results. 12. Anderson, H. L., E. Fermi, A. Wattenberg, and G. L. Weil, Phys. Rev., 72: 16, 1947. 13. Schulz, Z. G., and M. Goldhaber, Phys. Rev., 67: 202(a), 1945. 14. Seren, L., H. N. Friedlander, and S. H. Turkel, Phys. Rev., 72: 888, 1947. 15. Rainwater, L. J., W. W. Havens, Jr., J. R. Dunning, and C. S. Wu, Phys. Rev., 73:733, 1948. 16. Brand, E., Annals N.Y. Acad. Sci., 47: 221, 1946. 17. Edwards, R. R., and T. H. Davies, Nucleonics, 2, No. 6, 1948. 18. Sherwin, C. W., Phys. Rev., 75: 1799, 1949. 19. Gest, H., R. R. Edwards, and T. H. Davies, Plutonium Project Record IXB, Chap. 3, quoted in reference 15. 20. Seaborg, G. T., G. Friedlander, and S. W. Kennedy, /. Arji. Chem. Soc, 62: 1309, 1940. GENERAL REFERENCES Bethe, H. A., Elementary Nuclear Theory, John Wiley, 1947. Edwards, R. R., and T. H. Davies, Nucleonics, 2: 44, 1948. Goldsmith, H. H., H. W. Ibser, and B. T. Feld, Revs. Modern Phys., 19: 259, 1947. Goodman, Clark (ed.), Science and Engineering of Nuclear Power, Addison- Wesley, 1947. Livingston, M. S., and H. A. Bethe, Revs. Modern Phys., 9: 245, 1937. Siri, W. E., Isotopic Tracers and Nuclear Radiations, McGraw-Hill, 1949. DISCUSSION ZiRKLE : Solomon has pointed out that, when thermal neutrons bombard average animal tissue, about eight are captured by hydrogen for each one captured by nitrogen. I should like to add a note about the relative amounts of radio- biological action due to these two capture processes. The additional factors to be considered are: (a) The relative amounts of energy carried by the ionizing agents. The gamma photon emitted by H^ (n, 7) H^ is 2 mev; the proton and the C^^ nucleus re- sulting from N^^ (w, p) C^'* share about 0.6 mev. Accordingly the hydrogen reaction predominates in energy by roughly 4 to 1. (6) The relative biological effectiveness {RBE) of the ionizing particles. This varies widely among various radiobiological actions, but, as a rough average. DISCUSSION 53 we might set the effectiveness of the protons as 4 times that of the gamma rays. Accordingly, this factor approximately cancels factor (a). (c) The relative fractions of the emitted energy absorbed by the biological object. Since the range of the proton is of the order of 10 microns, the fraction of its energy absorbed is practically 100 per cent even in very small objects. On the other hand, the fraction of gamma-ray energy absorbed depends greatly on the size of the object and is quite low even for an object 1 cm in diameter. Accordingly, the relative biological action due to the two capture reactions is the product of the relative number of neutrons absorbed (8 in H to 1 in N) times the fraction of the gamma energy absorbed in the particular biological object. This product, even for an object as large as a mouse, indicates that the N capture predominates radiobiologically over the H capture. In larger objects, of course, the H reaction increases in importance because of increase in the fraction of gamma energy absorbed. Kamen : The analysis of beta recoil processes based on the simple picture of an isolated nucleus is not adequate for complex molecules. The main difficulty is ignorance about what it is that recoils: thus, a P^^ atom in a nucleoprotein is linked in a variety of ways through oxygen bridges to organic moieties, and it is not certain whether only one or two oxygen atomSj or also a portion of the nucleotide and protein, recoils along with the residual S atom. Thus, the mass effective in the recoil energy is unknown. It must also be noted that the recoiling S atom is imbedded in an atomic matrix with innumerable degrees of freedom, so that by a collision of the second kind, or by internal conversion, a large amount of the initial recoil energy can be degraded or transferred through a large portion of the protein, involving a general excitation of the whole molecule. Another diffi- culty, namely uncertainty about the neutrino distribution, has been mentioned bj' Solomon. Perhaps an adequate analysis is not available at the present time. Never- theless, it is badly needed because, as will be pointed out in the panel on bio- chemical processes, data are available which would permit conclusions about the radiosensitivity of specific sites in biologically important molecules to be drawn, provided such an analysis were possible. Magee : Two questions have particularly bothered me in connection with "hot" atom effects following the (n, 7) process, such as are being studied in our Radiation Chemistry Laboratory at Notre Dame by Hamill and Williams. (a) Is the gamma energy given off in one quantum or several? This bears directly on the energy given the recoiling atom, since the resulting momentum of several quanta will result in partial cancellation. The assumption has ap- parently been made in Solomon's paper that the energy is given off in one quantum in all cases. 54 NEUTRONS AND THEIR SPECIAL EFFECTS (6) What is the probabihty for the conversion of a gamma ray in these proc- esses? The chemical effects resulting from this process may well be more important than the mechanical effects of the recoil. Solomon: With middle- and high-atomic-number atoms, frequeiit multiple processes yielding more than one photon are likely; with low-atomic-number atoms, a single photon is more probable. Morrison: Very recent work at Chalk River indicates that, at least with N^^, capture results in a mixture of gammas, principally 4 gammas from two cascades, all between 4 and 6 me v.* Platzman : Magee is certainly correct in calling attention to the fact that further com- plexity in the spectrum of recoil energies associated with capture of thermal neutrons is contributed by internal conversion of the capture gamma rays. There is no reason, of course, why capture gamma rays should exhibit internal conversion phenomena at all different from those of ganmia rays of any other nuclear origin. [Except for the high conversion associated with the slow radia- tion of isomers. A rather special circumstance — that capture gamma rays commonly involve the consecutive emission of several "cascade" photons from the same nucleus — will not introduce any special effect, because, just for a gamma-ray transition which has appreciable conversion coefficient, the average time elapsing before emission of the photon will be longer than that required for refilling a depleted inner electronic shell of the atom; the inner part of the atom, which is the only portion relevant for the internal conversion, reverts to normalcy after each stage of the cascade. Quite obviously, complexity of the gamma-ray spectrum, and angular correlation between some of the successive photons, will lead to a most intricate spectrum of recoil energies. Ed.] Only very little information on the spectra of capture gamma rays has at the present time been attained. The few cases which have been studied show \'ery complex capture gamma-ray spectra, apparently even for atoms of fairly low atomic weight. The only instance in which internal conversion of capture gamma rays has been studied involves bromine, f and here the effect was indeed found. This experiment is, incidentally, a difficult one. The results indicate at least 0.15-0.40 conversion electron per neutron captured, but they cannot be interpreted adequately because the spectrum of the capture gamma rays is not fully known. On general grounds, one must expect that the average conversion coefficient of all capture gamma rays for any one nucleus will in most cases be small, because the nucleus, in its transformation from the initial, highly excited state following neutron capture to its ground state, will pursue a path from one * S. Wexler and T. H. Davies, Report BNL-C-7, p. 82, 1948. t B. B. Kinsey et al, Phys. Rev., 77: 723(L), 1950. DISCUSSION 55 energy level to another involving just the fastest transitions, and these are the ones with the smallest internal conversion. Even if there should be a slow step in the sequence, this would usually have appreciable probability of conversion only if it were also a low-energy transition, so that the recoil energy even from internal conversion would be small and would influence the final spectrum of recoil energies (which almost always include the effects of some high-energy gamma rays) in only a minor way. To summarize, internal conversion of capture gamma rays is a factor which will certainly affect the distribution of recoil energies resulting from neutron capture; whether it is an importarit factor for any atom is not yet known. 5- General Statements about Chemical Reactions Induced by Ionizing Radiation ROBERT LIVINGSTON * Chemistry Department University of Minnesota Minneapolis, Minnesota Introduction Molecules, in order to react, must be activated. If a certain system reacts only very slowly, its rate of reaction will be greatly increased by a moderate increase in the temperature of the system. In such a system of thermal reactions, the energy of activation is obtained from the ordinary thermal energies of the molecules. Only that small fraction of all the molecules, which (by the Boltzmann distribution) have energies greatly in excess of the average, will be able to react. When such a slowly reacting system is illuminated with light of wave lengths which are absorbed by one or more of its components, there is a chance that the rate of the reaction will be accelerated. The energy of activation of such photochemical reactions is provided by the energy of electronic excitation of the molecules which have absorbed photons. In radiation chemistry there are two paths of activation. Some of the mole- cules are electronically excited whereas others are ionized. Both the excited molecules and the ions are capable of undergoing further reaction. As a consequence of electronic excitation, the molecules may dis- sociate directly into radicals or may undergo a process of internal con- version (1), that is, transfer of the excitation energy of the electronic system into oscillational energy of the atomic constituents of the mole- cule. Thus the molecule will oscillate like a "hot" molecule and will undergo chemical reactions in some respects like a molecule at high temperatures. These reactions may involve other molecules, or the original excited molecule may break up into radicals or stable molecules. * The author is greatly indebted to Professor James Franck for his encouragement and advice. The general outline of the paper and any new concepts which it may contain are due entirely to Professor Franck. 56 INTRODUCTION 57 The thermal reactions of these radicals and molecules will then de- termine the course of the overall reaction. Ions similarly produce radicals or molecular products, either directly or as a result of recombination. At the present time it is generally believed that the results of all radiochemical reactions (at least for non-vital systems) can be explained as the consequence of a set of reaction steps, similar in nature to those observed in ordinary photochemical and thermal reactions. Since 1936 (2), when this view was first clearly stated, there has been such rapid progress (3) in the field of radiation chemistry, especially during and subsequent to World War II, that we are sometimes inclined to over- look the important contributions which Lind and Mund made before 1936. It should be worth while, therefore, to review briefly some of these earlier results and the hypotheses which were suggested to interpret them. Bragg (4) in 1907 noticed that the number of molecules of liquid water decomposed by radon was equal to the number of ions which the same amount of radon would produce in air and referred to this relation as "a curious parallelism in numbers." Three years later LeBlanc (5) interpreted this parallelism as an analog to Faraday's law, and referred to the radiochemical decomposition of water as "electrolysis without electrodes." These ideas were extended and systematized in 1918 by Lind (6), who presented the formal or "stoichiometric" cluster hypothesis in an attempt to explain the constancy of the ion-pair yield observed for the formation of w^ater from its elements. As experimental evidence accumulated, it was found that the measured values of the ion-pair yields of the oxidation of carbon monoxide and of methane (including the sensitizing action of inert gases) , of cracking reactions of simple saturated hydrocarbons, and (possibly) of the decomposition of nitrous oxide were all consistent with the simple or formal cluster hypothesis. However, the large ion-pair yields observed in the polymerization of unsaturates (especially acetylene) demanded the assumption of large clusters, and accordingly a modified "physical" or "dynamic" cluster hypothesis was suggested by Mund (7). The results of the measurements of the decom- position of ammonia required the addition of an arbitrary (but not unreasonable) assumption to render these data compatible with the cluster theory. Finally, the experimental results for the formation of hydrogen chloride and of hydrogen bromide from their elements, as well as results for the or^/io-para-hydrogen conversion, were completely divergent from predictions based upon the simple cluster theory. Eyring, Hirschfelder, and Taylor (2) published in 1936 the first attempt to inter- pret the observed rates of radiochemical reactions in terms of ordinary reaction kinetics. Their analysis of the o/1/io-para-hydrogen conversion 58 REACTIONS INDUCED BY IONIZING RADIATION data, and the interpretation of the results on the oxidation of carbon monoxide, which was pubHshed by Hirschfelder and Taylor (8) in 1938, appear entirely satisfactory and prove, at least for these cases, that it is unnecessary to assume that there is any special or unusual character- istic of the kinetics of radiation chemistry. To sum up, information which was not available when the cluster hypothesis was first introduced by Lind is sufficient to enable us to reject the assumption that the formation of clusters plays any important role in the kinetics of radiochemical reactions. We are not justified in believing, however, that the part played by ions is necessarily a minor one. Although the historical reason for reporting the results of radio- chemical reactions in terms of ion-pair yields is certainly untenable, the use of the ion-pair yield, at least for gas reactions such as the oxidation of CO, is a convenient and reasonable one. It is, of course, for just these systems that the ion-pair yield and the energy yield (for example, the yield per 100 ev) are closely proportional to one another. It must be remembered that, when the ion-pair yield is used for reactions occurring in condensed systems, the number of ion pairs is not directly determined but is extrapolated from gas-phase measurements. The similarity between the ion-pair yield and the quantum yield of photo- chemistry should not be too heavily stressed, since it is likely to bias one's thinking about such reactions. The earlier classical work on radiation chemistry was largely a study of reactions involving simple molecules in the gas phase. Recently, because of the obvious biological implications of complex molecules, for practical reasons, and on account of the greater simplicity, in at least one sense, in interpreting the results, the tendency amongst radiation chemists has been to study chiefly reactions involving complex molecules and, frequently, condensed systems. Although undoubtedly these studies are of vital importance, it is somewhat to be regretted that the investigation of gaseous systems of simple molecules has been allowed to lie dormant. The newer viewpoints and the additional information which has been gained, partly from mass spectrographic studies, about the nature of the reactions involving simple ions should permit much more rapid and sound progress to be made in the interpretation of these simple processes. For example, the published data (6) of the water- formation reaction still exist as a challenge to any theoretical student of radiation chemistry. As Franck has stated several times in the last few years, it should also be interesting to study the products of radiochemical reactions involving simple gaseous compounds of carbon, hydrogen, and nitrogen. The mystery of the origin of complex organic material upon the earth might be solved by such experiments, since it is reasonable to EXCITATION 59 assume that the first complex organic ring structure (such as porphyrins) might have been formed in the atmosphere of the primitive earth by- brush discharges. In photochemistry it has long been recognized (9) that it is useful to separate the reaction steps which constitute the overall process into primary and secondary reactions. The primary reactions are those which the light-absorbing molecule undergoes immediately after cap- turing a photon. The secondary reactions are thermal steps involving the radicals or other products of the primary reaction steps. Very few, if any (10), chemical reactions are truly simple in nature. With few exceptions, all those which have been analyzed are the result of a com- bination of a number of consecutive and simultaneous reaction steps. Most commonly, these steps are bimolecular reactions between stable molecules, a molecule and a radical, or two radicals. Less frequently, monomolecular reaction steps, consisting of spontaneous rearrangements or dissociations, are involved. In some cases, chiefly the recombination of atoms or small radicals, reaction steps may be termolecular. Reaction steps of higher order than third probably never occur. In radiation chemistry reaction steps may involve ions. Chain reactions are of special interest. They are distinguished by high quantum, or ion-pair, yields. During the course of these reactions, some of the reaction steps, in addition to producing the final products, return to the system the radicals or atoms which were formed by the primary process. In this way a single excitation or ionization act may induce the reaction of many thousands of molecules. Excitation The following review of electronic excitation is chiefly a restate- ment of well-known classical principles. However, in a few in- stances speculations about the nature of specific processes have been introduced. If no chemical process such as delayed dissociation or rearrangement takes place, an isolated excited molecule will have a mean life of not less than 10~^ sec, and it will lose its energy of excitation by emitting a photon. Commonly this fluorescent light will have a longer wave length than that of the absorbed radiation. The longer the normal life of the excited state, the smaller will be the absorption coeflficient for the light. For instance, the direct photochemical excitation of an ordinary stable molecule (with a ground singlet state) to an excited metastable state (with a triplet state, which would have a lifetime of about 10~" sec) is so improbable that the coi-responding absorption can be detected only 60 REACTIONS INDUCED BY IONIZING RADIATION under quite special conditions. The transition probabilities for both absorption and emission are determined by selection rules and by the Franck-Condon principle. For large or even moderately complex molecules the selection rule which holds most generally is that which "forbids" transitions between states of different multiplicities. It is, for instance, the reason that a transition from a singlet to a triplet state is 10"^ times less probable than otherwise similar transitions which do not involve changes in multiplicity. The Franck-Condon principle states that during an electronic transition the positions of the heavy nuclei and their kinetic energies practically cannot change. Thus the absorption spectrum permits conclusions about the position of the nuclei at the moment of the absorption act. The heavy nuclei can, how- ever, gain potential energy by the electronic transition, and will there- fore start to oscillate if their equilibrium position in the excited state is different from that in the ground state of the electronic system. Usually excitation weakens the binding energy between nuclei and thereby enlarges their equilibrium separation. If this difference is sufficiently great, as it is for the hydrogen iodide molecule, an application of the Franck-Condon principle shows that photochemical excitation results almost exclusively in the formation of an electronically excited molecule with oscillational energy greater than its energy of dissociation. Ac- cordingly, it will dissociate after a single vibration (about 10~^^ sec) into radicals. Under these conditions the gas is, of course, non-fluorescent and photochemical reactions are very probable. A process called predissociation (1) is of importance for complex molecules, including moderately complex compounds, such as nitrogen dioxide. Whenever two electronic states of a molecule have the same nuclear configuration and total energy, there is a finite probability that a molecule which is in one of these states will "cross over" into the other. The value of this probability depends on certain selection rules and upon the time the molecule spends in the configuration which is common to both states. As a result of this process, a molecule, in an excited state in which it does not have enough oscillational energy to dissociate, may after a lapse of time cross over into a second excited state in which it is unstable. This second state either may be less stable than the first or may be a completely repulsive state. Depending upon the probability of transfer, the mean life of the excited molecule may be anything between the period of a single vibration (10~^^ sec) and the normal life of the excited state (10~^ sec). The occurrence of predissociation is marked by the appearance of photochemical action, by a decrease in the intensity of fluorescence, and by the disappearance of the rotational structure of the absorption bands. EXCITATION 61 An excited molecule may also lose energy (11) by a collision of the second kind. In such a process the excited molecule or atom gives up its electronic energy of excitation to its collision partner, and this energy may appear as electronic or oscillational energy of the second molecule or even as translational energy of the system. The amount of energy which appears as either oscillational energy or as kinetic energy of the system is small (not much greater than ^kT) ; and, therefore, the bulk of the energy has to be transferred into the electronic system of the collision partner. If, as a result, the coUision partner has an excited electronic system which is able to emit light, the process is called sensitized fluo- rescence. If, on the other hand, changes of the electronic system occur like transitions of electrons from bonding to antibonding, that is, a singlet-triplet transition causing dissociation, we speak of a sensitized photochemical process. If the atoms in the colliding molecules come into positions during the collision in which by an electronic transition an atom can switch from the first to the second molecule, such processes will occur with great probability, provided the energy originally absorbed by the first molecule is sufficient. One of the most thoroughly studied examples of this type is the interaction of a normal hydrogen molecule with an excited (6 ^P\) mercury atom, resulting in the formation of a H atom and a HgH molecule. In addition to emission (fluorescence), direct optical dissociation, simple predissociation, and collisions of the second kind, complex mole- cules may lose energy of excitation as a result of the occurrence of internal conversion (1). This process, like predissociation, depends upon the existence of two electronic levels having in common the same total energy and nuclear configuration. Since there are so many more atoms and degrees of freedom in a complex molecule, it should be expected that the time required for the excited molecule to attain the appropriate configuration will be much greater than the minimum times observed for predissociation in simpler molecules. The usual result of an act of internal conversion will be the transfer of the electronic energy of excita- tion (in all or in part) into generalized oscillational energj^ of some lower electronic state. The occurrence of internal conversion is very probably the explanation of the absence of fluorescence of many complex mole- cules, even when they are in dilute solution or the gaseous state. If the molecule were completely isolated it would, if it did not dissociate, even- tually return to its original state and emit (fluorescent) light. In practice, complete isolation is never attained, and during the long time it takes to reverse internal conversion, the molecule will lose energy by impact with others, thereby losing its ability to come back to the fluorescent state. Momentarily after occurrence of internal conversion the molecule 62 REACTIONS INDUCED BY IONIZING RADIATION is thermally activated; that is, it is a "hot" molecule. As such it can undergo chemical changes typical of thermal reactions. It may dis- sociate into two radicals or into two stable molecules. Decarboxylation is a reaction of the latter type. In giant molecules, such as proteins, it is probable that the oscillational energy would not be evenly distributed over all the degrees of freedom of the molecule, but would for a time be confined to one segment of the molecule. Although energy which is distributed over many degrees of freedom cannot break ordinary chemical bonds, it could be sufficient to break weak linkages such as hydrogen bonds. In this way internal conversion might easily be responsible for reversible denaturation of proteins. The fate of an excited molecule can be profoundly influenced by its environment. A simple molecule, such as hydrogen iodide, when dis- solved in a chemically inert solvent can be photochemically dissociated just as it is in the gas phase. However, the resultant atoms will be caged in by surrounding solvent molecules. Before they can escape from this cage they will undergo many collisions with one another. As a result there is a considerable probability that they will recombine before they can separate. This F ranch- Rabi now itch (12) effect can be responsible for a noticeable decrease in the quantum yield of a dissociation process occurring in a solution. The probability of escape from the cage is greater if the atoms are small and if they are initially endowed with high kinetic energy. When an excited complex molecule in a solution undergoes an act of internal conversion, the probability of its losing its oscillational energy is greatly increased, since it is constantly in a state of multiple collision with the solvent molecules. Should the excited complex molecule be dissociated, its radicals will be hemmed in by the solvent cage. The recombination of complex radicals frequently requires some energy of activation. Furthermore, complex radicals must be in a definite orienta- tion relative to one another before they can recombine. These steric and energetic requirements greatly reduce the rate of recombination of complex radicals and probably more than counterbalance their decreased rate of escape due to their size. It should be expected, therefore, that the cage effect will be less efficient in preventing the separation of complex radicals than of atoms or simple radicals. The dominant factor influencing the decomposition of excited complex molecules in a con- densed system is most likely the rapid removal of their oscillational energy after the act of internal conversion. Excitation energy may migrate through many molecules in a crystal in which the binding forces are strong and in which a very good resonance exists between the neighboring fundamental units of the crystal. This EXCITATION 63 process of exciton migration probably plays an important role in ionic crystals, but is of less importance in crystals made up of organic mole- cules. Crystals in which a strong exciton migration occurs have absorp- tion spectra which differ typically from the spectra (13) of the compound in the gas phase. The normal molecular spectrum will be replaced by a much narrower, strong absorption region. This criterion leads to the conclusion (14) that transfer of energy of excitation by exciton migration is important in the micelles of cyanine dyes but is of little consequence in crystals like naphthalene. The well-known fact (14) that energy given to the lattice of naphthalene by absorption of radiation is largely re-emitted by naphthacene (present as a trace impurity) has probably little to do with exciton migration. It may be better explained by the process of sensitized fluorescence or, to use a more general term, "classical resonance" (15). This process allows transfer of excitation energy be- tween molecules separated by distances which are great relative to their collision diameters. It has been discussed chiefly in connection with the self-quenching and the depolarization of the fluorescence of solutions of dyes. Its occurrence is most probable when the emission spectrum of the excited molecule overlaps the absorption spectrum of the receiver. However, there is a finite probability of its happening even when the excited molecule is non-fluorescent. It appears to be of importance (14) for intramolecular, as well as for intermolecular, transfer of excitation in complex molecules. From this viewpoint of radiation chemistry, the most important mode of excitation is the impact of charged particles upon molecules. The specific characteristics of the excitation by the several charged and uncharged particles have been discussed by the physics panel. Direct excitation by impact is in many respects similar to photoexcitation. The Franck-Condon principle still applies, but some of the selection rules are different. The direct transition from a ground singlet state to a repulsive triplet level due to the absorption of a photon is forbidden, but the corresponding transition may be produced by some types of impacts of charged particles. Impact excitation can produce this type of direct dissociation in addition to the other methods of dissociation which are also produced by the absorption of a photon. In some cases the impact excitation of the molecule may be followed by light emission together with a dissociation process. The continuous emission spectrum of a hydrogen arc is an example of this kind (16). The hydrogen mole- cule, normally in a singlet state, is raised to an excited stable triplet state by electric impact and then falls to a lower repulsive triplet state, emitting a photon and dissociating into atoms. Either predissociation or internal conversion may follow excitation by impact just as it follows 64 REACTIONS INDUCED BY IONIZING RADIATION photoexcitation. Indeed, internal conversion appears to be Of dominant importance in determining the course of the radiation chemistry of complex molecules (17). Excited molecules may also be formed by the recombination of ion pairs. Since the energy of ionization exceeds that required for the dis- sociation of a chemical bond, it should be expected in most cases that dissociation will follow the recombination of an ion pair. The behavior of certain complex molecules (18), particularly aromatic hydrocarbons, is an interesting exception to this general rule. A positive ion may be neutralized by either a negative ion or an electron. There are probably no important restrictions upon the recombination of positive and nega- tive ions, in either condensed or gaseous systems. The capture by an isolated simple molecule of an electron accompanied by the emission of a photon is a very inefficient process (19). However, in gases even at relatively low pressures (say, 50 mm), the system, ion and electron, is on the average so coupled with neighboring molecules that recombination to a highly excited state of the molecule should occur with a high yield by what is effectively a triple collision. Ionization As has been discussed by the physics panel, molecules can be ionized by impact (3) with charged particles such as alpha particles, beta par- ticles, protons, and electrons. Molecules are also ionized by interaction with high-energy photons, x-rays, or gamma rays. High-velocity neu- trons also indirectly induce ionization. In addition to impact ionization, a molecule containing high enough excitation energy may spontaneously ionize by a process (20) analogous to predissociation. This phenomenon is called preionization when it involves the valence electrons and the Auger effect when it is in the x-ray region. Molecules may also be ionized by thermal impact with an excited molecule or atom (for example, 2 ^*S He), provided the energy of excitation is greater than the energy of ionization of the molecule concerned. Processes such as the combination of two excited atoms (for example, 6 ^Pi Hg) to form an ionized molecule (Hg2'^) and an electron have also been observed. A wide variety of ionized molecules can be produced from a single compound (21) by electron impact if the energy of the electron is sufficient. As has been shown by mass spectrographic studies, single ionization of the original molecule is usually the most probable process at reasonably low electron energies. However, multiple ionization, as IONIZATION 65 well as ionization accompanied by dissociation of the molecule, also occurs in a variety of ways. Some ions are metastable and dissociate spontaneously after a short lapse of time. Negative ions (22) can be formed by the capture of an electron by a neutral molecule or atom. Whereas most atoms and radicals have positive electron affinities and can therefore form stable negative ions, many simple molecules (especially those which have a S ground state) cannot form stable negative ions. Other molecules, particularly those which are strongly polarizable or have permanent dipole moments, form stable negative ions but usually have small electron affinities. In addition to the simple capture of an electron, negative ions may be formed by capture accompanied by dissociation. For example, a hydrogen bromide molecule can interact with a slow electron to yield a hydrogen atom and a bromine negative ion. Ionization may migrate through a system either by simple exchange of charge between molecules or because of the conductivity of the medium. Measurements of the effect of traces of impurities upon the mobility of positive ions in gases (23) (such as helium) demonstrate that the exchange of charge between unlike molecules occurs effectively at ordinary pressure. Lind's demonstration (6, 24) that radiochemical reactions can be sensitized by inert gases is additional independent evidence that the exchange of ionization takes place readily, at least under the conditions of the experiments. A similar exchange of charge between a negative ion and a neutral molecule is to be expected. Failure to take the exchange of ionization into account is likely to invalidate any analysis (25) of the kinetics of radiochemical reactions. In solution, particularly aqueous, the solvation of the ions may influence profoundly the rate of exchange. Fortunately the rates of such solution reactions involving electrolytic ions are subject to direct study. Migration of either positive or negative charges in a crystalline solid (26) may occur readily by electron exchange or by electron migration in the conductivity bands of the crystal. Although such conductivity is observed most readily in ionic crystals, it should also occur in atomic lattices (like diamonds) and in molecular lattices. However, in crystals made up from organic molecules there will be httle electron migration on account of the frequent disturbances by thermal vibrations of the lattice and the molecules. Electron conductivity is also to be expected in liquids such as water, but here it should be limited to quite short dis- tances corresponding to the short range order of liquids. Reactions between ions and molecules are of importance in radiation chemistry. Except for the existence of the charge on one of the reactants, these reactions are in every way similar to the kinetic reaction steps of 66 REACTIONS INDUCED BY IONIZING RADIATION ordinary reactions. The following three equations (8, 25) are possible examples of such reactions. The third of these may require some energy of activation. Br2+ + H -^ HBr+ + HBr H2+ + Bra -^ H + Br+ + HBr C0+ + CO -^ C+ + CO2 Similar processes undoubtedly occur in solution, but here their energies are greatly affected by the solvation of ions. Solvent Effects in Aqueous Solution Radiation chemistry of aqueous solutions differs in many important respects from the corresponding chemistry of gaseous systems. The energy of solvation greatly changes the energy of ionic reactions in aqueous solutions. For example, the heat of recombination of hydrogen and hydroxyl ions in a gas is about 350 kcal per mole. For the same reaction of the solvated ions in aqueous solution, the heat is about 14 kcal per mole. Ions which are formed by impact in a solution will ordinarily exist long enough to come into equilibrium with the sur- rounding solvent molecules, before they can diffuse together and neutral- ize one another. For this reason they have some of the properties of ordinary electrolytic ions. The following two equations (27) represent the reactions which largely determine the chemical characteristics of irradiated aqueous solutions. H2O -}- H2O+ -^ H3O+ + OH H2O + e- -^ OH" + H The resulting H and OH radicals react readily wuth oxidizing and reducing agents, respectively. However, in the absence of such reagents, recombination of the radicals greatly reduces the ion-pair yield. Hydro- gen peroxide can be formed by combination of two hydroxyl radicals or, more efficiently, if dissolved oxygen is present, by a reaction between a hydrogen atom and an oxygen molecule to form the perhydroxyl radical. Since hydrogen peroxide is a relatively stable but reactive oxidizing agent, its presence undoubtedly influences the properties of irradiated water. However, the primary radicals, H, OH, and HO2 are very probably of greater importance. REFERENCES 67 It is convenient and informative to consider hydrated ions as complex molecules. As Franck has pointed out (28), the photochemical and radiochemical properties of such ions are to a large extent determined by the acts of internal conversion which they can undergo. For example, when a hydrated ferrous ion absorbs a photon, an electron is ejected into the shell of water molecules which surround the central ion. The resulting electronically excited complex molecule can undergo a process of internal conversion and so produce a complex molecule with a large amount of oscillational energy. It may lose this energy as heat to the solvent or, less probably, eject a hydrogen atom leaving a stable hydroxy ferric complex ion. As may be predicted on the basis of this mechanism, the absorption of more energetic photons increases the probability of the escape of the hydrogen atom. High concentrations of oxidizing agents also favor the production of the ferric ion. It is an interesting fact, and one which is consistent with a more detailed analysis of this reaction, that the quantum yield of oxygen production due to the illumination of a solution containing ferric ions is less than 10~^, but in the presence of certain reducing agents the photochemical reduction of ferric ion approaches unity. When an alpha particle is absorbed in liquid water a concentrated column of ions is formed. Momentarily, the core of this column consists of positive ions surrounded by a cylindrical shell of negative ions pro- duced by electrons which were ejected with relatively high velocities. The localized concentrations of hydronium and hydroxyl ions which are so produced exceed the concentrations of the same ions which are obtainable in even concentrated basic or acidic solutions. Franck has recently suggested the interesting idea that these short-lived regions containing high concentrations of hydroxyl or hydronium ions could well be responsible for the denaturation of a protein molecule or the breaking of a chromosome chain which suffers a near miss by an alpha particle. REFERENCES 1. Franck, J., and H. Sponer, Contribution a Vetude de la structure moleculaire, p. 169, Liege, 1948. 2. Eyring, H., J. Hirschfelder, and H. S. Taylor, /. Chem. Phys., 4: -179, 1936. 3. Burton, M., /. Phys. Colloid Chem., 51: 611, 1947. 4. Bragg, W. H., Phil. Mag., 13: 333, 1907. 5. LeBlanc, M., Z. physik. Chem., 85: 511, 1913. 6. Lind, S. C, The Chemical Effects of Alpha Particles and Electrons, Chemical Catalog Co., 1928. 7. Mund, W., Ann. soc. sci. Bruxelles, B51, 1931. 68 REACTIONS INDUCED BY IONIZING RADIATION 8. Hirschfelder, J., and H. S. Taylor, /. Chem. Phys., 6: 783, 1938. 9. Noyes, W. A., and P. A. Leighton, The Photochemistry of Gases, pp. 153-155, Reinhold, 1941. 10. Semenoff, N., Chemical Kinetics and Chain Reactions, p. 5, Oxford, 1939. 11. Herzberg, G., Atomic Spectra and Atomic Structure, Prentice-Hall, 1937. 12. Franck, J., and E. Rabinowitch, Trans. Faraday Soc, 30: 120, 1934. 13. Jelly, E. E., Nature, 138: 1009, 1936. Scheibe, G., Z. angew. Chem., 50: 51, 1937. Mattoon, R., /. Chem. Phys., 12: 268, 1944. 14. Franck, J., and R. Livingston, Revs. Modern Phys., 21: 505, 1949. 15. Perrin, F., Compt. rend., 180:581, 1925. Pringsheim, P., and I. Wavilow, Z. Physik, 37: 705, 1926. Forster, T., Ann. Physik, 2: 55, 1948. 16. Herzberg, G., Molecular Spectra and Molecular Structure, pp. 425-426, Prentice- Hall, 1939. 17. Burton, M., /. Phys. Colloid Chem., 52: 810, 1948. 18. Burton, M., J. Phys. Colloid Chem., 51: 786, 1947. 19. Magee, J., and M. Burton, J. Am. Chem. Soc, 72: 1965, 1950. 20. Reference 11, pp. 167, 173. 21. Hustrulid, A., P. Kusch, and J. T. Tate, Phijs. Rev., 54: 1037, 1938. 22. Glockler, G., and S. C. Lind, The Electrochemistry of Gases and Other Dielectrics, Chap. XV, Wiley, 1939. 23. Reference 22, pp. 355-357. Also Tyndall, A. M., and C. F. Powell, Proc. Roy. Soc, A134: 125, 1931. 24. Lind, S. C., and M. Vanpee, J. Phys. Colloid Chem., 53: 898, 1949. 25. Eyring, H., J. Hirschfelder, and H. S. Taylor, J. Chem. Phys., 4: 570, 1936. 26. Mott, N., and R. Gurney, Electronic Processes in Ionic Crystals, Oxford, 1940. 27. Lea, D., Actions of Radiations on Living Cells, pp. 47-52, Cambridge, 1947. Weiss, J., Nature, 153: 748, 1944. 28. Zimmerman, G., Ph. D. thesis, University of Chicago, 1949. Platzman, R., Ph. D. thesis, University of Chicago, 1942. DISCUSSION Burton : In amplification of points raised by Livingston we may note that in photo- chemical reactions energy absorbed electronically at a particular locus may be internally converted to vibrational energy at another favored locus. In such event rupture of one particular bond, or a particular rearrangement decom- position, may be specially favored. In thermal reactions the energy is initially distributed in many degrees of freedom, and, in general, the most probable primary reaction is that most favored by the frequency factor and particularly by the activation energy. In contrast, in photochemical reactions the process of lowest activation energy is not necessarily the most likely to occur. In radi- ation chemistry there is evidence for a great variety of possible products, related undoubtedly to the fact that primary ionization and primary excitation (both of which are produced by the ionizing radiation) are not restricted to one part of the molecule. Resultant chemical events are shaped by the nature of the initial physical events peculiar to radiation chemistry. The situation is not comparable to the photochemical case. In radiation chemistry one must reckon DISCUSSION , 69 with ionization transfer between molecules and also with ionization transfer within the molecule. Before a process of thermal degradation of energy occurs there is perhaps a situation not too different from that in a photochemical process. However, the process of thermal excitation is not to be compared with that in reactions induced photochemically or by ionizing radiations. Livingston: There are cases in radiation chemistry where the products are like those produced in thermal reactions. Examples of this type are the decarboxylation of organic acids and the splitting of hydrocarbons into two stable molecules. On the other hand, no one can deny that there are photochemical and radiation chemical reactions whose products are predetermined by the manner of acti- vation. The interesting thing is not that there are these differences, but that the reaction products of complex molecules are so often independent of the manner of activation. Magee : It is somewhat unfortunate that there is so much talk of ionization of the individual atoms of a molecule. Ultimately the charge always resides in the valence electrons and must belong to the molecule as a whole, not a single con- stituent atom, except in special cases. However, there are differences in the distribution of charge in a molecule ion characteristic of the particular energy state involved in the ionization. It is this average depletion of electron charge in a part of the molecule to which reference is properly made by the expression that a particular group or atom in the molecule is ionized. Concerning the breakdown of selection rules in impact processes it is, of course, true that almost all selection rules are violated for very close collisions of any fast particle. However, most of the effect on the matter in any irradiation is due to the relatively slow secondary electrons. Here exchange effects must be considered between the secondary electron and the electrons of the molecule with which it collides. The ordinarily forbidden singlet-triplet transitions occur with high probability. Solomon : I should like to point out that the process of "flow" of ionization is an im- portant one in the operation of Geiger counters, where it is relied upon in order to quench the pulse. Perhaps one could use the quenching time in a Geiger counter to obtain further information on how quickly the ionization flows. Dale: Is the theory that the hydroxyl and hydrogen ions are responsible for the denaturation of proteins meant to substitute for the theory of radicals? Livingston : It is probable that both means of denaturation are effective. Their relative importance is not known. 6- Chemical Reactions in the Gas Phase Connected with Ionization MERRILL WALLEXSTEIN, AUSTIN L. WAHRHAFTIG, HENRY ROSENSTOCK, AND HENRY EYRING Department of Chemistry University of Utah Salt Lake City, Utah The previous papers in this symposium have dealt with the physical theory basic to the study of the interaction of high-velocity particles with matter. We wish to take the first step toward the consideration of complex systems, to consider processes sufficiently simple so that the application of approximate quantum mechanical and statistical calcula- tions is possible, yet sufficiently complex to point the way toward a correct discussion of systems of biological interest. Thus, this report will deal largely with the effect of bombardment of isolated molecules by electrons, under such conditions that no secondary reactions occur between the products of the bombardment. Most of the subsequent discussion will be based upon data obtained with a mass spectrometer. Let us consider just what information can be so obtained. Almost all the mass spectrometric data now available have been obtained with instruments of the Dempster-Nier type. Here the substance to be studied, which must have a vapor pressure of at least a millimeter or so at a reasonable temperature, is introduced into the ionization region through a capillary leak to give a pressure of about 10~* mm. It is there bombarded by a beam of electrons of known energy, commonly variable over the range 0-100 volts, and the positive ions formed are accelerated by a small field, a few volts per centimeter, toward a slit. Those that pass through are accelerated by a "high potential" of 300-5000 volts and focussed on a second slit, the entrance slit to the analyzer section. Depending upon the type of instrument, 180, 90, or 60°, the ions diverging from the entrance slit pass through a magnetic field in which they follow circular paths of radius C m H \ e 70 MON ATOMIC GASES 71 for the angular deviation given above. Tliose ions having the appropriate radius are refocussed on an exit sUt behind which is a collector electrode connected to a sensitive electrometer circuit. The current in the electron beam is usually about 10 microamp. Total ionization is the order of 10~^ or 10"^ amp. A very strong peak might give an ion current of 10~^^ amp. The size of the minimum peak detectable will depend upon the sensitivity and the noise level of the electrometer circuit; 10~^^ amp is a reasonable lower limit for most instruments. As is indicated by the above, in the modern mass spectrometer the current density in the electron beam and the gas density in the ionization region are sufficiently low to assure that all processes are the result of single electron impacts upon isolated gas molecules and that all sub- sequent breakups and rearrangements of the molecules are unimolecular [only exceptions reported: formation of Hs"^ and HC02"^ (1)]. Proper location of the filament and the pumping lead essentially prevents the diffusion of products formed by thermal cracking at the filament back into the ionizing region. Thus, by varying either the accelerating voltage or the magnetic field, one can successively collect and measure the ions of each tn/e ratio formed from a given molecule by collision with electrons of known energy, without the complications that arise from secondary reactions in gases at higher pressures or in condensed phases. One major trouble in the interpretation of data arises from the com- plete lack of direct information regarding the neutral fragments formed along with the ions. This matter will be discussed in some detail in subsequent sections of this report. A second serious complication is that the simple "single-focussing" mass spectrometer is designed to focus and collect efficiently only those ions formed with essentially zero kinetic energy (of the order of translational thermal energy at 200° C, 0.06 volt). By applying a retarding potential to the final ion collector one can determine the amount or distribution of excess kinetic energy possessed by the ions of any given mass number, but the geometry of the spectrometer tube is such that the vast majority of ions with appreci- able (over about 1 volt) translational energy will strike the sides of the tube long before reaching the ion collector (2). MoNATOMic Gases The simplest substances for study in the mass spectrometer are the monatomic gases. Here, all the data obtainable can be represented by a set of "ioriization-efficiency" curves for the ions obtained by removing one or more electrons from the atom. These curves, in which the magnitude of the ion current is plotted against the energy of the electron 72 CHEMICAL REACTIONS IN THE GAS PHASE beam, are all of the same general shape; a curved "foot," a very nearly linear section, and a flat maximum after which the ion current drops off inversely with the increase in the electron voltage. The minimum electron potential which yields a given ion should correspond to the spectroscopic ionization potential. However, this minimum potential cannot be measured directly, as there are always contact potentials of unknown and appreciable magnitude associated with a hot filament. In addition, the electrons emitted by the filament have a thermal energy spread of several tenths of a volt. Also, the electron beam is of finite thickness, and in the ionization region there is an electric field perpen- dicular to the electron beam. These factors contribute to the shape of the foot of the ionization-efficiency curve, but if such experimental factors were alone responsible all curves should have identical feet. Experi- mentally, this is not the case (3). Moreover, the values for "appearance potentials," the minimum electron voltages at which given ions are formed, obtained by noting the first upward breaks from the axis, give differences in agreement with the spectroscopic ionization values for A+ and Ne+ and for A+ and A++. If instead one assumes that the initial curved portions of the curves arise from experimental factors and so extrapolates to zero ion current the linear portions of the curves, the resulting differences are not in agreement with the spectroscopic data. This is unfortunate, since the "linear extrapolation" is usually simple and objective whereas the point of the "initial break" is subjective (6) and also in some cases depends greatly upon the sensitivity of the instrument (8). The approximations usually made in obtaining ioni- zation cross sections by quantum mechanical calculations break down completely near the appearance potential, so that the theoretical shape of the curve is unknown. Several articles in the literature discuss the significance of these two methods of determining appearance potentials for molecules (3, 4, 5), and other methods of determining appearance potentials have been proposed in which a correction is made for the electron energy spread (6, 7). The situation is not satisfactory. The best method at present seems to be the initial break or "vanishing current" method, with the voltage scale corrected for contact potentials by mixing a gas of known ionization potential, usually neon or argon, with the gas under investigation. Diatomic Molecules The effect of electron bombardment on diatomic molecules has been . carefully studied for many such molecules (2, 4, 9, 10, 11). We shall here make no attempt at completeness but only consider those aspects useful DIATOMIC MOLECULES 73 in the discussion of the processes occurring in more complex molecules. The velocity of an electron accelerated by a voltage V is approximately 6 X IQpVv cm per sec, or 6 X IO^^a/f A per sec. Thus the time of interaction of even a 1-volt electron with a small molecule is of the order of 10~^^ sec, and for electrons of the voltages generally used the time of interaction will be much less than this value for molecules of molecular weight up to several hundred. The highest vibrational fre- quencies of molecules (other than the H2 molecule) are of the order of 3000 cm""^, or 9.10^^ sec~^; most frequencies are one-third this value or less. It is seen that the time of interaction of an electron of energy greater than 10 volts with a molecule is at most one-thirtieth of the shortest period of vibration of the molecule. The electron is too light to transfer appreciable energy directly to the nuclei. We can safely apply the Franck-Condon approximations (12) and state that the first direct result of the electron impact is to raise the molecule to an excited electronic state without change in either the internuclear distance or the nuclear momentum. If we are to observe the results of this collision in the mass spectrometer, it is, of course, necessary that the excited state be an ionized state. It can be either the ground state or an excited state of the ion. A set of possible potential functions for a diatomic molecule is given in Fig. 1. Here, curve I represents the ground state of the molecule, curves II and III are attractive states, and curve IV is a repulsive state which dissociates to the lowest state for the separated atom plus ion; curves V and VI represent two of the many possible states formed in first approximation from excited states of the atom and ion. In accord- ance with the Franck-Condon principle, transitions are probable only to those parts of the potential curves between the lines a-a and h-h. We note several possibilities. Transitions to state II will give only molecule ions, and the minimum electron potential at which such ions appear should be an accurate measure of the ionization potential of the molecule. Transitions to state III can yield either molecule ions AB"^ or atom ions A"*"; the appearance potential for AB"^ will be definitely larger than the ionization potential of AB (to give the ion in this particular state), but the appearance potential for A^ should give an accurate value for /(ab) + ^(AB+) = ^(AB) + -^(A); and the ions A^ will be formed with low^ kinetic energy irrespective of the magnitude of the electron energy. To obtain transitions to state IV will require an electron energy several volts greater than 2)(ab) + ^(X), and the resulting ions A"^ will have the fraction W(B) W(A) + m(B) 74 CHEMICAL REACTIONS IN THE GAS PHASE of this excess as translational energy. The efficiency of collection of such ions will be very low in most mass spectrometers (2), and those that appear will come at an accelerating potential or magnetic field Fig. 1. Potential energy functions for a few possible states of a diatomic molecule, indicating some of the dissociation processes and products which can result from a single electron impact. corresponding to a mass slightly greater than the true value. Transi- tions to state V, if the Franck-Condon rule is strictly applicable, can yield directly only molecular ions, but as this state is above the asymp- tote for formation of A"*" + B, secondary reactions can occur: (a) there i DIATOMIC MOLECULES 75 can be radiative transitions to states such as II (with formation of stable AB"^) and III (with formation either of AB"^ or A"^ with low kinetic energy); the half life for state V for such transitions, by the usual spectroscopic selection rules, will be of the order of 10~^ sec; (6) there can be radiationless transition to state IV, with formation of A"*" ions with some intermediate amount of kinetic energy ; the half life for this transi- tion will depend greatly on the magnitude of the interaction between the two states and can be anything between <10~^^ sec to >1 sec. The time spent by an ion in the ionization region before collection by the ion "draw-out" potential is the order of lO"*" sec. Hence, ions initially formed in a state such as V will normally undergo an electronic transition, with or without dissociation, before acceleration and collection. Lastly, transitions to state VI will be followed immediately by dissociation to give A"^ with excess kinetic energy. A set of potential-energy curves such as those indicated therefore gives rise to AB+, A+ (low KE), and A+ (high KE). Certainly there will also be states yielding B"^, both without and with kinetic energy. Also, to be complete, one must include double ionization, the formation of AB"'"^, Avith possible subsequent dissociation most likely to A"^ + B"*", but also sometimes to A"^"^ + B and A + B"*""*". There is also the possibility of forming an excited neutral molecule AB* in such a state that it dissociates: AB* — > A"^ + B~". All these possibilities have been observed (2). In H2"^, the two lowest states are located relative to the ground state of H2 in similar fashion to states III and IV of Fig. 1. The observed mass spectral data agree completely with those expected from the above discussion in regard to appearance potentials and kinetic energy of the H"*" ions. Also, a calculation based upon the simple application of the Franck-Condon principle leads to a value of the rates of 'ii^/Yi.2^ to D~^/D2"^ in essential agreement with experiment (13). A detailed discussion for diatomic molecules of the dependence of peak shapes as observed with the mass spectrometer upon the relative shapes of the ion and ground state curves has been given by Hagstrum and Tate, with special reference to CO, NO, N2, O2 (2). They find, for instance, that both C"^ and 0"^ are obtained from attractive states of CO"^, the state yielding C"*" having its minimum a little further out than the curve for state III, Fig. 1, so that some C"^ are formed with appreci- able kinetic energ^^, and the state yielding O"^ similar but with its minimum much further out, so that practically all the CO^ ions formed in this state dissociate with appreciable kinetic energy. It is probably not necessary to point out that an appearance potential for an ion, since it corresponds to a "vertical" transition on a potential- 76 CHEMICAL REACTIONS IN THE GAS PHASE energy curve diagram, gives only an upper limit for the amount of energy necessary for a given process. That is, referring again to Fig. 1, ^(A+) >:-D(AB) + -^(A) = -^(AB) + -D(AB+) In recent years, workers in mass spectra have succeeded remarkably well in building instruments in which second-order reactions involving ions and molecules are almost completely absent. This was not the case in older instruments, and indeed some instruments were constructed especially for the study of secondary reactions by differential pumping arrangements which permitted independent control of the gas pressure in the analyzer regions (14). The essential result of such work is that the probability of charge exchange reactions, A+ + B -^ A + B+ varies inversely with the size of /(a) — 1(B), and only on the absolute value of this difference if A+ has much kinetic energy. It may be very large for 7(A) — /(b) ~ 0. For the special case of charge exchange where A and B are the same, as N2+ + N2 -> N2 + N2+ the cross section for the reaction can be orders of magnitude greater than expected from the kinetic theory diameters. Interesting applications of mass spectrometric data in combination with thermal and kinetic data are contained in several papers by Eyring, Hirschf elder, and Taylor (15). In the ori/io-para-hydrogen conversion by alpha particles, 700-1000 molecules are converted per ion pair formed (16). The ionization by alpha particles is actually largely due to the secondary electrons of energies comparable to electron energies in the mass spectrometer. From the ratio of ion pairs produced to alpha- particle energy, and the mass spectrometric data on hydrogen, it is deduced that the primary effect of the secondary electrons is to produce approximately equal amounts of (2H) and £[2"^, and much smaller amount of H+. Absolute reaction rate calculations indicate that the reaction H2+ + H2 = H3 + + H is very rapid, and that the reaction H2 + H2+ = H2 + H + H+ is of no importance. The formation of "clusters," the formation of H~ and Il2~, and the ortho-para conversion directly by alpha particles and by H2"^, H3+, and H are shown to be negligible. Recombination of H3+ POLYATOMIC MOLECULES 77 with an electron will give an average yield of between two and three H atoms per Hs"^. This is the principal neutralization reaction. Com- bining it with the previous arguments, one obtains a total H-atom yield of about 6 per ion pair produced. The remaining reactions to be con- sidered are H + H = H2 and H + H2(p) = H2(o) + H The first reaction requires a third body and occurs essentially only on the walls of the vessel. The second reaction is very rapid and effective in causing the conversion. Calculations based upon the known diffusion constant of H atoms in H2 and the rate of the last reaction above give results in good agreement with experiment. The synthesis and decomposition of hydrogen bromide by alpha particles in hydrogen-bromine-hydrogen bromide mixtures were treated in similar fashion (17). The primary ionization processes were obtained from mass spectral data. Of the large number of secondary reactions possible, many were immediately discarded as too slow to be important, and an analysis of the kinetics of the system was made in terms of the remaining reactions. It was found that the data could be satisfactorily explained with the assignment of reasonable values to the few unknown rate constants. A similar treatment of the alpha-particle-induced re- actions in carbon monoxide-oxygen-carbon dioxide systems has been made by Hirschf elder and Taylor (18). Polyatomic Molecules One is naturally inclined to attempt to extrapolate from the reasonably well understood actions of diatomic molecules to polyatomic molecules (19). There are many points of similarity. Each ion formed from a polyatomic molecule has a well-defined appearance potential, and the ionization-efficiency curve for the production of an ion is of the same general shape as similar curves for diatomic molecules. With small polyatomic molecules such as CO2 and CH4, one finds ions formed with appreciable amounts of translational energy. However, when we examine the mass spectra of hydrocarbons the size of propane and larger, we find the spectra show certain distinctive features different from small polyatomic and diatomic molecules. The mass peaks for ions obtained from large molecules are almost invariably symmetrical and of a shape determined only by the character- istics of the instrument. This fact, important in permitting the determi- 78 CHEMICAL REACTIONS IN THE GAS PHASE nation of relative intensities by the simple measurement of peak heights, also indicates that the production of ions having appreciable kinetic energy is far less frequent here than in diatomic molecules. Very careful measurements (20) have shown that all the members of any one isomeric series of molecules, such as the octanes, show the same total ionization when measured under the same conditions. This can be interpreted in two ways. Either there are essentially no products formed with kinetic energy, or every isomer in the series produces about the same amount of products with kinetic energy. In view of the striking differences in mass spectra of a series like the octanes this second alternative seems far less likely. (See Table 1.) When an attempt is made to correlate appearance potentials with thermal data on bond dissociation energies, the agreement is generally fair (21, 22). The discrepancies, as might be expected, are all in a direction which indicates that the products of dissociation have a certain, amount of excess energy of the order of a few tenths of a volt. This energy could conceivably be either vibration-rotation energy or kinetic energy of translation. In view of the previous discussion it seems likely that it is mostly vibrational, and indeed the apparent magnitudes of the excess energy for heavy molecules seem never to be too large to be interpreted in this way. It is frequently necessary in small molecules such as methane to assume such dissociations as (21) CH4+ -^ C+ + 4H in which the uncharged fragments come off as atoms. However, in a paper by Delfosse and Bleakney (22) on the mass spectra of propane, propylene, and allene it is shown that here no such assumption is necessary for any of the ions measured, which include all the important ions. Every appearance potential measured is best explained by as- suming that the minimum energy process possible occurs. That is, wherever possible, the uncharged fragments come off as molecules. Also, in the mass spectra of all large hydrocarbon molecules one finds a number of metastable peaks (23, 24). These metastable peaks con- stitute a large fraction of the diffuse peaks occurring at non-integral masses. These peaks have been shown to be formed by delayed dis- sociations which occur after the original ion has been accelerated but before or immediately after its entry into the analyzer (25, 26). Exami- nation of these peaks in a very large number of hydrocarbons (23, 24) shows that the uncharged fragments of the dissociation almost always are in the correct ratio to form a stable molecule. For most of these molecules, we have no way of knowing the state of association or dis- sociation of the neutral part, but the fact that the fragments are of such POLYATOMIC MOLECULES 79 < ^ O w o h-] H u » cu Ct! T3 C V > s 'S 3 03 c S a) lO iN-OOOiNi-iOO MINiNIN(NiN,-H,-irHr^f-(,-i IN'-HININ.-I 35 iot^t^T)i-HOoor-c^coo-*cqooooooco -t CO CO t> -t CO lO (N CI IN C-l CO C-1 ■>) CI C) C^I CI (N 00O0:OOOt^MM'«OO>0t^-fH>0 05-f 10 rt t^ rt ^ t o Tt( CI -H c) CI rt CO 10 CO 00 t^ — 1 Tf rH CO ^ rt rt CO cs 00 -- r- OOOOO -OOOiOClCOt^OOOOO CI— icioiO>05o>t-icaoooooot^'racDooo ^^ — iio — ico^»OTfi w 1005— M>nuii^u5r--*Mcooeot^'*03 cocjcoTttoo— ieo-*ooooot~«ocio— 1 Cl^ — l^H ^^c C2H4 + C2H6''" is not observed, either metastable or other- wise. (e) A few small peaks that must be due to doubly charged ions are found; a much larger fraction of the doubly charged ions formed surely immediately dissociates to give two singly charged ions. Although the number of ions so produced will be but a small fraction of the total ioniza- tion, it probably accounts for much of the relatively constant amount of CH3 produced from all large hydrocarbons. It is obvious that the complexity of the situation makes these rules have only the barest qualitative significance and that they are only of limited applicability. The mass spectrum of cyclohexane (23) can reasonably be interpreted in terms of these rules, but the mass spectrum of benzene cannot without special consideration of the effect of the large number of electrons. To sum up, the essential idea in the foregoing discussion is that the process of ionization of the molecule is accompanied by the simultaneous transfer of excess energy to the other electrons of the molecule, and that instead of an immediate dissociation occurring this energy undergoes a process of rearrangement, being transferred from the electronic to the vibrational states of the molecular ion. As has already been mentioned, we have at present no way of knowing the distribution function for this excess energy. Moreover, we cannot be certain of how much of this energy is transferred to the vibrational states of the molecule. It is likely that at least some of the radiationless transitions are very rapid, but there may, as already mentioned, be some transitions which are much slower than the total lifetime of a molecule in the mass spectrom- eter. The fact remains, however, that there will be some sort of dis- tribution function for the vibrational energy and that in view of the very large number of electronic states this function will approach a 86 CHEMICAL REACTIONS IN THE GAS PHASE continuous one. Even without this distribution function in explicit form it is possible to formulate a fairly simple theory of the decompo- sition of these molecules which, although it oversimplifies the situation, serves to provide a reasonable picture of the processes involved. As has already been done by Kassel (36), we shall use as a model to represent the molecule a collection of harmonic oscillators coupled by forces which are sufficiently large to allow the energy to pass freely from one to the other but small enough so that the energy of a group of such oscillators may be expressed as a sum of squares of their coordinates and momenta. Also to further simplify the problem we shall assume that the harmonic oscillators all have the same frequency, v. We shall let P{Ei) be the probability that after ionization the molecule has acquired, in the manner discussed above, an amount of vibrational energy Ei and shall assume that this energy is randomly distributed among the vibrational degrees of freedom of the molecule. It now becomes necessaiy to formulate an expression for the specific rate of a particular reaction in which molecules, each with energy Ei, decompose. A molecule with total vibrational energy Ei will contain Ei n = ~ (1) hv quanta. The molecule, as already mentioned, will be represented by harmonic oscillators corresponding to the vibrational degrees of freedom of the molecule. Consider a single possible way of arranging these n quanta in the s oscillators such that there are rii quanta in the first oscillator, 712 in the second, and so on up to Ug quanta in the last oscillator. This set of n's obviously must conserve energy, that is, J^^r = n (2) r We shall consider that the slow step in the decomposition process is the transfer of energy to the oscillator corresponding to the reaction co- ordinate which is to rupture in a particular reaction. In the following formulation we shall consider the reaction to be governed by accumula- tion of a critical number of quanta, n*, in a single oscillator. The number of quanta, Ikj, which when transferred from the kth. to the jth oscillator will cause a break must satisfy the relation Ikj > fij* — rij, where rij is the number of quanta in the reacting oscillator at the start of the re- action. We now define a transmission coefficient, 7(n, 7li, 712, • " Ur, " ■ W«-b hj) POLYATOMIC MOLECULES 87 and a frequency, v, such that vy{n,ni,7i2, ••• ns-i,hj) (3) is the rate at which molecules transfer Ikj quanta from the kth to the jth oscillator. The total number of ways that n quanta can be distributed among s distinguishable oscillators is (n + s - 1) ! n\(s - 1)! The reciprocal of this quantity will be indicated by the letter C. Thus C 21 n{n, rii, W2, • • • fis^i, hj) (4) represents the number of reactions per second due to the transfer of Ikj quanta from the kth. to the jth oscillator. The specific rate for this distribution can then be written as rik k(n,ni, n2, ■■■ n._i)i = <^ 2] £ n(.n, 7li, 112, ' ' ' ^^s-l, kj) (5) k lki = nj* — nj where 22 sums over the s — 1 oscillators which can feed energy into the k reacting oscillator j. This method of expressing the rate omits the possibility that the I quanta might come from two or more different oscillators into the jth one. Such occurrences will be neglected. For a given n the total rate of decomposition at the ^'th oscillator will be rik knj = C J2J2 2 nin, ni, 7i2, • • • Ws_i, kj) (6) Tlr k IkJ = Itj*— llj where ^ is the summation over all sets of n's consistent with the con- servation of energy, ^ rir = n, and the condition that no oscillator have r enough quanta to dissociate them, that is, Ur < rir*, where iir* is the number of quanta required for dissociation at the rth oscillator. We may define an average 7 such that ^ (w-nfc-ny + s-3)!_ 2^ y{n, 7ii, 712, •'•ris-i, hj) = — rrr 7(^1, %, rij, hj) (jl - Uk - 7lj)l{s - 3)\ where the summation includes all ways of distributing n quanta in the molecule such that the j and k degrees of freedom have the fixed values Uk and 7ij. The rate then becomes 88 CHEMICAL REACTIONS IN THE GAS PHASE Knj=C2^ 2^ 2^ 2^ — ~vi{n,nk,nj,lkj) k nk = 0 7iy = 0 lki = nj*-nj (Tl — Uk — Uj) .{S — 6)1 (8) Actually we should exclude from the terms (n — nk — Uj -{- s — 3) ! {n — Uk — nj)\{s — 3)! those states which correspond to dissociation, that is, n^ > rir*. How- ever, this would only decrease the expression slightly at the price of great complication. Expression 8 is apparently quite complicated and in this form would be difficult to evaluate. Actually, however, it is almost certainly true that the sums involved will contain a large number of terms equal to or very near zero. First of all, 7 will be strongly dependent uopn Ikj, the number of quanta which are transferred from oscillator k to oscillator j in a single period of the frequency factor. In fact it seems likely that the probability of transferring more than one quantum in time 1/v may be negligible. This means that all terms are essentially zero unless Ikj = 1. If this is true then w^e must have Uj* — Tlj — I SO that only those molecules having Uj = Uj* — 1 can decompose in time 1/v. This reduces the summation over hj to a single term kj = 1. Since in the summation over Uj the lower limit is rij = Uj* — kj, this now becomes ny = rij* — 1, and the summation over rij is thus also reduced to a single term. In the molecule, energy transfer is principally between adjacent bonds; the summation over A; can be taken as zero except for three or four terms. The rate then reduces to ->^^[n -Uk- (uj* - 1) + s - 3]! _ / * ix n knj = C E E 1 r^^ T^TTT ^ n[^> ^k, {uj* - 1), 1] k nk^o [n - Uk- (rij* - l)]!(s - 3)! For quanta corresponding to j'osc = 1000 cm~\ a reasonable value for a hydrocarbon molecule, the factorial term will decrease rapidly in size. For example, if n - n^ - (wy* - 1) = 12 and s - 3 = 52 (values which are approximately correct for a molecule like hexane), the factorial term is 3.3 X 10^^; and for the next term, where n — Uk - (rij*) = 11, its value is 6.2 X 10^ ^ The ratio of the second term to the first is approxi- mately 0.2. The transmission coefficient 7 will increase as rik becomes larger. There will therefore be a maximum term in the summation over Uk. Assuming this maximum to be strong allows us to discard the POLYATOMIC MOLECULES 89 summation over rik except for the term where Uk = Uk^ . The rate then finally becomes rr^^^ ~ ^^mnx " K"* - 1) + S - 3]! ^ k [n- m^,^ - {nj* - l)]!(s - 3)! "^^ ' ''°^'" ^ ' ^' ^ (10) In order to evaluate knj it will be necessary to find a value for 7. As- suming V = 10^^ (approximately the vibration frequency of one of the oscillators), one term of knj would be of the order of 10^ to 10^^ if 7 = 1. The half life of an ion in the mass spectrometer is of the order of 10~^ sec, so that 7 can be no smaller than about 10~^ to 10~^. These values for 7 seem reasonable when we consider that it is the probability of transferring energy from an oscillator having lower energy than the one which is to receive the energy. It is now possible to discuss the general nature of the mass spectrum of a molecule. In a molecular ion with a given amount of vibrational energy a number of different reactions may be possible. Moreover, if the energy is sufficient the products of the initial dissociation of the ionized molecule may undergo one or more successive breaks. The probability that a given molecule will break at two points simultaneously is small. We shall therefore consider that the mass spectrum results from a number of successive decompositions and that the problem of calculating the spectrum can be discussed in terms of a set of competing reactions which have reached a steady state. For a given molecule the fraction fnj of products of decomposition which will be formed by initial break of the parent ion will be fnJ = P{E,) -^ (11) } where X) is the summation over all possible single decompositions of the j parent ion. (We shall ignore the uncharged fragments here, although their number and energy distribution could also be calculated.) The fraction fnj represents the fractional number of ions of species j resulting from the break of a parent ion with energy nhv = Ei. The total fraction of all parent ions which yield this species will be L- = i:fnj = j:p(n)^;^ (12) n , n / y Kif^j J where now P{n) has been written for P{Ei) and the summation extends from the minimum energy necessary to produce species j to the maximum energy of the impacting electrons. The sum over j will, in general, be 90 CHEMICAL REACTIONS IN THE GAS PHASE different for different ranges of n, since knj is zero for molecules having n < Uj*. The fraction fj given by expression 12 does not represent the number of species j ions which will be found in the mass spectrum. For values of n sufficient to break two or more bonds in the molecule the chance exists that the initial break will leave the rij ion with enough energy to break a second time. The resulting fragment may also de- compose further. In order to simplify the discussion it will be convenient to break the energy distribution into a number of more or less arbitrary ranges. We shall suppose that there is some definite range Ei = E'-^^ to Ei = E^^^ within which the energy is sufficient to break only one bond in the parent ion. A second range E^^^ to E^^^ is defined such that the products of the first dissociation of the parent ion will have between them enough energy to cause one more dissociation but not enough for two. Higher ranges can be similarly defined. Now the fraction of parent ions which do not dissociate at all is h = Z ^^;^^ (13) and the fractional amount of a product j resulting from parent ions having energies in the first range is //■>-<^'= V P(«)^ (14) 3 A similar expression can be written for the j ion produced in the second energy range, but now the situation becomes more complicated. For each j ion produced in this range we must consider the chance that at the time of the initial break enough of the energy not concentrated in the breaking bond will be in the charged product of this break to cause subsequent dissociation of this fragment. That is, if the j ion which is formed by the initial break has q oscillators, then we must calculate the chance that these q oscillators will, at the instant of the first break, contain a number of quanta rij*' or more, where 7ij*' is the minimum energy necessary for the j ion to decompose to give a fragment /. This chance is given by {n - Uj* - n' + s - g - 2) ! {n' + g - 1) ! ^ in - Uj* - n')\{s - q - 2)\ 7i'\{q - l)\ A„ = E ^ — — (15) n' = nj*' {n — llj* + 8 — 2)! {n - ny*)!(s - 2)! POLYATOMIC MOLECULES 91 Such a term as this should multiply each term of 23 in ^nj, but since k ^A;max will be only slightly different in each term it will probably be sufficient to write for those j ions which have sufficient energy for a second break //^)-(^^ (unstable) = E ,^ P(n) ^^ a,„,, A„ (16) „ = „(-) Z^ knj i where ^^^^ is the number of terms in the sum over k in knj. It follows immediately that the fraction of stable j ions in this energy range is /y(^^-^3) (Stable) = f P(n) ^ «,„,, (1 - A^) (17) " = "'"^ Z^ knj j We can now calculate the amounts of the various breakdown products of the j ion. To do this we require the energy distribution of the j ions formed in the first break. This distribution is given by the terms in An, since each term gives the fractional number of j ions having energy n'. Thus the interesting fact emerges that the distribution function, P{n), once determined, is sufficient to determine also the energy dis- tributions for all secondary and later ions. The expression for the fractional number of j' ions from j ions formed from parent ions in the initial energy range n' to n is /,.<^'-«'= E A„.^?^ (18) "' = «;* 2^ kn'j' J' The further calculation of the spectrum is simply a repetition of the calculations indicated before. In spite of the simple model employed in the discussion it is obvious that, even if accurate data were available for the dissociation energies and if the values of 7 were known, the calculations would be extremely involved. Such calculations would also be expected to yield answers with only the general outlines of the experimental spectrum. A better approximation would require taking two or three different frequencies for the oscillators, but this would even further complicate the formula- tion. It appears that numerical calculation must await further refine- ment of the theory. It remains to consider how much decomposition will occur in a mole- cule in solution when struck by radiation. The well-known formula T = e-'-'/^'" (19) 8pc(7r/c0^ 92 CHEMICAL REACTIONS IN THE GAS PHASE gives the temperature t at a distance r from the point of absorption of the energy after a lapse of time t. Here k, the diffusivity, is the thermal conductivity, k, divided by the heat capacity per cubic centimeter of substance, p and c are the density and the heat capacity per gram, respectively. For a hydrocarbon such as pentane k = 0.00032 while pc ^ I. For water k = 0.00143 and pc « 1. Thus the rate at which temperature drops off is approximately the same for different substances. If we substitute the constants for water into expression 19, it becomes immediately obvious that for periods of 10""^^ sec or greater and for molecular distances of r = 3.3 X 10~^ or less the exponential factor is unity. If it be further assumed that 100,000 cal per mole is delivered by the radiation, one finds that after 10~^^ sec the temperature of the struck molecule is 6900° C above its original temperature. After 10~^° sec it has dropped to 7° above the original temperature. A C — C bond oscillates about 3 X 10^^ times per second, while a CH bond oscillates about 9 X 10^^ times per second. Thus, in the very unusual circum- stances that enough energy is delivered to a bond to break it, dissociation will ensue. The earlier considerations of the mass spectrographic data indicate that 10~^ sec is a more probable half life for molecules. These processes with half lives of 10~^ sec are completely quenched in the liquid state. Even if the molecule should decompose, it still must escape the Franck-Rabinowitch cage before recombination or there will be no reaction. Thus the viscosities of pure liquids are given with some accuracy by the equation V Vk' where 77, A^, h, V, AH^^p, R, T, and k' are the viscosity, Avogadro's number, Planck's constant, molal volume, heat of vaporization, gas constant, temperature, and rate of jumping out of the cage, respectively. The constant 2.5 in this equation is the ratio of the heat of vaporization of the liquid to the free energy of activation of the flow process (30). Remembering that pure liquids have a viscosity at these melting points of about 0.02 poise, one finds that a molecule has a half life inside the Franck-Rabinowitch cage of about 2 X 10~^^ sec at room temperature. Hot molecules or molecules dissociating with large kinetic energies can escape from the cage even more quickly. Thus the rapid chilling of molecules has much more to do with preventing decomposition than does the entrapment in a cage of their neighbors. The main source of destruction of large molecules by direct hits will be by the ejection of an electron and the reaction of the positive ion GENERAL REFERENCES 93 with the solvent. Much more frequently decomposition of the enzymes will come from reaction with the H, OH, and HO2 formed from water and oxygen, as is discussed elsewhere in this volume. REFERENCES 1. Aston, F. W., Mass Spectra and Isotopes, Edward Arnold, 1942. 2. Hagstrum, H. D., and J. T. Tate, Phys. Rev., 59: 354, 1941. 3. Stevenson, D. P., and J. A. Hippie, Phys. Rev., 62: 237, 1942. 4. Tate, J. T., P. T. Smith, and A. L. Vaughan, Phijs. Rev., 48: 525, 1935. 5. Vought, R. H., Phys. Rev., 71: 93, 1947. 6. Honig, R. E., J. Chem. Phys., 16: 105, 1948. 7. Mitchell, J. J., and F. F. Coleman, J. Chem. Phys., 17: 44, 1948. 8. Fox, R. E., and A. Langer, J. Chem. Phxjs., 18: 460, 1950. 9. Blewett, J. P., Phys. Rev., 49: 900, 193G. 10. Hogness, T. R., and R. W. Harkness, Phys. Rev., 32: 784, 1928. 11. Bleakney, W., Phys. Rev., 40: 401, 496, 1932. 12. Condon, E. U., and H. D. Smyth, Proc. Natl. Acad. ScL, 14: 871, 1928. 13. Stevenson, D. P., ./. Chem. Phys., 15: 409, 1947. 14. Kallmann, H., and B. Rosen, Z. Physik, 61: 66, 1930. 15. Eyring, H., J. Hirschf elder, and H. S. Taylor, /. Chem. Phys., 4: 479, 1936. 16. Capron, P. C, Ann. soc. sci. Bruxelles, 55: 222, 1935. 17. Eyring, H., J. Hirschfelder, and H. S. Taylor, J. Chem. Phys., 4: 570, 1936. 18. Hirschfelder, J., and H. S. Taylor, /. Chem. Phys., 6: 783, 1938. 19. Vought, R. H., Phys. Rev., 71: 594, 1947. 20. Mohler, F. L., Phys. Rev., 78: 332(A), 1950. 21. Smith, L. E., Phys. Rev., 51: 2G3, 1937. 22. Delfosse, J. M., and W. Bleakney, Phys. Rev., 56: 256, 1939 23. Catalogue of Mass Spectral Data, Am. Petroleum Inst. Research Project 44, National Bureau of Standards, Washington, D.C. 24. Bloom, E. G., F. L. Mohler, J. H. Lengel, and C. E. Wise, J. Research Natl. Bur. Standards, 40: 437, 1948. 25. Hippie, J. A., R. E. Fox, and E. U. Condon, Phijs. Rev., 69: 347, 1946. 26. Hippie, J. A., Phys. Rev., 71: 594, 1947. 27. Fox, R. E., and A. Langer, J. Chem. Phys., 18: 476, 1950. 28. Roberts, J. S., and H. A. Skinner, Trans. Faraday Soc, 45: 339, 1949. 29. Eyring, H., J. Walter, and G. E. Kimball, Quantum Chemistry, p. 232, John Wiley, 1944. 30. Glasstone, S., K. J. Laidler, and H. Eyring, Theory of Rate Processes, McGraw- Hill, 1941. GENERAL REFERENCES 31. Hippie, J. A., /. Appl. Phys., 13: 551, 1942. References to molecules studied up to 1942. 32. Tate, J. T., and W. W. Lozier, Phys. Rev., 39: 234, 1932. 33. Lozier, W. W., Phys. Rev., 46: 268, 1934. This article and that in reference 32 deal with the kinetic energy distribution of ions from diatomic molecules. 34. Smyth, H. D., Revs. Modern Phys., 3: 347, 1931. Review of molecules studied to that date. 94 CHEMICAL REACTIONS IN THE GAS PHASE 35. Mott, N. F., and H. S. W. Massey, The Theory of Atomic Collisions, Second Ed., Oxford, 1950. 36. Kassel, Louis S., Kinetics of Homogeneous Gas Reactions, American Chemical Society Monograph, Chemical Catalog Co. DISCUSSION Burton : It is important to emphasize the tremendous gap which separates such funda- mental studies of isolated molecules as that presented by Eyring, and an understanding of reactions in condensed phases, which include biological mate- rial. In attempting to bridge this gap, if only crudely, several questions are significant. What is the quantitative role of the cage effect? A molecule may be in one particular cage for less than lO"^*^ sec after excitation, but the life of that cage in such a case is not of immediate importance. For practical purposes, the molecule is caged until it is deactivated or until it decomposes. If the latter process occurs, the life of the cage thereafter is of great importance. If the radicals can recombine, or otherwise interact, during a time approximating the life of the cage, we have a cage effect. Otherwise, there is no cage effect and the reaction with the solvent dominates. The hfe of the cage evidently depends not only on the statistical behavior of the radical products and the surrounding molecules but also, as Eyiing notes, on the energy with which the radicals are formed. How fast does internal conversion of energy occur as compared with external loss (for example, collisional deactivation)? Can a chemical reaction involving an excited molecule occur? Does internal conversion of energy occur fast enough so that a particular degree of freedom acquires sufficient energy for rupture, or does the molecule, as it were, cool off before this happens? One must consider both the rate at which heat is lost externally and the probability of escape of the radicals from their cage before recombination. In the theory of the radiation chemistry of solutions such factors must be considered in addition to those discussed by Eyi'ing. In the case of a water molecule, caging of the decomposition products need not be considered, since the hydroxyl radical and ionic hydrogen primarily formed do not back-react. Eyring: Are there examples of reactions studied in both the gaseous and liquid phases of water? Are the products of reactions in these two phases much the same? Platzman (Communicated) : There are no examples of aqueous reactions so studied. Radiolysis of water vapor has not yet been investigated, and interest in aqueous solutions has focussed on non- volatile solutes. Lind: Eyring will undoubtedly recall the classical experiments of Schoepfle and Fellows in which liquid hydrocarbons were irradiated with high-speed electrons. These experiments showed that the greater the branching of the hydrocarbon, DISCUSSION 95 the more methane was obtained. Since Eyring states the opposite to be true in the gaseous state, I beUeve we have here an instance of the distinction that Burton has discussed. Eyring: We are unable to explain this difference. Certain differences in reaction in the liquid and gaseous states are due to the cage effect and to the fact that the energy has not time to migrate when the irradiation is carried out in the gaseous phase. In the gas the molecule may fly apart when it is hit by the bombarding particle, whereas in the liquid state there is time for some of the energy to be degraded into heat. Burton: The products of irradiation of both liquid and gaseous organic compounds are characterized by their complexity, but there is some indication, as Eyring sug- gests, of a smaller diversity of products in the liquid phase. Allen : Previous talks might give the impression that radiation chemistry is such a hopelessly complicated subject as to be of little use to radiobiologists. However, experiments in radiation chemistry frequently give reaction rate daws which appear to be reasonably simple and rational. In solution, especially, the ob- served phenomena can be correlated in a sensible fashion and certain valid predictions made. As in other fields of chemistry, a good deal can be understood about reactions without attempting to ascertain the complete details of the molecular dynamics of each reaction. In radiation chemistry, the attempt to discuss in full detail the nature of all the types of activation, though of interest to radiation chemists, may well tend to give other people too pessimistic an impression of the values and possibilities of this field of study. Burton (Communicated) : The techniques, disciphnes, and speculations of radiation chemistry are similar to those of other branches of chemistry. Efforts toward detailed understanding are common to all branches of science, and the existence and relation of such efforts should prove a source of encouragement to those not actively engaged in the field. Such efforts are in the direction of simplification and unification. Detailed understanding and well-developed theory Hmit the number of facts which must be remembered and indeed make a subject more attractive to the uninitiate. Platzman : If chemical reactions — thermal, photochemical, or radiation-chemical — seem to be simpler in the liquid than in the gaseous phase (a debatable impression), it is probably because we know so much less about them that we have inadequate empirical or theoretical information about their complexities. 96 CHEMICAL REACTIONS IN THE GAS PHASE Hart: The complexity of reactions in aqueous solution is illustrated by the work of Gordon and myself at the Argonne National Laboratory. We have irradiated acid solutions containing dissolved deuterium gas and find that deuterium is converted first to HD and then to H2. In other words, there is a gradual re- placement of the deuterium gas by hydrogen gas. On the other hand, at pH 12 there appears to be direct conversion of the deuterium to hydrogen without the formation of HD as an intermediate. This has been interpreted as indicating a reaction between the OH radical and D2 to yield HOD and D ; the free D atom then reacts with an 0H"~ ion to form HOD". The latter ion then exchanges with the hydrogen in H2O, forming HOH~, and this decomposes to give free hydrogen atoms which combine to produce molecular hydrogen. In the basic solution at pH 12 there is less than 10 per cent as much HD as is obtained in the acidic solution. Solomon : We have been using a mass spectrograph in the determination of the abun- dance of deuterium in biological samples, and read the H peak ratio with an electron beam at 75 volts. Because of the formation of H3, it is necessary to extrapolate the readings to zero pressure, as the H3 is pressure dependent. However, th^ slope of the curve of pressure dependency does not appear to vary in a discoverable way with the pressure, with the electron density, or with any other known variable. In other words the Hs-ion formation does not seem to be proportional to any known factor in the mass spectrograph. Is there an explanation for this? Eyeing : No information is available, as far as I know. /• On the Primary Processes in Radiation Chemistry and Biology ROBERT L. PLATZMAN Department of Physics Purdue University Lafayette, Indiana Introduction The first stage in the interpretation of chemical or biological changes which result from the exposure of any material to radiation of high en- ergy must be the elucidation of the primary physical effects of the radi- ation. Radiation chemistry and radiobiology bear a close relationship to photochemistry and "photobiology." In both, radiation from an exter- nal source (less commonly, from an internal one) is allowed to impinge upon an aggregate of molecules in stable, or almost stable, states, thereby raising some of the molecules to excited states and permitting chemical interactions which could not proceed between normal mole- cules. The incident energy functions as activation energy, or as requisite energy for endothermal processes, or both, and is ultimately partitioned between degraded energy (heat), altered chemical (potential) energy of the material, and occasionally also emitted radiation. The primary processes of "high-energy radiation" effects, however, dif- fer from those of photoeffects in their tremendously greater complexity. This distinction is incisive and affects even the simplest reactions in- duced by high-energy radiations. Its origin lies in the fact that ea;Ch particle of high-energy radiation has energy of from thousands to many millions of electron volts (ev) * — magnitudes very much greater than the energies of the more probable transitions of a molecule, which are usu- ally less than 15 ev. On the other hand, the energy units absorbed in * This discussion is for the most part restricted to radiations of energies not greatly in excess of those of common nuclear radiations, for which (with the excep- tion of neutrons) specific nuclear interactions are so rare as to have negligible in- fluence on chemical or biological effects. The problems are therefore ones of atomic and molecular physics and of chemistry, and not at all of nuclear physics. 97 98 PRIMARY PROCESSES photochemical reactions, commonly those of photons of visible or ultra- violet light and therefore in the region of 2-10 ev, lie just in the range of the most likely transitions of molecular electronic systems. Whereas in photoeffects the quanta are absorbed in single events, in high-energy radiation effects the energy loss occurs gradually, in hundreds or many thousands of steps. Hence use of monochromatic light for a photochemi- cal reaction ensures, in general, the excitation of a unique excited state, but monoenergetic high-energy radiation — for example, homogeneous al- pha or beta rays — always produces a very great number of different types of excited and ionized molecules of quite widely varying energy. Another contribution to the complexity displayed by the primary processes of radiation chemistry and biology arises from the fact that many, and usually the majority, of the products are formed, not directly by the incident radiation, but indirectly by secondary, tertiary, etc., radiations which the primary radiation produces. It would evidently be extremely difficult to gain full knowledge of the products of absorp- tion of even the simplest high-energy radiation. The nature of the primary processes has already been discussed by the first panel of this symposium. These primary processes are almost all separate excitation or ionization events in isolated atoms or molecules of the medium. The processes can be described in a general way, and such a description is invaluable background for understanding chemical and biological effects of radiation. We may be permitted to emphasize, however, that quantitative prediction of the physical effects is within the realm of possibility, at the present time, only for gaseous media com- posed of monatomic molecules, because a necessary basis for comprehen- sive treatment of the physics of a radiation process is the knowledge (that is, of constitution, energy, stability, and other specifications) of the possible stationary states of the system. Such information on sta- tionary states, obtained principally from spectroscopic investigation, is available in detail only for atoms: excited and ionized states of diatomic molecules are far less extensively known, owing to the overwhelmingly greater difficulty in interpreting the spectra; in the case of polyatomic molecules our knowledge is hardly more than in its infancy, and great progress in the near future is not to be anticipated. Some knowledge of ionized states, but not of excited states, is obtainable from mass spectrographic studies for both atoms and molecules; however, it is of highly restricted content. It would be a sophistry to deny that contem- porary radiation physics, although it provides most of the information ordinarily required by the -physicist, customarily disregards consequences of chemical binding, and is scarcely more than a general guide to the understanding of the details of the primary processes in chemical and PROCESSES OTHER THAN SIMPLE 99 especially in biological systems, the latter being invariably composed of molecular species of more or less great complexity. Much the same difficulty, indeed, also underlies the interpretation of the effects of light on all but the simplest gaseous systems, and this fact has inhibited serious study of such effects in complicated systems, particularly those in the liquid state. For this reason trustworthy infor- mation available from photochemistry is, for complex systems, regretta- bly meager. Hence the important task of interpreting results of radi- ation effects — that is, of analyzing the extremely intricate stages be- tween initial absorption acts and ultimate chemical or biological trans- formation— must usually proceed with inadequate aid (from physics, on the nature and distribution of the primary products, and from chemistry, on their interactions), and is usually exceedingly difficult. In all too many instances in which interpretations have been advanced they in- volve suspicious radicals or ions, endowed with mainly ad hoc properties, which effect remarkably convenient actions under almost no discipli- nary control except perhaps an occasional admonition from the laws of thermodynamics. Despite this discouraging basis, some success has been achieved in the understanding of at least a few of the chemical effects of radiation, and notable progress in the field is now being made. It is even possible to understand some of the less intricate reactions in complex systems — at least semiquantitatively, if not in the same satisfying detail achieved by Eyring, Hirschfelder, and Taylor in their uniquely important studies of several gaseous systems some fourteen years ago. Indeed, some striking inferences may often be drawn from an analysis which penetrates no more deeply into the nature of the primary effects than simply to dis- tinguish between excitations and ionizations. These matters are dis- cussed elsewhere in this volume. We may merely note here the familiar conclusion that the experimental evidence thus far interpreted appears to support the identification of excitation and ionization acts with the predominating primary processes. Primary Processes Other Than Simple Excitation AND Ionization Nevertheless, it would be incautious at the present time to presume that chemical and biological effects of high-energy radiation are caused exclusively by isolated excitation or ionization events in which energy transfers lie in the neighborhood of 10 ev. Whereas these primary proc- esses are surely the ordinarily dominant ones for media composed of simple molecules, complex molecules of high molecular weight may well 100 PRIMARY PROCESSES be found to differ in important respects. Especially if a complex mole- cule should be exceptionally resistant to the effects of a small energy transfer, or should have ability to recover from such effects, must al- lowance for other possibilities be made! (That such behavior may not be uncommon is suggested by both empirical evidence and theory.) Great energy transfer to such a molecule arises, according to the point of view commonly held, only from the accumulated effect of a number of small energy transfers to individual atoms of the molecule considered as isolated entities. The probability of great energy transfer would thus be intimately related to the specific ionization at the appropriate point along the path of the incident particle. In the case of densely ionizing particles, such as alpha particles and especially heavier positive ions like recoil atoms or fission fragments, rather great energy transfer to a single large molecule may be achieved, and under favorable circum- stances much greater permanent changes than would result from equiva- lent dosages of less densely ionizing radiations can be anticipated. Of course, the effects of specific ionization or, expressed somewhat more significantly, of the spatial distribution of excited and ionized atoms and molecules at the instant after the incident radiation has penetrated the medium, and of the fluctuations in this distribution, are well appreci- ated and are an important factor in the interpretation of ultimate chemi- cal and biological effects of radiations, although they have not yet been analyzed in any quantitative detail. However, the fact that the individual component of incident radiation has energy that is orders of magnitude greater than the average energy transfer per excitation or ionization act should always be borne in mind. Relatively great energy transfer from a single particle to a single mole- cule is certainly infrequent, but it is not impossible. The smallness of the average energy transfer results from the particular nature of the chief interaction between incident particle and molecule, and other types of interaction, usually much less probable, could conceivably lead to energy transfers of different magnitude. [One should not regard as being in this category the great primary energy transfers which certainly occur when a very fast secondary electron (delta ray) is produced by a charged particle, or a photon of bremsstrahlung is radiated by a fast electron, for this great quantity of energy is subsequently dissipated in a number of secondary events to many molecules, the average energy transfer per molecule still being in the 10-ev region.] It therefore seems not without importance to examine possibilities of energy transfer other than simple excitation and ionization, and several such are considered below. The discussion is deliberately rather general, but is supplemented by calculations for an illustrative case. The processes all have rather low NUCLEAR COLLISIONS 101 probability compared to simple ionization, and thus would seem to of- fer promise of importance (in the absence of chain reactions in inter- mediate stages) principally in the case of reactions of low yield per "ion pair" or low "target area." Of the processes one, especially, affords a mechanism of great energy transfer to a small volume — perhaps a single molecule— and appears to have quite appreciable probability in many instances. Nuclear Collisions Some fraction of the energy of any high-energy radiation penetrating a medium is lost in direct momentum transfers from charged particles to atoms as whole units. The primary process here is customarily called a nuclear collision, because the effective interaction is that between the (screened) Coulomb fields of the particle and the nucleus of the atom. An atom experiencing an impact of this type is often ejected from its original position in the medium, and will then usually come to rest else- w^here. If the struck atom acquires sufficiently great kinetic energy, some of this may be lost in excitation and ionization; the remainder, and practically the entire energy of more lightly ejected atoms, is ex- pended in further nuclear collisions. Much of the energy that is trans- ferred by this mechanism goes directly or indirectly into excitation of molecular vibrations and is for the most part ultimately dissipated into heat; some of the original energy loss, however, is preserved as aug- mented potential energy of the medium, deriving from the altered atomic arrangement. The nuclear collision is known to be the effective process in the observed disordering of the structure of a solid substance by heavy charged particles (the latter being either the primary radiation or second- ary particles projected under neutron irradiation). Indeed, this effect is apparently not brought about, at least with appreciable yield, by sim- ple ionization events. For most of the range of an energetic charged particle this mode of energy loss contributes a very minor fraction — for protons or alpha parti- cles in media composed of light atoms roughly 0.05-0.10 per cent — of the total. For very slow heavy particles, and therefore for initially en- ergetic particles near the end of their ranges, it is the predominant process, but the energy of these particles is so low (of the order of 1 kev for alpha particles) that the total energy transferred to nuclear mo- tion is small. Thus, to cite an example, rough estimate shows that a 10-Mev alpha particle absorbed in air will lose 8 kev in nuclear colli- sions, of which about 1 kev will be lost at the very end of the range. In light media about two-thirds of the nuclear-collision energy loss will be in collisions violent enough to remove the struck atom from its mole- 102 PRIMARY PROCESSES cule, and for, say, a 10-Mev alpha particle the total number of ejected atoms (including ejections by the ejected atoms themselves) will be of the order of 100. For high-energy particles the number of ejected atoms is invariably very much smaller than the total number of excited and ionized atoms (as in the example cited, where the ratio is of the order of 10~^), and since the latter entities are usually chemically effective, nu- clear collisions are usually unimportant and are properly neglected in considering the primary processes. Whether cases exist in which elec- tronic energy transfer is so ineffectual in causing chemical or biological change that the nuclear collisions are of consequence is not known. It is certainly not inconceivable that in some instances energy transfer to nuclei might play a decisive role in effecting changes in very large, stable molecules, for the ejection of an atom, particularly if this in turn ejects other atoms from the same molecule, will severely and probably perma- nently damage the molecular structure. It is evidently much less likely, in general, that a molecule will recover from loss of an atom than from loss of an electron. Fast electrons also may lose energy in nuclear collisions, but even if they have very great energy (their mass then significantly exceeding the small rest mass) the number of collisions sufficiently violent to eject atoms from molecules is extremely small — much smaller, relative to the number of ionizations produced, than for energetic heavy particles. The nuclear-collision mechanism is therefore certainly negligible for electron or gamma-ray irradiation. For heavy ions, such as fission fragments, the energy loss in nuclear collisions is relatively greater than for bare nuclei like protons or alpha particles, because, compared to the ionic charge, the nuclear charge of the particle is higher and the internuclear Coulomb interaction there- fore of more consequence. This suggests that for these radiations the process might be more likely to achieve importance in determining chemi- cal and biological transformations. The same conclusion applies for slowly moving recoil ions — for example, some of the recoils from fast neutron collisions — and the effect would seem to merit study, for in- stance in evaluating dosages for these recoil ions. In order to provide quantitative illustration of some of the matters discussed above. Figs. 1-3 (pp. 111-112) present information on the nuclear collisions of protons in water, which was chosen for convenience in computation and also because it is a representative medium (in this respect) for radiobiology. The figures, and the calculations upon which they are based, are explained in the appendix below. Similar calcula- tions could be made readily for any medium the atomic composition of which is known. MULTIPLE IONIZATION 103 Multiple Ionization A primary process not often considered in radiation chemistry and biology is the formation by the incident particle, in a single collision, of a doubly (or, in general, multiply) excited or ionized atom or molecule. Such a process, it will be shown, is probably not of importance in most instances. Nevertheless, it certainly ought not to be completely ignored, for although undoubtedly infrequent it could in some special cases con- ceivably have much greater permanent effect than a number of isolated single excitations or ionizations. (The latter are usually chemically effective for simple molecules, but, as mentioned above, are perhaps less commonly so for complex molecules.) Multiple processes produced by consecutive impacts of two different particles of the radiation are, of course, utterly negligible in all cases. Multiple excitation or ionization in a single event finds perhaps its most conspicuous physical manifestation in the excitation of non-dia- gram, or satellite, x-ray emission lines, some of which owe their origin to multiple ionization of inner shells of the target atoms by fast electrons. Unfortunately, contemporary theory does not offer much possibility of accurately calculating the yield for this type of process — for example, the relative probability that a charged particle will in a single collision produce a doubly or singly charged ion — except for the simplest of atomic systems. This is because the approximation that one is ordi- narily obliged, for reasons of tractability, to use for the possible station- ary states of the affected atom (the so-called single configuration, cen- tral-field approximation) is such that multiple excitation and ionization events automatically have zero probability, that is, are inherently neg- lected. The use of a more realistic atomic model imposes the greatest calculational difficulties, even for single processes; and, moreover, su- perior models are not generally available for substances of chemical or biological interest. The only theoretical treatment of a multiple col- lision process thus far accomplished is one for the excitation of some of the x-ray satellite lines mentioned above. It should be permissible, however, to disregard multiple excitation, since this process seems less likely to play a significant role than does multiple ionization. (By multiple excitation is normally meant the simultaneous excitation, by a single passing charged particle, of several electrons in a single atom or in atoms closely coupled together in a mole- cule. Excitation by a single particle of two or more widely separated atoms in a molecule will more often be important. It can be treated by theory as a special case in the consideration of the spatial distribution of the energy loss. Since the collision time for a fast particle and a not 104 PRIMARY PROCESSES too large molecule is of the order of 10~^^ sec, while roughly 10~^^ sec is required for significant internal reorganization of atoms in a molecule, such multiple excitations are also effectively "simultaneous." This dis- tinction, although not always well defined, is a useful one. Either type of multiple excitation could be important if more energy than is provided by a single excitation is required to disrupt the molecule.) There does exist some empirical information on multiple ionization by impacts of electrons of low and intermediate speeds, almost all of it for monatomic gases. (Slowly moving charged particles are known, on general grounds, to be much more effective than rapidly moving parti- cles in producing multiple processes, the yield of the latter being weighed relative to that of single processes.) The results on the noble gases, which are the simplest to interpret, indicate a yield for the production of an (n + 1) positively charged ion roughly one-tenth that of the cor- responding -{-n ion for electron energies from several times the ioniza- tion potentials up to 500 ev, the upper limit of the experiments. (For helium the relative yield of He"^"*" to He"*" is much smaller — of the order of 10~^.) The yields of multiply charged ions are, however, very much lower at smaller energies (and are, of course, zero below the respective ionization potentials), and this is the region in which most of the second- ary electrons ejected by high-energy particles fall. For the few other gases investigated, the relative yields, although not exactly similar, are at least of the same order of magnitude. Thus the over-all yield of initially doubly charged ions produced by high-energy radiation would be expected to be very small — less than 10~^ and perhaps less than 10~^ of all ions. The contribution of the primary ionization should not alter this conclusion, although experimental information on the question is unsatisfactory and is in fact entirely lacking for beta or gamma radi- ation. Experiments performed some 30 years ago failed to detect multi- ple ions, either primary or secondary, in yields greater than about 1 per cent, produced by alpha particles in a variety of gases. (Some re- sults indicating 5-15 per cent yield of He"*""^ relative to He"'' in helium gas, by alpha particles at various speeds, attracted much attention at the time. They have been interpreted as proving that in helium half of the primary ionization acts near the maximum of the Bragg curve create double ions. If true these results are remarkable and merit further study. They are not relevant in the present considerations, however, and the evidence on other gases is clear.) Virtually nothing is known about multiple processes in molecular sys- tems of chemical or biological importance. A conservative estimate suggests 10~^ as an upper limit for the relative yield of doubly ionized atoms or small molecules produced directly in single events by most CAPTURE AND LOSS OF ELECTRONS BY POSITIVE IONS 105 varieties of radiation. For radiations of high specific ionization the rela- tive yield will be greater, but it would be difficult to treat this situation even semiquantitatively. That multiple ionization must be extremely effective chemically can be concluded from the fact that even doubly ionized molecules tend to be highly unstable; they are, for example, observed only rarely in mass spectrometric studies. The basis for this instability is clear: even if the doubly ionized molecule should be formed in a stable state, the potential surface for this state will in general cross that of the repulsive state formed from two singly ionized radicals. The latter state always lies lower than the former at great nuclear separation because more energy is required to doubly ionize one atom than to singly ionize two. This instability can be pictured very crudely as resulting from the capture by a doubly ionized atom of an electron from an adjacent neutral atom in the molecule, the two singly ionized atoms then dissociating the mole- cule by their repulsion. (Recall that two electronic charges separated by a distance of 1 A, the C — H bond distance, repel each other with an energy of 14 ev.) The problems posed by multiple ionization of com- plex molecules are obviously much too elaborate to permit their dis- cussion here. (For the lines along which an analysis should proceed, cf . the contributions to this volume by Eyring et al. and by Livingston.) We note finally that multiple ionization is to be expected for all high- energy radiation and for all media except hydrogen and helium as the result of Auger processes follow^ing ionization in inner electron shells. The yield relative to single ionization events, for biological media, should be of the order of 10^^ to 10~*, and will usually exceed that of direct multiple ionization. These effects and their consequences are discussed in detail below. Capture and Loss of Electrons by Positive Ions Another mode of energy loss, possible only for a positively charged particle, is the capture of an electron from a molecule of the medium into a discrete orbit about the particle and its subsequent loss in a later collision. This pair of events occurs a few thousand times, for example, in the absorption of a single alpha particle. _ It is important, for light ions, only when they are slow — and thus for an energetic particle only near the end of its range. Indeed, it is a principal mode of energy loss for an alpha particle of energy between (roughly) 1 and 500 kev. The net effect of such a pair of events is simply the ionization of a single molecule of the medium, the positive ion and electron being formed, how- ever, at a distance from each other. This should be an unimportant dis- 106 PRIMARY PROCESSES tinction, which fact — together with the small total energy loss by this mechanism — will make the process unimportant in the interpretation of most chemical and biological effects. A possible exception to the last conclusion might arise in the case of irradiation with rather slow neutrons, for which the recoil ions will often have velocities in the capture-loss region. Nevertheless even here the spatial distribution of ions will not be very abnormal, because just for those particle velocities for which capture and loss predominates, the cross sections for the two processes approximate geometrical cross sections, so that positive ion and electron originate at almost the same place. The total ionization in this velocity region, however, may differ considerably from that anticipated on the basis of extrapolation of knowledge for high velocities (the partition of the total energy loss between excitation and ionization may be quite different at low from that at high energies, and nuclear collisions have an important effect). There is as yet very little information on this question, and the pos- sibility of peculiar effects should not be discounted. Auger Disruptions Although most of the energy lost by high-energy radiation is trans- ferred to valence electrons of molecules of the medium, a not inappreci- able portion is absorbed by inner electrons of the atoms. The remark- able effects to be anticipated for this part of the energy loss apparently have not been pointed out before. The media of importance in radiobiology, and in much of radiation chemistry as well, are composed almost entirely of two types of atoms: hydrogen, having only a valence electron, and carbon, nitrogen, and oxygen, which have inner (K) electrons in addition to the outer ones. (Presence in the medium of small amounts of heavier atoms will not affect any of the conclusions to be drawn.) An important and repre- sentative medium is water, and, as an example, Fig. 2 illustrates the total fraction of the energy of penetrating protons which is transferred in primary collisions to the K electrons of oxygen atoms when the pro- tons are completely absorbed in water. (Figure 1 shows the manner in which this portion of the energy loss is distributed along the range of the protons.) The fraction, although small, is by no means negligible. In the case cited, for example, it is 4 per cent at 3 Mev and increases with the proton energy. Transfer of energy to a K electron almost in- variably ejects that electron from its atom, an ion with an inner vacancy thus being formed. The average energy transferred in such processes is several times the K ionization energy (which is 531 ev for oxygen) AUGER DISRUPTIONS 107 and varies somewhat with the velocity of the particle. Ionization of K levels by secondary electrons is entirely negligible. The ejection of inner electrons by swift charged particles — both electrons and heavy particles — has been verified experimentally in the observation of characteristic x-rays emitted by atoms during irradiation with these particles. Where a K shell of a C, N, or O atom has been ionized, an energy transfer of some hundreds of electron volts to the molecule containing the atom has occurred. (The exact values are 284 ev for C, 400 ev for N, and 531 ev for 0.) If the molecule is moderately or very large, it will retain all this energy! Creation of a 7v-shell vacancy in these very light atoms is not followed by emission of an x-ray photon, as is the case for a heavy atom. There ensues instead a radiationless transition in which an L electron drops into the vacancy and a second L electron is ejected from the atom. Thus a doubly ionized atom results. (Before emitting a photon of K radiation, an atom persists in its excited state for a period of the order of 10~^Z~^ sec, where Z is its atomic number. For very light atoms this is so much longer than the time required for radiationless transition that the latter almost always occurs first. The yield of radiation from C, N, or O is certainly smaller than 1 per cent.) Such a process, commonly called an Auger transition, takes place within a time interval of about 10~^^ sec. It is so much faster than any possible motions of atomic nuclei in the molecule that, as follows from the Franck-Condon principle, the nuclei cannot respond appreci- ably until after the second electron has left. Whether the ejected elec- tron must be one belonging to the same atom that suffered K ionization, or may originate in an atom bonded to it, is not known, and the question, moreover, is not free from ambiguity. In any case the distinction is not very important, for the molecular ion will have ample time for elec- tronic readjustments before dissociation proceeds. It is also worthy of mention that in some instances not only one but several Auger transi- tions might follow a single K ionization, for very much more than suf- ficient energy is available to ionize several valence electrons at or near the site of the original vacancy. The great quantity of energy transferred to a single atom when one of its inner electrons is ejected will not remain in that atom, of course — except for the minor portion retained by virtue of the ionization (and perhaps also excitation) of the valence shell. Where the atom is bound in a large molecule, however, the energy carried away by the Auger electron (s) will not escape, for such electrons dissipate their energy in excitations and ionizations within a distance of less than about 10 A. (Indeed, it may be proper to raise the question whether, in the case of polyatomic molecules, it is valid to consider the Auger transition and 108 PRIMARY PROCESSES the reabsorption of the Auger electron (s) as distinct events: these proc- esses may in fact be coupled together to some extent, at least for that portion of the energy reabsorbed by an adjacent atom. In this connec- tion it is of interest to note that an Auger electron of 100-ev energy has a speed of about 10^ cm per sec, and therefore is "reabsorbed" by the molecule in a time of the order of 10""^^ sec or less. This is very much shorter than the time required for molecular rearrangement. Regretta- bly little is known about Auger transitions in very light atoms, experi- mental investigation being hampered by extreme difficulties of a practi- cal nature. The structural changes consequent to Auger effects in light atoms bound in molecules can, however, be investigated more readily. Such studies have only recently been initiated. They should ultimately provide much information of interest.) Although the exact series of mechanisms cannot be mapped with cer- tainty, there can be no doubt that, wherever a K ionization occurs, a relatively great amount of energy is communicated to a small region of space — perhaps a single molecule or a portion of a very large molecule. The "primary process" embraces the effects of the initially ejected K electron, of the Auger electron (s), and of the multiple ionization, all centering at the atom originally affected. This energy, transferred in a single primary encounter, will soon be converted to molecular jwtential energy by electronic "rearrangements" (including internal conversion) and will then usually shatter the molecule by a complex polyatomic dis- sociation, the latter occurring rather slowly, that is, not as a direct pri- mary effect. (Indeed, the process could result in the disordering of a solid — even by electron bombardment, for which the amount of dislo- cation by direct momentum transfer is very small.) The concentration of absorbed energy will in fact exceed that arising on the average from direct energy transfer to electrons in the penetration of the medium by a beta or gamma ray, and may even exceed that sustained in penetra- tion by radiation of such high specific ionization as an alpha particle. Its possible importance for effects not induced by small energy transfer is therefore impressive. Such effects are customarily ascribed exclusively to delta rays, which transfer relatively much energy to a small volume. The relative importance of the two mechanisms cannot be elucidated without a much more detailed analysis; however, an approach to this analysis could readily be based on a simplified model, and such a study would doubtless prove most interesting. It is evident that the relative contribution differs for different types of irradiation and different media. The mechanisms are intrinsically distinct from the physical view: in one case energy is transferred only as direct momentum transfer to an elec- tron; in the other, it is transferred to the electronic system of a single AUGER DISRUPTIONS 109 atom and is subsequently communicated to the neighborhood of the atom. The connotation for interpretations in radiation chemistry and biology is obvious. Many effects are known which have yields, per "total" number of ionization acts, of magnitude 10"" or less. The possible role of "Auger disruptions" should merit earnest consideration in some of these cases. Effective dosages, in any instance where this mechanism is operative — if any such be found — would be illusory if computed on the basis of total ionization. There the number of K ionizations of C, N, or 0 would be the relevant measure. It is possible to calculate this number purely from theory. Figure 3 presents the results of calculations, for protons penetrating water, which, it is hoped, may be useful for comparison with practical cases for which this mechanism may be considered. For non-aqueous media studied in radiation chemistry more elaborate calculations would have to be made, account being taken in the case of atoms of intermediate or high atomic number of electron ejection and subsequent Auger effects in other inner shells as well. With increasing atomic number the yield of ejections from any given inner shell decreases, the fraction of ejections which lead to Auger transitions also decreases, but the energy transfer per ejection increases; indeed, a cascade of suc- cessive Auger effects should occur, leading in some instances to an atom or molecule with many electrons missing. Because of the importance, for very swift charged particles penetrating atoms of intermediate or high atomic number, of energy transfer to electronic shells other than the valence shell. Auger processes might well have conspicuous effects in media in which such atoms preponderate. For biological media the yield of Auger disruptions is greater than is suggested by the values given for water, for two reasons. First, the effect increases, other factors being the same, with the ratio of the total number of K electrons in C, N, and 0 to the total number of electrons other than K electrons in atoms of the medium. This ratio is greater for biological media than for water: for water it is 1 : 4; for carbohydrates and for glycine it is 1:3; for dry virus it is about 1:2.9. Second, and more important, the probability for ejection of a K electron from an atom of atomic number Z by a charged particle increases rapidly as Z decreases: for high particle energies this increase is as Z~^; for very low (heavy) particle energies it is as Z"^^. The total number of Auger dis- ruptions for any medium can be computed approximately from theory if the atomic composition of the medium is known. It is notable, and merits emphasis, that the effect for the K shell is relatively great (the ratio of number of Auger disruptions to total number of ionization acts no PRIMARY PROCESSES being, in favorable cases, as high as 10~^ to 10"^) just for atomic num- bers in the region near C, N, and 0 : for larger Z the probability of K excitation is very small, and also the probability of x-ray emission com- petes more favorably with the Auger process. Summary The primary processes in the absorption of high-energy radiations by matter are considered in relation to the chemical and biological effects of the radiations. The importance of achieving a detailed understanding of these processes is discussed, and the reasons for the extreme com- plexity of the problem analyzed. Although simple isolated excitation and ionization events are the preponderant primary process, the pos- sibiHty of greater chemical effectiveness, especially in complex molecules, of rarer events — particularly those involving greater-than-average en- ergy transfer — suggests examination of less probable primary processes. Several relatively infrequent processes are, therefore, investigated. Of these, the nuclear collision may well be of chemical consequence in some cases; direct multiple excitation or ionization is probably unimportant; capture-and-loss of an electron is certainly unimportant as a distinct process, although it may well influence the partition of the energy loss between excitation and ionization; ejection by a swiftly moving charged particle of an inner atomic electron, followed by Auger "disruption" of the molecule, is a process, in effect one involving a great energy transfer, which is distinct from great energy transfer to a secondary electron, and, although no specific apphcation is offered, it is concluded that this process might in some instances play a significant role. The results of detailed calculations of the extent of some of these processes in a typical case of interest (protons penetrating water) are presented. SUMMARY 111 ton. Mev Fig. 1. Various modes of energy loss (stopping power) of protons in water. 112 PRIMARY PROCESSES ^2 ^ Fraction of total energy lost in >^ ejecting K electrons of oxygen ^^>^ ^ 1 / Jx ^ / Fraction of total energy 'lost in nuclear collisions for which AE^ 20 ev 1 i — 0.5 1.0 1.5 2.0 2.5 3.0 £n Mev Fig. 2. Fraction of total energy of protons penetrating water which is lost in ejecting K electrons of oxygen, and in "violent" nuclear coUisions. 80 60 40 20 / K electrons ejec ed from oxyg ;n-^^ ^ V Nuclear collisions for which A£>20 ev 0.5 1.0 1.5 2.0 2.5 3.0 ' proton Mev Fig. 3. Total number of K electrons ejected from oxygen atoms, and of "violent" nuclear collisions, for protons penetrating water. APPENDIX. EXPLANATION OF THE FIGURES 113 APPENDIX. EXPLANATION OF THE FIGURES Fig. 1. Various Modes of Energy Loss of Protons in Water (a) The total electronic energy loss (stopping power), newly recalculated, is plotted as a function of proton energy. For more suggestive reference in inter- pretations of radiation chemistry and biology, the stopping power is expressed in units of electron volts of energy loss per angstrom of distance traversed, for water of density 1.00. (The distance between nearest neighbor molecules in water is about 3 A.) The basis for the calculation of these values of the stopping power is discussed in detail in reference 7. Briefly, it is: 1. The stopping power of water is assumed to be equal to the sum of the stopping powers of its constituent atoms. 2. The Born approximation is presumed to be valid, and the theory of Bethe (6) is therefore applied. 3. The simple Bethe stopping-power formula is, however, corrected for its premise that the orbital velocities of the various atomic electrons are negligibly small compared to the proton velocity: for the K electrons of oxygen, by the accurate method developed by Bethe (6) and recently modified (2), and for the electron of hydrogen, and the L electrons of oxygen, by a prescription of yet unestablished validity proposed by Hirschf elder and Magee (5). 4. The empirical constants representing the mean excitation potentials of hydrogen and oxygen atoms are redetermined from data on the stopping powers of the elements and some of their compounds. 5. At low energies — specifically, for proton energies less than about 0.3 Mev— no competent theory exists. The stopping power in this region is therefore estimated as well as possible, using experimental data on water vapor obtained by Crenshaw (3). The results cannot be considered trustworthy in this region. Because of some of the approximations used, and the absence of adequate experimental infor- mation on the stopping power of water itself, the data are presented with no assurance that they are more than a qualitative guide (7). Values of the stopping power of water for alpha particles can be obtained from those for protons by use of the famihar relation: — ■ ) (for alpha of energy E') ^^ ' (dE\ = 4 ( — ) (for proton of energy E = 0.2517^') \E' > 1 Mev] \dx/ For proton energies greater than about 3 Mev (but not so great that relativ- istic corrections are demanded) the energy loss may be computed directly from the formula: \ dx) ^^ (1.525 + logio E) ev/A [E in Mev] E 114 PRIMARY PROCESSES (b) A second curve presents the contribution to the stopping power of energy loss to the K electrons of oxygen, and is calculated from results of Brown (2). Values for the corresponding energy loss for alpha particles may be obtained from those for protons by a relation similar to that given above. For extremely great values of E, the energy loss to the K electrons approaches 19 per cent of the total energy loss; for the energy region treated here, however, this contri- bution is smaller, being 7 per cent at 3 Mev, for example. (c) A third curve gives the energy loss in nuclear collisions, calculated by methods developed by Bohr (1). Since this treatment uses a Thomas-Fermi approximation to the screening, it will be somewhat inaccurate for these light atoms; however, the error should be sUght. (d) Finally, there are presented values of that contribution to the stopping power which derives exclusively from the more violent nuclear coUisions. By a "violent" coUision is meant (here) one in which the struck ("recoil") atom is ejected from its molecule. For convenience in calculation, violent collisions are assumed to be those in which more than 20 ev is transferred to the (H or 0) recoil atom. Such a model contains the effect of chemical binding on atom ejection — very crudely, to be sure. The W ev is an estimate; the conclusions, however, are not very sensitive to the value of this quantity. It will be seen that such violent collisions contribute about one-half of the total nuclear- collision stopping power. (Although less numerous, the events involve greater energy transfers.) Fig. 2. Fraction of Total Energy of Protons Penetrating Water Which Is Lost in Ejecting K Electrons of Oxygen, and in "Violent" Nuclear Collisions This information is obtained by numerical integration of appropriate data from Fig. 1, and is subject to the same Hmitations mentioned above. Fig. 3. Total Number of K Electrons Ejected from Oxygen Atoms, and of "Violent" Nuclear Collisions, for Protons Penetrating Water (a) The cross section for ejection by a proton of a K electron from an oxygen atom is computed from results of Henneberg (4). Since he calculated the effect of screening for a case somewhat different from that of oxygen, values for the number of K ejections computed from his cross sections and presented in Fig. 3 are in error, but are too low. However, they are still trustworthy approximate values. A more accurate calculation could be made readily by numerical in- tegration of the transition probabilities given by Bethe (6), which are valid for oxygen. The total number of K ejections from oxygen atoms is computed by numerical integration from values of the cross section for K ejection and of the total stopping power of water (from Fig. 1). (6) The total number of violent nuclear colUsions (cf. above for definition of "violent") is computed by numerical integration from the simple Rutherford DISCUSSION 115 cross section (which is vahd for such coUisions) and the total stopping power of water (from Fig. 1). Note, however, that this gives only the number of violent primary coUisions; the total number of atoms ejected will be greater — often as much as twice as great — because some of the recoils eject other atoms. The figure shows clearly that at low proton energy (for water, below 1 Mev) most of the ejections occur at the end of the range, whereas at high energy most of them are distributed along the range, in approximately constant proportion to the total (that is, electronic) energy loss. REFERENCES (FOR APPENDIX) 1. Bohr, N., Kgl. Danske Videnskah. Selskah, Mat.-fys. Medd., 18: No. 8, 1948. 2. Brown, L. M., Phys. Rev., 79: 297, 1950. 3. Crenshaw, C. M., Phys. Rev., 62: 54, 1942. 4. Henneberg, W., Z. Phy.uk, 86: 592, 1933. 5. Hirschfelder, J. O., and J. L. Magee, Phys. Rev., 73: 207, 1948. 6. Livingston, M. S., and H. A. Bethe, Revs. Modern Phys., 9: 245, 1937. 7. Platzman, R. L., "Influences of Details of Electronic Binding on Penetration Phenomena, and the Penetration of Energetic Charged Particles through Liquid Water," Paper No. 9, p. 139 of this volume. DISCUSSION Fano: I sympathize very much with the general idea of emphasizing the limitations to the help that physics can give in these matters. At the same time I wonder whether Platzman's remarks might not cause undue concern in the opposite direction. On the whole, it would seem that physical theory does provide a reliable guide on how to analyze most of the practical questions relating to the physical action of radiation. True enough, the theory does not yield quanti- tative predictions as accurate as one might wish, but this lack of accuracy does not seem to me to be too critical at the present time. Platzman : I quite agree with Fano that radiation physics has contributed immensely by making possible the treatment of most of what he terms the "practical questions" of radiation action. There was no intention of depreciating this contribution. Rather, I have sought to stress how radiation physics has been inadequately developed, thus far, as an aid in understanding the fundamental chemical mechanisms of radiation effects. Magee: I should like to say a word about the chemical effect following the Auger process. It is my opinion that to consider the chemical effect as the result of electrostatic repulsion between the two charges is viewing the situation too simply. Considerable work has been done at Notre Dame on the isomeric tran- sitions in hydrogen bromide and deuterium bromide. Theory indicates that the 116 PRIMARY PROCESSES atoms may receive an average charge exceeding 4; nevertheless, it is found ex- perimentally that as many as 60-70 per cent of the molecules will not rupture their bonds. According to a simple electrostatic repulsion model, the bromine ion in this case would capture all available electrons, and, as a result, the positive hydrogen and the still positive bromine would repel each other with 100 per cent probabiUty of decomposition. The model obviously fails- for this simple case, and there is no reason to beUeve that it applies in any more compUcated one. Morkison: Platzman has brought out in interesting detail the role of what he has termed the nuclear coUision in the energy loss of charged particles moving through matter. By this designation he referred to the way in which energy was trans- ferred to the mass motion of the atom as a whole. That is to say, the collision arises from an interaction between the charged particle and the electrostatic field of the nucleus. In other words, the nucleus is here regarded not as a sticky point, but as center of an electrostatic field. This kind of reaction does occur, and it is to be clearly distinguished from the sort of collision discussed by Tobias and Wilson, in which there is a nuclear-force interaction between the nucleus and very high-energy protons or deuterons. It is interesting to compare the two for 200-Mev deuteron beams. Most such particles traverse their range without making a single nuclear coUision of the sort I have described, that is, a nuclear- force collision. There is about one chance in three that such a particle will make a nuclear-force collision. Wlien it does, it transfers a considerable amount of its energy, giving rise to a many-pronged star from which several columns of heavy ionization start out in several directions. If any large-scale structure of micron size responsible for racUobiological effects is located in one of these stars of ion- ization, the stars might give rise to biological effects. But the sort of nuclear collision that Platzman has been discussing, that is, the electrostatic field col- hsion, might occur in the order of 100 times, along the deuteron path, while each energy transfer would involve only enough energy to disrupt a few^ molecules. The difference betw^een these two types of characteristic nuclear events is worth mentioning. 8- Elementary Chemical Processes in Radiobiological Reactions * MILTON BURTON Department of Chemistry University of Notre Dame Notre Dame, Indiana The elementary chemical processes of radiobiology are the elementary chemi- cal effects of high-energy radiation on aqueous solutions containing oxygen, on pure organic matter, and on organic matter suspended in aqueous solution. In the aqueous layer the important primary physical process is ionization; in the organic portion both ionization and excitation must be considered. In biological systems the active entities in the aqueous layer are principally OH and HO2. The presence of the latter radical increases the volume of the effective aqueous layer around the biological particle, f The effectiveness of a hit in the organic material is determined in part by the properties of the surrounding cage and depends, among other factors, on the size of the biological particle. The effect of a hit may be propagated by ionization transfer, by free-radical diffusion, by a chain reaction, or by change in local pH. Furthermore, free H and resultant HO2 are formed in the ambient hquid even when the hit is in the particle itself. In view of these elementary processes the biological particle cannot be uniformly sensitive to radiation over its entire volume, the target of "target theory" is not to be identified wdth the biological particle, a single ionization act is not neces- sarily lethal, and the target dimension is not simjDly related to the true size of the biological particle. From the chemical viewpoint the elementary processes of radio- biology are those which can be expected in aqueous systems containing dissolved and suspended organic compounds. For simplicity we may consider first the general nature of the elementary processes which can occur. * A contribution from the Radiation Chemistry Project, operated by the Univer- sity of Notre Dame, under Atomic Energy Commission Contract No. AT(ll-l)-38. t In this paper the term biological particle refers to the microscopic unit of in- terest in target theory, such as a cell or a virus unit. 117 118 ELEMENTARY CHEMICAL PROCESSES Elementary Chemical Processes Characteristic of Water In pure water the primary processes are usually written H2O ^ H2O+ + e (1) H2O+ + aq -> H3O+ + OH (2) H20 + aq + e -^ H + OH--aq (3) Processes involving excitation of water in the primary physical act are usually neglected because it is the general opinion that the Franck- Rabinowitch cage either prevents formation of free hydrogen atoms and hydroxyl radicals or causes their immediate recombination without con- sequent secondary chemical effects; that is, H2O ^ H2O* -^ H + OH ^ H2O (4) A feature peculiar to the set of reactions 4 is that, unlike the first three listed, they occur without any local changes of hydrogen- or hydroxyl- ion concentration. The reactions ensuant on reactions 1 to 3 depend on the relative proximity to each other of the products of reactions 2 and 3. In fast-particle irradiation (that is, high-energy electrons, x-rays, etc.) the processes of reaction 1 may occur several hundred molecules apart, whereas in slow-particle irradiation (that is, the usual alpha and neu- tron processes) the distances between adjacent hydroxyl radicals, formed by the successive reactions 1 and 2, may be less than 10 molecules. Reaction 3 usually occurs a considerable distance, perhaps as much as 10 molecules, from the locale of origination of the electron involved. Thus, we may expect a distribution of atoms and radicals somewhat isotropic for a fairly homogeneous beam of fast-particle irradiation, definitely anisotropic for slow-particle irradiation.* In the latter case each ionization column consists essentially of a core of hydroxyl radicals and oxonium ions surrounded by a sheath of free hydrogen atoms and hydroxyl ions. * Note added in proof {August 29, 1951): In a rapidly developing field, new facts are found and new ideas develop in the course of a year. This picture and its conse- quences have now been greatly modified. In both fast-particle and slow-particle irradiation, about three-quarters of the primary physical effects are in spurs which contain approximately the same number of ions approximately similarly distributed in both cases. The important parameter, which must account for the difference in the effects of the two types of radiation, is, therefore, the distance between spurs, which is relatively large for fast particles. In further development of the theory this fact must be carefully considered (cf. forthcoming paper in Nucleonics by Burton and Magee). ELEMENTARY PROCESSES OF WATER 119 Thus, reaction between hydroxyl radicals OH + OH -^ H2O2 (5) is quite probable when they are formed close together, as in slow-particle irradiation. The reaction OH + OH -> H2O + 0 (6) might also occur under such conditions. However, although reaction 6 is '■^9 kcal mole~^ exothermal, the difference in activation energies Eq — E5 may favor reaction 5. An unpublished estimate indicates that £"5 < 4 kcal mole~^ The reactions xl + M -^ II2 (7) H + OH -^ H2O (8) occur readily under any circumstances, and the reaction OH + H2 -> H2O + H (9) which is -^9 kcal mole~^ exothermal, is an important back reaction which serves to decrease the yield of electrolytic gas in pure water. According to Allen (1, 2), reaction 9 is one step of a two-step chain, of which re- action 10 H + H2O2 -^ H2O + OH (10) is the second, which in general reduces gas production under gamma, x- ray, and fast-electron irradiation to barely detectable levels. Ghormley and Allen (3) have shown, however, that with slow-electron irradiation, as with 5.6-kev betas from tritium, the yield of electrolytic gas resembles what might be expected from our knowledge of effects of alpha-particle irradiation. Introduction of impurities into the water may have a considerable effect on the yield of gas. Such anions as SO4"", P04~, and Cl~~ are without effect, but Br~ and I~ are successively more effective in inducing production of gas. The obvious point noted by many acquainted with these facts is that the electron affinity of the negative ion involved relative to hydroxyl ion is the decisive factor. Where we can write, for aqueous systems, X- + OH -^ X ^- OH- (11) the anion X" is effective in production of electrolytic gas to an extent related to the weakness of its electron affinity. Of course, in reaction 11 the electron affinities involved pertain to the solvated ions. However, the 120 ELEMENTARY CHEMICAL PROCESSES explanation of the effect was not so readily apparent. Many efforts were made to explain the result in terms of a chain involving X and X~. Allen (1,2) suggested the explanation that the redox potential of reaction 11 controls the steady-state concentration of free radical OH and thus determines the effectiveness of the back-reaction sequence 9 and 10. It is, as a matter of fact, known that with sufficient Br~ or I~ present the rate of gas production is proportional to the intensity of irradiation and that the concentration of Br~ or I~ determines the maximum rate of such production, I~ being the more effective. So far as this author is informed, Allen's theory of this effect has not been subjected to quantitative tests. For the purpose of this presentation it is sufficient to note that the presence of anions of low electron affinity reduces the free-hydroxyl-radical concentration at any intensity level of irradiation and thus decreases the effectiveness of steps 9 and 10 for the back re- action. As a result, H2 gas escapes from the liquid. The H2O2 which escapes reaction 10 may, however, decompose by an overall reaction we note simply as H2O2 -^ H2O + i02 (12) Allen (1, 2) has suggested as the chief mechanism of this reaction the chain H2O2 + OH -> H2O -\- HO2 (13) H2O2 + HO2 -> O2 + H2O + OH (14) OH + HO2 -^ H2O + O2 (15) Presence of oxygen also assists production of electrolytic gas under fast-particle irradiation. In this case the effective reaction presumably involves formation of free hydroperoxyl radical H -t- O2 -> HO2 (16) The process reduces the free-hydrogen-atom concentration (and thus the effectiveness of the back sequence 9 and 10) and also provides a substance itself capable of capturing hydrogen atoms. HO2 + H -^ H2O2 (17) The simplest satisfactory explanation of the effectiveness of dissolved * oxygen in assisting production of electrolytic gas and hydrogen peroxide lies in those two facts. However, as Allen has noted, the hydroperoxyl radical enters also into its own back-reaction sequence (13 and 17) and the total effect consequently depends in quite complicated fashion on intensity of irradiation, concentration of dissolved oxygen, pressure above the liquid, and relative volumes of gas and liquid. BEHAVIOR OF ORGANIC COMPOUNDS 121 In summation, irradiation of water results ultimately in formation of hydrogen, oxygen, and hydrogen peroxide. With slow-particle irradia- tion, the yields are determined by the dosage. With fast-particle irradi- ation, yields are barely detectable without special provision for study. With such irradiation, yields of the order of those obtained under slow- particle irradiation may be obtained when oxygen or anions of suitably low electron affinity are present. The results are understood in the framework of a mechanism involving primary formation of free hydrogen atoms and hydroxyl radicals and, when oxygen is present, secondary formation of free hydroperoxyl radicals. According to Allen, the number of moles of hydrogen gas formed per 100 ev in water is very much the same with various dissolved anions [Br~, I~, N02~, SeOs^, AsOs", Fe(CN)6~] present but is affected by the velocity of the impin- gent particle. Obviously, any dissolved substance substantially affected by free atomic hydrogen or by free radicals will change the nature of the gaseous product and may, at the same time, be itself permanently affected.* Fricke, Hart, and Smith (5) studied the products formed by irradiation of aqueous solutions of organic compounds, and a number of workers (6-11) have been investigating the mechanisms of these processes in a detailed way. In the early work of Fricke and his -coworkers the results were ascribed to the action of "activated water." Presently, the "activated water" is believed to be free hydrogen atoms and hydroxyl radicals. When oxygen is present, the free hydroperoxyl radical may be added to this list. Behavior of Organic Compounds Elementary processes of radiation chemistry in organic compounds may be understood in the light of the Eyring, Hirschfelder, and Taylor (12) (EHT) mechanism as it has been interpreted by this writer (13-16) and by ]\Iagee and Burton (17, 18). We may write for the processes involving ionization f A ^ A+ + e (I) A+ + e -^ A* (IIg) * Such an effect has been known for many years. For example, A. Kailan (4) noted that during the fumaric-maleic acid isomerization reaction in water there oc- curred a simultaneous reaction which he at that time ascribed to intermediary of hydrogen peroxide. t Processes involving excitation have already been discussed in this symposium by Livingston. Here, we dismiss them temporarily with the statement that they are akin to the phenomena of photochemistry. 122 ELEMENTARY CHEMICAL PROCESSES or M + e -^ M- (116) A* + M (lie) A+ + M- < Other reactions ■ • 0-^d) , R + X (Ilia) A* <( ^ B + C (III&) In this scheme A"*" as it reacts in reaction Ila is probably in a more stable configuration than the ion in the instant of its production in step I; A* is an excited molecule; M may be the same as or different from A; R and X represent free atoms or radicals; B and C designate stable molecules formed in one elementary act. In an interpretation of part of this mechanism based essentially on a careful study of the reasonably cal- culable aspects of the reactions of hydrogen, Magee and Burton (17) concluded that (in those cases where reaction lib and its ensuant re- actions can be ignored) the most probable path of reaction involves pro- duction of free atom or radical partners Ilia, one of which is excited. In the liquid state, probability of rearrangement of an excited particle to yield two ultimate molecules is considerably increased (16, 17). Since, of all the possible rearrangements which may occur, one usually goes by an energetically lowest path, it may be expected (16, 17) and it has actually been found in certain cases (19, 20) that a particular rearrange- ment decomposition is so highly favored as to preclude the probability of any other such process. In the EHT mechanism as just outlined we have omitted considera- tion of possible decomposition of the ion A+ itself. Since this subject is discussed in this symposium by Eyring et al, we note only that two paths of decomposition are open R+ + X (Ifl) A" + /■ \ B+ + C (16) where the letters have the significance already noted. Of the two re- actions, one involving decomposition into a free radical and a free-radical ion and the other decomposition into a molecule and a molecule ion, the first may usually be expected to require more energy. Thus, unless the initial ionization is to a point on an attractive curve above the dissocia- tion limit for rupture as indicated in la, decomposition of type 16 alone occurs, even though it may take a considerable time (of the order of BEHAVIOR OF ORGANIC COMPOUNDS 123 10~^ sec or more). However, the initial ionization may be to an elec- tronically excited state of the ion A"^. Ensuant internal conversion to a lower state of A"*" and decomposition (that is, predissociation), as by either of the reactions la and 16, may likewise take a long time. The time involved will govern the phenomena observed. For example, in mass spectrometry we detect all manner of peaks with masses less than the parent peak. Some must have resulted from rearrangement in a time short compared with that required to move from the slit system to the magnetic field (< H + OH--aq (3) of significant importance. In general, it is improbable (though not impossible) that some dissolved or suspended species can capture thermal electrons to form negative ions. Since water preponderates, we may once again expect that on this basis alone it will provide the major trap for free thermal electrons. Possibility of the competitive reaction H3O+ + e -^ H + H2O (19) must not be ignored. However, its effect, like that of reaction 3, is to yield free hydrogen atoms in the ambient layer and to increase the local pH of the solution. / Biological Systems The major conclusion from our brief consideration of elementary physical processes is that in aqueous systems of interest to radiobiologists the initial radiation chemical processes are much the same as they are in pure water. We may now review the major important features. DISTRIBUTION OF EFFECTS For the most part the radiant energy affects the water itself. The first chemically important elementary processes are reactions 1, 2, and 3. Reaction 3, or tts equivalent {19), ynay occur in the aqueous layer even when the primary effect of the radiation is in the biological particle itself. The distribution of primary products depends on the nature of the incident radiation. For particles of the same charge and energy, velocity and mass are related by the expression velocity oc mass~^-^ The frequency of production of excited molecules or ions by action of the impinging particle is inversely related to its velocity. Thus, slow particles produce reactions such as (1) relatively close together, whereas fast particles tend to produce a relatively isotropic distribution of free hydrogen atoms and hydroxyl radicals. High-energy gamma and x-radi- 126 ELEMENTARY CHEMICAL PROCESSES ation (which produce Compton recoils) and electrons are included among the fast group. On the other hand, alphas, deuterons, and protons of energies usually employed belong in the slow group. Since in hydroge- nous material the major effect of an incident neutron is the liberation of energetic protons, the neutron may also be placed in the slow group. In summation, slow particles produce relatively high densities of ionization and of hydroxyl radicals along the ion track. A beam of fast particles produces a relatively isotropic distribution of a mixture of free hydrogen atoms and hydroxyl radicals. pU. EFFECTS It may be remarked also that pH is markedly changed in the neighbor- hood of an ion track. In the track itself pH decreases; in the envelope immediately surrounding the track pH increases. The pH values locally attained may be very much less or very much greater than those which are common to biological systems. In the neighborhood of the track of a heavy particle this effect may be very much exaggerated. In a private discussion with the participants in this symposium Franck has pointed out that since biological systems are notoriously sensitive to variations in pH this pH effect itself can have profound significance for radiobiology. EFFECT OF OXYGEN It is usually maintained that in the radiation chemistry of aqueous systems the principal competitive processes are the diffusion of free H and OH toward each other and their diffusion toward the other reactive species present. Biological systems are marked principally by the presence of impurity, the outstanding example of which is oxygen. When the latter is present, an important fate of the free H atoms is the formation of hydroperoxyl radicals, HO2, as by reaction 16. Although these radicals may themselves react with free OH, reaction 15, we may expect that such a process will have a distinct activation energy and steric factor. Meanwhile, since both H and H2 concentrations are reduced, probability of reactions 8 and 9 decreases. The effect of the presence of oxygen is thus not only to convert a reducing agent, free H atom, into a persistent radical, HO2, but at the same time to increase the life span of free OH radicals. It must he emphasized that reaction 3 or 19 very probahly occurs in the water even when the primary ionization effect of the radiation is on the biological material. Thus, there is a high prohahility, if oxygen is present in the system, that the entity HO2 will he formed sufficiently close to a biological particle to have a chemical effect on it even when the initial ionization does not. In such case, ionization of the biological particle is effectively destructive, whereas excitation may not he. BIOLOGICAL SYSTEMS 127 Thus, presence of dissolved oxygen in a biological system sensitive to oxidizing agents makes that system more sensitive to the effects of radiation. An antidote to oxygen in biological systems involves the incorporation in such systems of strong (non-toxic) reducing agents not quite capable, however, of direct reaction with oxygen. This latter requirement is not essentially a thermodynamic one (for example, the redox potential might even permit a direct reaction with oxygen) but in reality one of kinetics (that is, the reaction with oxygen simply should not proceed under the particular conditions). Choice of suitable com- pounds depends primarily on an experimental search. Oxygen, however, does not always play a special role. Imagine an alpha-irradiation process in which a hit is scored directly within the biological particle. Since the alpha is relatively slow moving, there will be a number of accompanying hits in, and close to, the particle. As a result, some free radicals are necessarily produced so close to the bio- logical particle that their only fate can be to react with it. Consequently, it would not be inconsistent with these simple concepts to discover that presence or absence of oxygen is without discernible effect on the results obtained in alpha irradiation of biological systems. CHAIN REACTIONS WTien a radical such as OH or HO2 reacts with an organic compound, it either breaks a single bond or opens a double bond. In either event a new free radical is produced, the fate of which depends on its specific chemical properties. It is important to appreciate that under suitable conditions (for example, when the initial process is distinctly exothermal) this free radical may itself enter into chain reactions. One such type of chain is common to pyrolytic reactions: R— + AH -^ RH + A— A > R— -f S The radical R reacts with species AH in a reaction which requires activa- tion energy. Decomposition of A into R plus a stable product S may also require activation energy. Such chains would tend to be very short near room temperature and would probably not be very important in biological processes. Another type of chain is characteristic of the growth of polymer molecules: R h A -^ RA— RA 1- A -> RA2— RAn 1- A — > RA„+i — 128 ELEMENTARY CHEMICAL PROCESSES with a final chain-termination step which involves another radical. Only the simplest type has been indicated. Polymer chains involving two or more kinds of molecules are also possible. In general, such free-radical chain reactions can proceed readily at room temperature and the chains may be 1000 or more molecules long. An interesting example of radia- tion-induced polymerization is afforded by the work of Dainton (26) on alpha-ray-induced polymerization of acrylonitrile in deaerated water. In that work he gave good evidence that the chain starter is free OH. The possibility of such chain-propagated effects of radiation-produced free radicals certainly may be quite important for radiobiological processes. A converse phenomenon, perhaps equally important for radiobiology, is suggested by the work of Grassie and Melville (27), who report free- radical-induced depolymerization. A simple illustration of such a phenomenon is afforded by unpublished work of Sworski, Gordon, and Burton which has led to speculation that acetylene production in benzene radiolysis proceeds via the steps CeHe + H -^ — CH2=CH2— CHa^CHa— CH2— CH3 / CH2=CH2 + — CH2=CH2— CH2— CH3 / CH2=CH2 + — CH2— CH3, etc. in a sort of "peeling-off" reaction. The first step may involve some con- siderable activation energy, but the interesting feature is that links in a chain of similarly linked units break successively so that a large molecule is degraded, as the result of one primary step, into a number of smaller ones. Such a process may also be operative in radiobiology and could account for sensitivity of biological particles to energetic free radicals. DIRECT HITS We have seen that radiation may act on biological material indirectly through its action on ambient water. Even when the primary effect of the radiation is on the biological material, free H and resultant IIO2 necessarily formed in the ambient layer (via reaction 3 or 19) may have an important chemical effect. However, direct action on such material is not necessarily precluded. Indeed, on desiccated biological material radiation must act directly. Consequently, it is important to note that the remarks concerning the effects of radiation on organic compounds may have considerable significance for radiobiology. Two classes of effects may occur. Either the primary chemical effect occurs at or near BIOLOGICAL SYSTEMS 129 the locus of energy absorption, or it may occur at a more remote region. The latter phenomenon occurs with aromatic compounds. Energy absorbed in the ring may split off a methyl hydrogen from toluene or mesitylene or a methyl group from ethylbenzene (22). Perhaps, in certain conjugated structures, an even more remote split is possible. Thus, "we may conclude that in biological material energy need not necessarily be absorbed in the prosthetic group in order for it to have a devastating effect there. On the other hand, there is no assurance that a purely random hit is necessarily damaging to the prosthetic group. Indeed, we may visualize the possibility that a significant fraction of the volume of a biological material may be damaged (either temporarily or permanently) without effect on the prosthetic group; that is, without lethal effect as we might measure it. We might expect, consequently, that the probability of a chemically effective hit increases with the number of nearly simultaneous hits made on a particle of biological material. Seemingly, our best evidence (28, pp. Ill ef seq.) is that in many cases a single hit is all that is necessary. A multiplicity of hits, as by an alpha particle, seems to be no more effective than a single hit, as by an electron. However, this conclusion is based on calculation and surmise — certainly not on direct visual observation — and there is no requirement that the hit be within the biological particle. A verj^ care- ful analysis of the implications of this so-called tai^get theory in the light of our knowledge of the elementary processes of radiation chemistry is necessary for an understanding of the data and of the attendant processes. SOME REMARKS ON THE TARGET THEORY It is by no means the function of this paper to attempt a critical e^•alu- ation of the target theory, details of which have been presented so ably by Lea (28). Rather, I would prefer to interpret certain aspects in the light of our general knowledge of radiation chemistry. In his presenta- tion Lea recognized quite clearly that the not-necessarily spherical target had dimensions which were not necessarily identical with the particle of biological material (28, pp. 93 et seq.). Since ionization in the water immediately surrounding the particle could produce free H atoms and OH radicals capable of reaction with the biological material, the target size according to L. H. Gray is effectively increased by an amount related to the diffusion distance of those active particles (28, pp. GG, 67). Lea stated that this distance could be 150 A for H atoms but only 20 or 30 A for OH radicals. He neglected mention of the diffusion distance of the very much more persistent HO2 radicals. However, perhaps the best way to see the target is to look at it as a whole, that is, the biological particle and its environment. 130 ELEMENTARY CHEMICAL PROCESSES Figure 1 is a schematic representation of a biological particle in its aqueous sheath. The region ABC within the solid line is the particle itself. The region D includes all that aqueous layer in which primary creation of ions has a resultant chemical effect on the particle. The region AB (not necessarily continuous) is the sensitive part of the particle. It includes (or may be) the group injury to which is made apparent by a change in the detectable behavior of the particle. The region C is effectively inert; that is, injury, or a hit, within it is not made Fig. 1. Schematic representation of a biological entity. A, Hits in this region are always effective; B, hits in this region are sometimes effective; C, hits in this region are never effective; D, ambient layer of fluid in which hits may be effective. apparent by a change in detectable behavior; as a matter of experi- mental fact it may be non-existent. The region A includes several portions : (a) That volume which may be directly affected by radicals produced in the layer D or by changes in the pH of that layer. (6) That volume which may transfer ionic charge to the layer D and thus make it chemically active. (c) That volume in which untransferred ionic charge leads every time to damage in the region A or B. In reference to this last point we must make some note of the elemen- tary processes of the Franck-Rabinowitch cage effect. Neutralization of an ion deep in the cage will not necessarily result in decomposition; that is, the ion-pair yield may be less than unity both because of energy dis- sipation (from the excited molecule A*) without primary decomposition and because of primary recombination of radical (as distinguished from molecule) products while still within their mutual range of influence. In the region B the ion-pair yield is less than unity. On the other hand, as intimated in (c) above, part of the decomposition in B may be the result of a primary physical effect in A ; cf . the effect of absorption of energy in an aromatic ring on decomposition in a side chain. A feature to be emphasized in consideration of Fig. 1 is the precise BIOLOGICAL SYSTEMS 131 meaning of a hit. Lea (28, pp. 66, 67) defines a hit as the production of ionization within the target. From the point of view of our knowledge of the elementary processes of radiation chemistry as they relate to organic compounds such a definition appears unnecessarily restrictive. Production of excitation within the biological particle, depending on the locale of such excitation, can produce chemical change just as ultraviolet radiation might. The number of such primary excitations within the target always exceeds the number of primary ionizations. Thus, the conclusion, based on preoccupation with ionization processes, that in many cases a single hit is all that is necessary for production of a lethal effect is fundamentally misleading and obviously in error. If our ideas of the elementary processes of the radiation chemistry of organic com- pounds are correct, such a computation emphasizes a fact not otherwise apparent; namely, in the target theory there is no special virtue in a single hit if the hit is presumed to be in the organic material itself. Indeed, the best data so far used to support that notion offer clear evidence that it cannot be correct for targets of the size assumed. On the other hand, there is almost inevitable formation of HO2 (via reaction 3 or 19) in the ambient layer whenever primary ionization occurs in the biological particle. Such HO2 may be more virulent in its effects than either a primary ionization or a primary excitation. Under such circumstance, we may expect that ionization deep within the particle may be more probably associated with damage than would similar excitation. A question that must arise regarding application of Fig. 1 to any particular case concerns the volume ratio of regions A -{- B to C. This is a matter regarding which the chemist certainly can make no general statement. Another question concerns a possibility that this same ratio may be a function of particle size ; for example, the larger the particle the greater is the probability that a significant portion of it (that is, C) is effectively inert to the radiation. From the purely chemical viewpoint such an assumption may be justifiable in a homologous series. I am unable to judge its worth biologically. However, it may be profitable to examine the more obvious consequence of this assumption. That consequence simply is that the larger the particle the greater becomes the probability of an ineffective hit. The further consequence is that with large particles an increasingly large negative deviation of the calculated size (as determined from naive target theory) from the true geometric size (as determined from electron diffraction microphotographs of the dry material) is to be expected. The significance of elementary processes in radiation chemistry for radiobiological reactions can be summed up by consideration of "target size" as affected by each one of the elementary processes. 132 ELEMENTARY CHEMICAL PROCESSES 1. If a spherical target have radiosensitive dimensions corresponding to its geometric dimensions and if each hit be lethal, the probability that a hit at any distance x from the center would produce a lethal effect would be unity within the radius r and zero outside. Figure 2 illustrates ^0 -r 0 r Distance from center of target Fig. 2. Lethality of a hit as a function of distance from center of target on basis of simple target theory. this situation. On the same model, geometric dimensions and dimension computed from radiobiological effect would be identical for all sizes of biological particle. The "ideal" line 1 of Fig. 3 shows this effect. ■(/) 3 2 1 T3 CO / /^ /y ^^' T3 (1) 3 / X ^ Q. ^ X y E ^ / ^^ o o //y .-^ "(5 ^ / y ^^ '5b o / /y --" o in //'<^' ^ y y ^■^ ^'y^- 1 /f' yi'^ y Geometric "radius" Fig. 3. Effect of various factors on biologically computed "radius" of target. 1, Ideal; 2, ideal + contribution from region D; 3, C/(A + B) = constant > 0; 4, CI {A + B) increases with size. BIOLOGICAL SYSTEMS 133 2. Free radicals as well as sharp variations of pH in the region D, both resultant from hits in that region, ionization transferred to that region, or electron capture therein, may (but do not necessarily) produce a lethal effect in the region A. The effect is to make the computed particle size larger than the geometric size. This contribution of the region D is shown cjualitatively by the difference between lines 1 and 2 in Fig. 3. The probability of lethality is shown by Fig. 4. 1 1 • X' 1 / 1 ^ \ 0 ,^ZL- 1 1 \ ^.V -r 0 r Distance Fig. 4. Lethality of a hit as a function of distance from center of target on basis of various modifications of target theory. 3. The existence of a region C decreases the biologically computed size below the geometric size. If the ratio C/(A + B) is constant, line 3 in Fig. 3 shows this effect. If the ratio increases with size, line 4 shows the effect. The probability curve now has no simple shape but depends on the distribution of C relative to A and B. If the ratio C/(A + B) is a constant greater than zero and C is isotropically distributed, the probability of lethality of a hit within the particle is less than unity and the effectiveness of a hit within the diffusion distance in the ambient layer T> may be decreased. The effect is shown roughly by line 2 in Fig. 4. If the ratio C/(A + B) increases with geometric size, the probability of an effective hit within the biological particle simply decreases with size. This statement means that for a large particle the plateau of line 2 in Fig. 4 would be lower than for a small particle. 4. The region A is confined to the surface of the particle. A hit within that region gives a rupture (or other decomposition) and the cage effect 134 ELEMENTARY CHEMICAL PROCESSES is nil. On the other hand, in B there is a cage effect and that effect becomes bigger the larger the size of the particle. This effect is also shown by a line like 4 in Fig. 3. The probability of lethality of a hit is a maximum in or near the surface. The effect is shown by line 3 in Fig. 4. The conclusion from these considerations is that the relationship between biologically computed target radius and geometric radius even for a spherical particle is not simple. For real particles of such ideal shape the qualitative nature of the relationship must be as shown in Fig. 5. The fact that the computed radius is so nearly like the geometric / / A Geometric radius Fig. 5. Relation between effective radius of target and actual dimensions of biologi- cal entity. radius transpires to be an interesting consequence of the elementary processes involved in radiobiological reactions. It is a relationship which has, for some, emphasized the naive features of target theory and really beclouded the processes involved. The happy fact is that investi- gators in the field were actually not led astray in spite of the terminology employed. Lea (28) himself emphasized the lack of a definite boundary. In a study of the eft'ect of deuteron bombardment on bacteriophage, Pollard and Forro (29) have shown that a target exists which is smaller than the phage itself but that nevertheless the computed target size is increased because a deuteron whose path actually misses the phage can nevertheless inactivate it. In conclusion one fact not heretofore mentioned, so far as I know, bears repeated emphasis. Free H and resultant HO2 are very probably produced in the ambient liquid around a particle even when the hit, as in x-irradiation, may be directly and exclusively in the particle. It is DISCUSSION 135 the formation of these active entities which may be largely responsible for effects heretofore attributed to the hit itself. REFERENCES 1. Allen, A. O., /. Phys. Colloid Chem., 52: 479, 1948. 2. Allen, A. O., The Science and Engineering of Nuclear Power, Chap. 13, Vol. II, Addison-Wesley, Cambridge, Mass., 1949. 3. Ghormley, J. A., and A. O. Allen, Oak Ridge Natl. Lab. Unclassified Rept. 128, Oct. 4, 1948. 4. Kailan, A., Sitzber. Kaiserlich. Akad. Wiss., 123: 1, 1914. 5. Fricke, H., E. J. Hart, and H. P. Smith, J. Chem. Phys., 6: 229, 1938. 6. Hart, E. J., J. Am. Chem. Soc, 73: 68, 1951. 7. Farmer, F. T., G. Stein, and J. Weiss, /. Chem. Soc, 1949: 3241. 8. Stein, G., and J. Weiss, /. Chem. Soc, 1949: 3245, 3254, 3256. 9. Butler, G. C, Can. J. Research, 27B: 972, 1949. 10. Minder, H., W. Minder, and A. Liechti, Radiologia Clin., 18: 108, 1949. 11. Mathis, A. L., R. E. Brooks, and H. Schneiderman, Atomic Energy Comm. Unclassified Rept. 170 (UCLA 11), Feb. 11, 1949. 12. Eyring, H., J. O. Hirschfelder, and H. S. Taylor, J. Chem. Phys., 4: 479, 1936. 13. Burton, M., /. Phtjs. Colloid Chem., 51: 611, 1947. 14. Burton, M., /. Phys. Colloid Chem., 51: 786, 1947. 15. Burton, M., J. Phys. Colloid Chem., 52: 564, 1948. 16. Burton, M., /. Phys. Colloid Chem., 52: 810, 1948. 17. Magee, J. L., and M. Burton, /. Am. Chem. Soc, 72: 1965, 1950. 18. Magee, J. L., and M. Burton, /. Am. Chem. Soc, 73: 523, 1951. 19. Breger, I. A., /. Phys. Colloid Chem., 52: 551, 1948. 20. Burton, V. L., /. Am. Chem. Soc, 71: 4117, 1948. 21. Hippie, J. A., R. E. Fox, and E. U. Condon, Phijs. Rev., 69: 347, 1946. Hippie, J. A., Phys. Rev., 71: 594, 1947; /. Phys. Colloid Chem., 52: 456, 1948. Hippie, J. A., and R. E. Fox, Rev. Sci. Instr. 19: 462, 1948. 22. Hentz, R. R., and M. Burton, /. Am. Chem. Soc, 73: 532, 1951. 23. Bichowsky, F. R., and F. D. Rossini, The Thermochemistry of the Chemical Sub- stances, Reinhold, New York, 1936. 24. Rice, O. K., Electronic Structure and Chemical Binding, McGraw-Hill, New York, 1940. 25. Cf. Sugden, T. M., A. D. Walsh, and W. C. Price, Nature, 148: 372, 1941. 26. Dainton, F. S., J. Phys. Colloid Chem., 52: 490, 1948. 27. Grassie, N., and H. W. Melville, Proc. Roy. Soc, A199: 1, 14, 24, 39, 1949. 28. Lea, D. E., Actions of Radiations on Living Cells, Cambridge, 1947. 29. Pollard, E. C, and F. Forro, Jr., Science, 109: 374, 1949. DISCUSSION KORNBERG : Burton differentiated between the production of OH radicals, etc., and the direct effect of radiation on solute molecules with resultant activation followed in certain cases by decomposition. Perhaps, if the ratio of the two effects were known in some simple but fairly representative biological medium, a reasonable 136 ELEMENTARY CHEMICAL PROCESSES basis, not wholly dependent on ionization in gases, for estimation of number of primary physical processes might evolve. Of course, this is in addition to the contribution such knowledge could make to the mechanism of damage. Burton : Study of the effect of radiation on a mixture of benzene and cyclohexane yields information which is of much general interest and which may also have some bearing on Kornberg's comment. The ionization potential of the benzene molecule is less than 10 ev; that of cyclohexane is somewhat higher. If a mixture of the two compounds is irradiated, the ionization will tend to reside ultimately in the benzene. One might therefore expect that only benzene will be chemically affected: the benzene should protect the cyclohexane. The experimental evi- dence, however, indicates appreciable decomposition of the cyclohexane. It would thus appear that not quite all of the energy is transferred to the benzene; some decomposition has occurred before the energy is transferred. Primarily excited molecules may not transfer their energy like the ions, and it may be such cyclohexane molecules that decompose. Perhaps the chemical data may give some clue as to the relative probabilities of ionization and excitation transfer. However, we should note the comphcating possibility of some transfer of energy from benzene to cyclohexane in a sensitization process. Lind: Is there transfer of energy in the liquid system? Burton: I should have stated that the experiments were performed on benzene-cyclo- hexane mixtures in the liquid state. Morrison: In a field as complex as the one under discussion at this symposium it is both useful and necessary to set up simple models. The target theory is such a model, and during the past two decades much useful experimental work has been based upon this model. It is important that future models should define a situation in an operational way, as the target theory has done. I wonder whether the model set up by Burton defines the situation from this point of view. Burton (Communicated) : Morrison is correct in implying that the proffered model is an oversimplifi- cation. The purpose of this model, as of any other, is to assist in correlation of results of research. Without question it will be modified as the facts demand. The mathematics of the picture I have suggested is. I believe, the mathematics of the target theory. All that I have done is to define a possible target in the light of our present knowledge of the radiation chemistry of aqueous systems. That target is clearly something different from the biological entity. The model is suggestive of the mechanism of the action of oxygen and of experiments which may be performed to test that mechanism. It also indicates why the DISCUSSION ' 137 apparent target size may differ from the size of the biological entity even in the dry state. Morrison : I do not believe that ion migration in the organic molecule is a very likely event. Burton : It is the electron migration, and not the ion migration, that is important. The electron migrates; and, when it is thermalized, if it is thermalized near the water, it is much more likely to be captured by water than by an organic molecule or by a positive ion. Morrison : Is there any available estimate of the probability of electron escape without damage to the organic molecule? Burton : Not that I know of. Applet ARD : I do not wish to defend a target theory necessarily as a main mechanism of radiobiological action. It can, however, certainly be of use as an experimental procedure. Pollard's group at Yale has tried to work under experimental con- ditions which give such a target theory an optimal chance of apphcation, namely, irradiation of dried materials with densely ionizing particles. Under these con- ditions, for both enzymes and viruses, "operational" target sizes are found which, while often less than the known sizes of these materials, appear to mean something in the sense of defining a region with a high degree of molecular or- ganization. Burton : I should have referred to the work of Pollard's group at Yale. Did not some of the experimental evidence indicate a target size somewhat larger than the geometrical size of the biological unit in question? Appleyard : No. To the best of my knowledge Pollard and Forro have never found target sizes for phage larger than the size of the phage as evaluated by other methods. Burton (Communicated) : My question was founded on a misinterpretation of some data in the literature. However, I should like to re-emphasize a point which should not be forgotten: when dry biological material is irradiated the biological particle and the target of target theory are identical. Any size discrepancy between calculated and geometrical target is a reflection of inertness of a portion of the particle. 138 ELEMENTARY CHEMICAL PROCESSES Abelson: Since the hit theory has come under discussion, I wish to point out a case in which the single-hit target theory seems to be relatively meaningless. Roberts at the Carnegie Institution of Washington has shown that small differences in biological manipulation can give large changes in the apparent multipUcity of hits. By small variations in time of incubation in a sahne solution after ir- radiation, either single-hit or multiple-hit curves may be obtained for the sur- vival of E. coll irradiated by ultraviolet light. Burton: The target theory in its original form identifies a hit with an ionization. The target theory does not demand that a single hit be effective. A single hit has been found, by analysis of empirical results, to be effective in certain cases (for example, viruses and chromosomes), but multiple hits may be required in other cases. Hart: Oxygen molecules play an important role in the radiolysis by x-rays of aqueous solutions of formic acid and hydrogen peroxide. In the absence of oxygen, a chain oxidation of formic acid takes place, resulting in the overall reaction: HCOOH + H2O2 = 2H2O + CO2 Oxygen is an excellent inhibitor for tliis reaction. The hydrogen atoms pro- duced during irradiation react with oxygen in preference to formic acid even under conditions where the ratio of formic acid to oxygen is 10,000 to 1. In the absence of oxygen and hydrogen peroxide, the hydrogen atoms do, however, react with formic acid to produce molecular hydrogen. This has been demon- strated by the irradiation of DCOOH in aqueous solution. HD is a primary product of this reaction. Therefore it is apparent that hydrogen atoms do not require oxygen in order to promote chemical changes in solute molecules. Burton (Communicated): I did not intend to imply, in my paper, that hydrogen atoms require oxygen in order to be effective. As a matter of fact, we can guess that the activation energies of processes (in biological systems) invohing atomic hydrogen will, in general, be lower than those of processes involving HO2. This fact is precisely why HO2 survives longer and diffuses farther in such systems, and serves to explain the role of oxygen in processes in which the hit may not be in or near the target. In the interesting case described by Hart the atomic hydrogen reacts with formic acid whenever it is not removed by some other process. 9- Influences of Details of Electronic Binding on Penetration Phenomena, and the Penetration of Energetic Charged Particles through Liquid Water ROBERT L. PLATZMAN Department of Physics Purdue University Lafayette, Indiana I. Introduction Liquid water is perhaps the most important inorganic chemical sub- stance and is certainly the fundamental biological material. The inter- action of high-energy radiations with water is thus a subject of the greatest consequence for studies of radiation action. Yet almost nothing is known directly about the phenomena attending passage of swiftly moving charged atomic particles through liquid water. The im- portance of this problem in radiobiology is emphasized by the reminder that the phenomena referred to are chemically non-specific: the pene- trating particle affects atoms and molecules encountered along its path simply in approximate proportion to the populations of the various species. Hence the 'primary effects in biological systems are intimately related to the effects in pure water and, indeed, are often roughly iden- tical with them. Although we have today a wide knowledge of, and deep insight into, high-energy penetration phenomena for gases, particularly monatomic gases, we know less about the corresponding phenomena for polyatomic gases, very little indeed about solids, and almost nothing concerning liquids. Admittedly, the quantitative differences in the phenomena for the last three cases — when the material is compared with a hypothetical mixture of monatomic gases of identical over-all chemical composition — have up to the present time usually been comparable to or smaller than the relevant experimental uncertainties or the accuracies demanded in common applications. Indeed, it is to this lack of obvious practical im- portance that neglect of the fundamental aspects of the question is 139 140 PENETRATION PHENOMENA IN LIQUID WATER probably to be ascribed. In the case of liquid water, however, the con- sequence in theoretical radiobiology and radiation chemistry of ap- preciable manifestation of m^mmolecular binding and miermolecular interaction is potentially so great that further disregard of the possi- bilities can no longer be tolerated. Formidable experimental difficulties are responsible for the fact that only very few experimental studies relevant to this problem have been made. Thus-, neither the primary nor the total ionization produced in liquid water by any type of radiation has ever been measured, no method having yet been advanced by means of which the ionization can be directly observed. The stopping power of liquid water for energetic charged particles has been determined only once: the difficulty here lies in preparing a section of water which is sufficiently thin (for the energy region hitherto accessible). There exist also three measurements of the range of natural alpha particles in water. The results are not in accord- ance with one another, and it is sufficient at this point to note that they do not much clarify the basic questions. Moreover, no theoretical treat- ment of penetration phenomena for the specific case of liquid water has heretofore been given. This inattention should be attributed to the opinion that liquid water must behave toward high-energy radiation very much like a gas, or to the barrier presented by lack of any de- tailed knowledge of the electronic properties of liquid water. In the present paper we shall review the appropriate experimental results, consider the theoretical problems involved, and also point out directions which future investigation might pursue. It will prove grat- ifying if the discussion should serve to emphasize our ignorance of many of the fundamental aspects of the subject and help to stimulate their study. The subject of high-energy radiation effects in water is, of course, a great one, embracing a number of diverse phenomena. It is therefore necessary to restrict the present discussion to a few aspects which are of foremost practical importance, or which seem most likely to shed light on the question of possible peculiarities of liquid water. Topics which merit particular attention are the stopping power and range of swiftly moving charged particles, effects of electrons and positive ions of inter- mediate and low velocity, and the mean over-all efficiency of ionization. II. Review of Experimental Information A. MICHL (1914) (1) In this first measurement on liquid water a thin platinum wire covered with a very thin layer of polonium rested on a photographic plate, the REVIEW OF EXPERIMENTAL INFORMATION 141 whole being immersed in Avater, and the range of the Po alpha particles was determined from the contour of blackening of the emulsion. A careful study under different experimental circumstances led to a value for the range of Po alpha particles (5.298 Mev) of 32.0 ± 0.5 microns in liquid water. Michl dealt as best he could with a number of possible complications— among them swelling of the gelatin and depression of the gelatin by the wire, as well as solubility of the Po in water— but it is virtually impossible today to assess the accuracy of his final result. Michl's value for the range in liquid water is 20 per cent smaller than that calculated for water vapor at (hypothetically) equal density using modern data (cf. Table 1, p. 143). The ranges of Po alpha particles in alcohol and in six other organic liquids were also determined. All ranges were smaller (by 10-20 per cent) than values predicted for the corresponding vapor, reduced to the same density. B. PHiLipp (1923) (2, 3) By means of visual scintillation observation, with the radioactive source mounted below a Avater surface and the fluorescent screen just above it, the range of RaC alpha particles (7.680 Mev) in liquid water was found to be 59.5 ± 0.8 microns, 16 per cent smaller than that cal- culated for the vapor (Table 1). Ranges in alcohol and in two other organic liquids were also determined. Philipp measured the ranges in the corresponding vapors as well. For water vapor of density 0.532 mg per cm^ he found an extrapolated range of 13.0 cm, which corresponds to a mean range of approximately 12.7 cm; the above value for liquid water, when reduced to the same density, is 12 per cent smaller than this. In contrast to the abnormally small range in the liquid, which also was found to occur for alcohol (for which the difference was 11 per cent), measurements on aniline and pyridine gave more closely equivalent ranges for the liquid and vapor. Philipp attributed this difference to the circumstance that, whereas the latter liquids are ''normal," both water and alcohol are "associated." Even if his results are correct, this is a correlation but certainly not an explanation. C. APPLEYARD (1949) (4, 5) In this ingenious experiment a very dilute solution of polonium in 0.5-1.5 N HCl acted as a thick source, the alpha particles being counted by a thin-window Geiger counter mounted at varying distances in air above the liquid. From the variation of the number of alpha-particle counts with distance, the stopping power corresponding to an energy close to the initial energy of the alpha particles could be determined. 142 PENETRATION PHENOMENA IN LIQUID WATER This is the only determination yet made of the true stopping power of Hqiiid water; the other measurements (1, 3, 6) cited all yield ranges. Appleyard found the stopping power corresponding to an alpha-particle energy of roughly 4.5 Mev to be 1.71 ± 0.05 (per molecule of H2O rel- ative to one "atom" of air). (The uncertainty in energy is unimportant, since the relative stopping power is known to be rather insensitive to energy variations in this energy region; cf. Fig. 1, p. 165.) This value is 15 per cent higher than the theoretical one for water vapor (cf. Table 1). D. DE CARVALHO AND YAGODA (1950) (6, 7) In this measurement, the photographic emulsion technique was again employed. The source of alpha rays was a tiny particle ("radiocolloid") of polonium or radium sulfate; many such particles were sprinkled over the surface of the emulsion, which was then immersed in water. The procedure was in a sense a modern refinement of Michl's method. Those alpha particles emitted by a radiocolloid particle almost tangentially to the emulsion struck the latter when they were very close to the ends of their ranges and hence delineated the range in water. The experiments yielded values of the ranges of Po and of RaC alpha particles in vapor, liquid, and solid water which, when reduced to the same density, agreed closely with each other (to within 1 per cent) and with the value calculated for the vapor (cf. Table 1). The results are thus in utter disagreement with those of the three other investiga- tions. It might be noted that de Carvalho (6) states that his emulsions are sensitive only to alpha-particle energies greater than 0.2 Mev. This should make his ranges too short in the liquid, but this difference is not great and he is able to correct for it. [Similarly, the scintillation method, employed by Philipp, has a visual threshold of between 0.13 and 0.17 Mev (8).] E. DISCUSSION OF THE EXPERIMENTAL INFORMATION The experimental results are summarized in Table 1. The semitheo- retical values for water vapor listed there are taken from a new calcula- tion, the results of which are presented in Figs. 1 and 2 (pp. 165 and 166); the basis for this calculation will be discussed in Section VI below. The quantity Sr should not be confused with s. Whereas s is the true stopping power, relative to that of air at the same particle energy and for the same density of atoms, 1/Sr is the average reciprocal stopping power (the average being over all values of particle energy as the par- ticle is progressively slowed down) relative to the corresponding average in air for a particle of the same initial energy. More simply, 1/Sr is REVIEW OF EXPERIMENTAL INFORMATION 143 the range of the alpha particle in water divided by its range in air, cor- rected for the difference in atomic densities. Thus, although Sr is more directly a relative range, it is often called the average or mean relative stopping power and sometimes even the relative stopping power. How- ever, Sr and s would be equal only if s were independent of particle TABLE 1 Summary of Experimental Information on Range and Stopping Power: Alpha Particles in Liquid Water Source of Data 4.5-Mev Alpha Particle Po Alpha Particle (5.30 Mev) RaC Alpha Particle (7.68 Mev) s R in fi Sr Rin fj. Sr Michl (1914) (1) 32.0 1.83 Philipp (1923) (3) .... 59.5 1.77 Appleyard (1949) (4) 1.71 .... de Carvalho and Yagoda (1950) (7) 38.1 1.54 67.2 1.57 From semitheoretical calcula- tion 1.49 ±0.05 [40] [1.48] [71] [1.47] Definitions: s = stopping power, per molecule of water, relative to that of one "atom" of air R = range in water of density 1 gm per cm^, in microns Sr = reciprocal of range in water, relative to range in air, reduced to equal numbers of molecules (H2O) and "atoms" (air) per unit volume = 1.525. 10-3i2^i,/i2H2O Note: for explanation of uncertainties in calculated values of R and Sr indicated by brackets, cf. Section VI. energy. This is occasionally approximately but never exactly the case (cf. Fig. 1). The quantities Sr and s are often confused in the literature. It is a striking fact that both the older measurements of Sr by Michl and by Philipp and the later one of s by Appleyard give values approx- imately 15-20 per cent higher for the liquid than those calculated theo- retically for the vapor. The approximate agreement between these 144 PENETRATION PHENOMENA IN LIQUID WATER three ''discrepancies" is impressive. (One would not expect them to be identical, even if the experimental errors were negligible, for the meas- urements yield three closely related but essentially different quantities.*) It merits mention here that the reality of the discrepancies found by Michl and by Philipp has never been generally accepted by workers in the field. Thus Rutherford (9), in 1930, dismisses the effect as ''small and difficult to account for." Gray (10), in an authoritative review pub- lished in 1944, also discounts the direct measurements on liquids, imply- ing that they are in error in some unknown way (s) ; generalizing from an analysis of the stopping powers of a number of compounds, mostly of C, H, and 0 and almost all in the vapor phase, he concludes that pos- sible effects of chemical binding and state of aggregation on the stopping power for fast particles are at most of magnitude ±1 per cent. As far as applications are concerned, it has always been the custom, in any con- siderations into which the stopping power or range of energetic charged particles in liquid water enters, to presume the water to have behavior identical with that of a mixture of hydrogen and oxygen gas having the same composition and density as the liquid. We now have two new and different measurements, performed with modern techniques. The work of Appleyard, particularly in its semi- quantitative agreement with the earlier work, undermines the com- placency sketched above, although a great difference between liquid and vapor is so remarkable in the light of theory, as will be discussed below, that still another, independent confirmation would appear to be de- manded, and more accurate data are necessary in any event. However, the conclusion of de Carvalho and Yagoda that Sr is the same for liquid and vapor (and solid) throws a cloud of uncertainty over the entire situ- ation. It is clearly imperative to have a vigorous attack on the experi- mental problem, preferably from several different directions, not onty to establish the correct results, but also to identify the nature of the various errors. In view of this situation, it does not seem propitious to investigate extensively the theoretical aspects of the problem. Only a qualitative * Some doubt may also be raised that the medium in any of the experiments was truly "water." Thus Appleyard used rather concentrated electrolytic solutions, in which the ions were separated, on the average, by only about 3-4 water molecules. And in all the experiments the distance traversed by the alpha particle in the liquid was small — of the order of magnitude of 10^ molecular diameters of water in the experiments of Michl, of Philipp, and of de Carvalho-Yagoda, and even less in that of Appleyard — and the water layer might have had structural abnormality (of sur- face in the experiments of Philipp and of Appleyard, of intersurface in those of Michl and de Carvalho-Yagoda). It has not been possible, however, to invoke a plausible reason why such peculiarities might account for the observations. STOPPING-POWER THEORY FOR MON ATOMIC GAS 145 (but, it is hoped, complete) survey of possible effects of the finer details of electronic binding will therefore be given. This suffices to demon- strate that these effects are by no means all negligible, and that some are of extreme importance. In order to discuss the stopping-power effects more meaningfully, it will first be necessarj^ to review and evaluate such aspects of contem- porary knowledge and understanding of stopping powers as relate to the problems under consideration. This critical evaluation will, indeed, be one major objective of the present study, and should, it is hoped, prove helpful quite independently of the problem of liquid water. III. Resume of Stopping-Power Theory for a Monatomic Gas The stopping power of a medium for a swiftly moving charged particle of energy E is defined as the ratio of the energy lost by the particle ( — AE) in penetrating a very small distance (Ax) into that medium, to Ax. Thus, stopping power equals —AE/Ax, and equals —dE/dx in the limit of infinitesimal penetration. (Treatment of £" as a continuously decHning function of x is a valid approximation because the magnitude of AE which corresponds to a Ax of atomic dimensions is of the order of electronic binding energies, that is, a few electron volts, and hence is extremely small compared to E for high-energy particles.) The stopping power is a well-defined parameter of the physical situa- tion and is a compound of the probabilities of numerous possibilities of energy loss. (The stopping power has in fact a probability distri- bution, but only its average value will be considered in this paper.) It depends in general on the nature (charge and mass) and velocity of the particle, and on some of the properties of the medium. The stopping power of a particular medium is often expressed (as in Section II) in dimensionless form by its ratio to the corresponding stopping power of air (that is, for the same particle at the same velocity, and for air at such a density that the number of air "atoms" per unit volume is the same as the number of molecules of the medium per unit volume). This relative stopping power is simply the stopping power of a single molecule of the medium divided by that of one-half of an "average" air molecule. Stopping powers of air, for various particles over a wide range of ve- locities, are well established, largely through the work of Bethe, and are available in convenient graphical form (11, 12, 30). The relative stopping power, which is denoted by s, is convenient in that it is highly insensitive to the charge and mass of the penetrating particle; indeed, it is known from both experiment and theory that for "fast" particles s depends only on the velocity of the particle and on the nature of the medium, and 146 PENETRATION PHENOMENA IN LIQUID WATER in fact varies rather slowly with the velocity. By a fast particle is meant in this paper one with velocity great compared to vq, the orbital velocity of an electron in the normal Bohr orbit of the hydrogen atom (vq is a fundamental atomic constant compounded of universal constants: vq = e^/h. = 2.188 X 10^ cm per sec = c/137, where the symbols e, h = 27rh, and c have their customary meanings). At the velocity vq an electron has energy 13.6 ev, a proton 25 kev, a deuteron 50 kev, an alpha particle 99 kev, and a fission fragment (of mass 120) 3 Mev. For fast particles the stopping power arises almost entirely from individual acts of energy transfer from the particle to electrons of atoms close to the path of the particle; these transfers range in magnitude from small ones, of roughly 5-15 ev, which excite the atom, to greater ones, of energy ranging from the ionization potential P upward, which result in a free electron and a positive ion — and so on, with decreasing probability, to very great energy transfers which ionize the atom and produce a very fast secondary elec- tron. The mean energy transfer is always of the order of magnitude of 20 ev. Theoretical treatment of the stopping power for fast particles has been developed in satisfactory detail, thus far, only for the case of a medium composed of isolated atoms of low atomic number. (In this respect it is fortunate that only light atoms are important for radio- biology.) Indeed, a variety of different modes of treatment is avail- able. A particularly illuminating approach is the so-called "method of impact parameters," which will now be sketched very briefly for later reference. (An alternative model will be demonstrated in Section IV.) We restrict the discussion to fast particles having small positive charge and mass of atomic magnitude, of which the only ones now experimen- tally accessible are alpha particles and ions of hydrogen (H, D, or T). The other practically important cases, namely fast electrons and fast heavij ions ("recoils," fission fragments), will be mentioned later. For these heavy particles the momentum is extremely great compared to the momentum change in any collision in which momentum is trans- ferred to an atomic electron of the medium. Therefore the motion of the heavy particle is only insignificantly affected by any one momentum (energy) transfer, and the excitation or ionization act can be treated as caused by a uniformly moving Coulomb center of force. If we consider two concentric cylinders with radii p and p + dp and axes on the path of the particle, we easily find from the laws of classical dynamics the energy transfer to electrons initially lying between the cylinders: ^TTZ^e^ p dp — dEry = :r-n— —dx ■'p mv^ p^ + R' STOPPING-POWER THEORY FOR MON ATOMIC GAS 147 where ze is the charge of the particle, — e that of an electron, ni the elec- tronic mass, V the velocity of the particle, and n the number of electrons per unit volume; R is a parameter called the collision radius which measures the "size" of the Coulomb field and is equal to ze^/mv^. To find the stopping power we simply integrate over all permitted values of The quantity /c, defined as 2'Kz'^e^/mv^, is called the stopping parameter. The factor 2/cn appears in all formulae for stopping power, for all media. It determines the order of magnitude of the stopping power; all details, however, are contained in the balance of the expression, in which our interest will therefore be centered exclusively. Many of the intricacies of the problem lie in the determination of p^ax and Pmin- Although superficial examination might suggest that Pmin = 0, ap- plication of the laws of quantum mechanics shows that, if one uses the above, essentially classical, formulation, one must set p^i^ = h/mv. (Thus Pyain is just the wave length Xg which an atomic electron has in the coordinate system in which the particle is at rest, and the electron there- fore moves with velocity v; the position of the electron is "uncertain" by Xe, which suggests that Pmin cannot be smaller than Xg, but the fact that Prain = ^e actually requires a careful justification which will not be given here.) Since Xe = h/mv = R{v/zvq), it follows that for fast particles Pnun » ^, and _^ = 2«ln^ (1) ax Pmin It is obviously absurd to set p^ax = °° , for this would predict infinite stopping power. Bohr pointed out in 1913 that an upper limit to p is established by a kind of "dynamic" screening arising from the binding of the electrons in atoms of the medium: a bound electron located at a very large distance p from the path of the particle is perturbed adiabat- ically and no energy is transferred to it. If we consider first that an atom contains one electron bound with frequency aj/27r (binding energy = e = hco), the condition for adiabatic perturbation is "duration of collision" ^p/v » l/oj, so that p^ax ~ v/oi. This value of p^ax is cor- rect in quantum theory as well as in classical theory except for a nu- merical factor, which detailed calculation determines as 2, so that p,nax = 2v/w. Hence, for fast particles, dE 2mv^ = 2Kn In (2) dx 8 148 PENETRATION PHENOMENA IN LIQUID WATER The considerations above were oversimplified in one important re- spect: it was assumed that there is a unique binding frequency w/27r. The quantum theory of dispersion shows, however, that even for the hydrogen atom, with its single electron, it is necessary to treat the atom as an assembly of an infinite number of different "virtual" oscillating electrons; each type has effective number (or oscillator strength) /„ and frequency 27rajn = ejh. Here, X) //i = 1 • the total effective number of n such oscillators corresponds to just a single electron. Hence, for the stopping power of a gas containing N hydrogen atoms per unit volume, we have dE ^^ / 2mv\ 2mv^ -_^ / 2wr\ X;/n( 2/ciVln j = 2KN\n In this last expression I is defined by In/ = X)/nlnen (3) n The sum should be understood as embracing both the summation over discrete and the integration over ionization states. The quantity / is called the mean excitation energy or mean excitation potential. It is in effect a geometrical mean of all possible excitation and ionization ener- gies of the atomic system, each weighted by the corresponding oscillator strength. For hydrogen atoms an exact calculation can be carried through and leads to the value I = 15.00 ev, some 10 per cent greater than the ionization potential (13.60 ev). For atoms with more than one electron the same treatment is appli- cable, although it must cope with the vastly more complex dispersion model for the electronic frequencies. There is one important restriction, however: in order for the final formula given below to be valid, it is necessary for the incident particle to be "fast" not only with respect to ^0, but also with respect to all orbital electron velocities in an atom of the medium — or, in effect, to the greatest of these, namely that of a K electron, approximately Zvq. This is a most severe restriction. For oxygen, as an example, Z = 8, and at a velocity of S^o a proton has energy 1.6 Mev, a deuteron 3.2 Mev, an alpha particle 6.4 Mev. If, how- ever, the particle is fast compared to Zvq, the stopping power is given by the celebrated formula of Bethe: dE 2mv^ = 2kNZ In (4) dx I where N is again the number of atoms of the stopping medium per unit volume, Z the number of electrons per atom (atomic number), and I STOPPING-POWER THEORY FOR MON ATOMIC GAS 149 the mean excitation energy for all the atomic electrons. Unfortunately, it has not yet been possible, except for Z = 1, 2, 3, or 4, to calculate / on the basis of theory alone, because of inadequacy of present knowledge of the dispersive properties of the atoms, and this constant must be de- termined for each value of Z from experimental stopping-power data.* Once / is so established for any atomic medium, the stopping power can be computed at once for any sufficiently swift particle by Eq. 4.t The restriction of the stopping-power formula to particle velocities great compared to Zvq robs it of much of its potential usefulness by re- moving from its domain of applicability the majority of cases heretofore of practical interest. (Work with particles in the 20- to 1000-Mev en- ergy region is only just beginning.) Thus, even for a medium composed of oxygen atoms, the formula is not valid for any natural alpha particles. Fortunately, it is possible to deduce from theory the necessary correction to the formula, at least in some instances. For intermediate and heavy atoms this is a highly complicated problem which has not yet been very much developed. For light atoms, for which, in the case of all but very slow particles, only the K electrons do not satisfy the velocity criterion, the correction for the modified contribution to the stopping power of the K electrons when v is not great compared to Zvq has been calculated quantitatively by Bethe (11) and by Brown (13). This means that the stopping power so corrected is valid for particle velocities great com- pared to the orbital velocity of L electrons in an atom of the medium, and this is a very much less stringent restriction, so that the formula thus corrected covers a much broader region of applicability. The above considerations apply strictly only to media which are monatomic gases. Experimentally, this entails limitation to the noble gases He, Ne, A, etc. Metal vapors, ivhen monatomic — for example, Hg or Na — would be interesting cases, but their stopping powers have not * It is possible, on the basis of the Thomas-Fermi model for the atomic frequen- cies, to deduce from theory an expression giving I of any atom, complete except for a single constant (which is calculable in principle but must at present be obtained empirically). The usefulness of this result has been limited by the fact that the Thomas-Fermi model is trustworthy only for heavier atoms, for which, at familiar particle energies, the Bethe formula does not apply because of the velocity restric- tion. t It must be stated that the proof of Eq. 4 has been accomplished only for the special model in which all atomic electrons are assumed to be hydrogen-like. This fact, together with the absence of a purely theoretical calculation of / for a complex atom, has in a sense reduced the formula, which is of the greatest fundamental im- portance and which must be very accurate for atomic hydrogen, to the status of a semiempirical formula. As such it has been extremely valuable in practice, but it cannot be said to have been rigorously tested as yet by accurate experimental data for a single particle and medium over a broad energy region. 150 PENETRATION PHENOMENA IN LIQUID WATER yet been studied. To this list might be added atomic hydrogen, which can be treated very accurately by theory but which is not amenable to experimental observation. To summarize, for all monatomic gases we have an accurate, simple formula for the stopping power, involving one empirical constant for each gas, which is, however, applicable to a suc- cessively smaller domain of velocities, the larger is Z. For light mon- atomic gases there are available numerical values of the stopping power, again requiring an empirically determined constant, which span prac- tically the entire useful range of velocities. For atomic hydrogen and helium we have an exact, purely theoretical formula of very wide ap- plicability. Empirically, however, we possess only a moderate amount of information about the stopping powers of He and A, and rather less about Ne, Kr, and Xe. IV. Stopping Power of a Polyatomic Gas No detailed theoretical treatment of the stopping power of a diatomic or polyatomic gas has yet been achieved. There is available, however, a wealth of experimental data on diatomic gases (notably H2, N2, O2, and air) and also some information on a few polyatomic gases. It must be stated at once that the binding of atoms in molecules does not alter their stopping powers to any great extent. Little is known about the changes that do occur. The problem has not been one of great interest as far as collision theory is concerned — it is at least as much a problem involving the electronic structure of molecules — and since the advance of our understanding of penetration phenomena has, histori- cally, stemmed largely from the practical needs of nuclear physics, the topic has been a neglected one. Yet it surely is important. Not only have such small alterations an intrinsic practical consequence for ac- curate work; they also may afford valuable clues to far greater effects of molecular structure on other aspects of penetration phenomena; and they are in themselves of much fundamental interest. In this section we shall first appraise the present status of the empirical information and then analyze the molecular problem theoretically. To illustrate the effect of chemical binding on the stopping power we need only consider the case of hydrogen; here we have experimental data for H2 and trustworthy theory for H. The empirical data can be fitted to formula 4, with the quantity N interpreted as the number of molecules per unit volume and Z as the number of electrons per molecule (that is, 2). The data are not at all certain enough to determine the value of I as accurately as one might wish, but values ii^ the neighborhood of 18 ev are usually obtained. However, the theoretical value for isolated hy- STOPPING POWER OF A POLYATOMIC GAS 151 drogen atoms is 15.00 ev, and this must be very accurate. The differ- ence between these two values of I is equivalent to a difference in the stopping powers for alpha particles of 3 per cent at 10 Mev, 4 per cent at 5 Mev, and 5 per cent at 1 Mev. In lieu of theory, interpretation of the stopping phenomena of poly- atomic gases has always been based on the so-called "Bragg rule," which supposes each type of atom to have a definite stopping power for fast particles which is a function of particle charge and velocity, and the stopping powers of atoms bound together in a molecule to be additive. In practice this rule works surprisingly well. Thus, for example, stop- ping powers of H, N, and 0 have been obtained by taking one-half of the empirical stopping power of the corresponding diatomic gas, and that for C by difference from the stopping power of any of a number of gases (or solids) containing carbon. In this way a basic table for H, C, N, and 0, and for other atoms as well, is compiled, and is tested with data from as many different compounds as possible, or is used to pre- dict the stopping power of a compound not yet studied experimentally. The Bragg rule can be expressed equivalently — and more conveniently — in terms of the relative stopping powers of the elements. However, ranges can in general be measured much more accurately than can stopping powers (although even for range determinations there has been an abundance of overoptimistic assessment of experimental error), and there have been many more measurements of ranges than of stopping powers. For this reason the Bragg rule has often been tested by the additivity of values of Sr, rather than of s. (The distinction between s and Sr was explained above.) But additivity of Sr is not the same thing as additivity of s, because of the variable dependence of these quantities on the particle velocity. The statement of the rule which we have adopted, that is, additivity of values of s, is the more fundamental, and is equivalent to additivity of Sr if, and (barring highly unlikely ac- cidental compensations) only if, the ratios of all s values for atoms in the molecule, at all velocities less than the initial particle velocity, are velocity-independent. This tacit assumption is never exactly valid, and is more in error, the greater the disparity in atomic numbers of the atoms in the molecule, and the smaller the initial particle velocity. However, it should always be borne in mind that the Bragg rule does not rest on any quantitative theoretical basis. As data on stopping power improve in accuracy we must anticipate ever more numerous and more consequential departures from the rule. At the present time, because of inaccuracies in stopping-power data, the observed departures are not certain enough to be considered as established, far less to have revealed any pronounced regularities. The smallness of the departures 152 PENETRATION PHENOMENA IN LIQUID WATER does not mean, of course, that binding of an atom in a molecule has no effect on its stopping power. Rather, it implies that the binding of an atom in different molecules has approximately the same effect on its stopping power in each. Since the demands of valence lead to an ap- proximate uniformity in conditions of binding, this is certainly reason- able. If one would search for a disagreement with the Bragg rule, he should investigate different compounds of an atom in which the nature of the chemical binding is known to differ greatly. Furthermore, the atom should be as light as possible, for binding affects only the valence elec- trons, so that it is for the lightest atoms that the effect will be most pro- nounced. How great the effect may be is easily estimated to order of magnitude but is not known with any certainty. As mentioned above, theory has thus far provided no quantitative information, and, more- over, experimental stopping-power data are not yet accurate enough to shed light on the question. As a rule, investigators have greatly under- estimated the systematic errors in their measurements; and in inter- pretations such vital factors as the velocity dependence of the relative stopping power, the distinction between relative range and relative stopping power, and necessary corrections to the Bethe stopping-power formula have all too often been disregarded or dealt with inadequately. The writer views with some skepticism the claims of many stopping- power data to an accuracy of ± 1 per cent or better. Even the accepted values for the stopping powers of air, surely the most carefully in- vestigated of any substance, have changed as much as 5 per cent be- tween 1937 (11) and 1950 (12). In perhaps the most thorough analysis of the validity of the Bragg rule yet made, Gray (10) concluded that the atomic Sr values are additive in molecules in almost all cases to within ±1 per cent. This conclusion, based upon data for alpha particles in the 5- to 9-Mev energy region, would probably be equally valid for the true stopping powers. However, it is, perhaps, also an underestimate. Whereas in many cases the Bragg-rule discrepancy may indeed be 1 per cent or smaller, in others it may ultimately be found to be somewhat greater — perhaps 2 or 3 or even 5 per cent. That it will ever be found to be very much greater than the last figure for any gas (except at very low particle velocities) is most unlikely. The entire problem is cer- tainly ripe for refined reinvestigation. It would seem that among the most promising cases are those for which large discrepancies have al- ready been reported. Thus, to cite only a few: Schmieder (14) found "the" stopping powers of N2O to be 92 per cent, of NO to be 109 per cent, and of NO2 to be 113 per cent, of the respective Bragg-rule pre- dictions; data of Gibson and Eyring (15) lead to a stopping power of STOPPING POWER OF A POLYATOMIC GAS 153 azomethane which is 4 per cent "too low"; Forster (16) found H2O vapor to have 3 per cent smaller stopping power than an equivalent mixture of H2 and O2. Such results are usually discounted [for instance, cf. Gray (10)]. Turning now to theory, we may recognize several more or less distinct factors which should cause the stopping power of a diatomic or poly- atomic molecule to differ to some extent from the simple sum of stopping powers of the isolated atoms of its constituents. They are: 1. Binding of atoms in a molecule changes the values of the possible excitation energies (£„) of the system, and also of the associated oscil- lator strengths (/„). 2. The incident particle may transfer small quantities of energy to vibrational modes of the molecule. 3. Energy may be transferred to rotational modes of the molecule. For the last two effects the existence of a permanent dipole moment of the molecule plays a decisive role ; for the first also it has, in a sense, an indirect influence. We shall consider each of these factors, briefly, in turn. The first factor is undoubtedly the most, and except at low particle energies is probably the only, important one. It affects the stopping power by altering the value for the molecule of the mean excitation en- ergy 7, as may be seen from Eq. 3.* Insight into the working of this in- fluence is gained by considering the Williams-Weizsacker method of treating the collision problem (which was discussed above in terms of impact parameters). For collisions in which the energy transfer to an electron is great compared to the ionization potential of the latter the * Mention must again be made that the Bethe stopping-power formula 4 has not been demonstrated to apply exactly in the case of a molecular medium. Conceiv- ably, two sorts of complication may exist. One, that the spatial distribution of atoms is not random, as inherently assumed in the derivation of formula 4, is almost al- ways without influence. (It might have an effect for radiations of extremely great specific ionization.) The other, that the valence electrons are necessarily far from hydrogen-like, is in a general way covered by the alteration of 8„, /„ values discussed above, provided that formula 3, as properly generalized for a many-electron system, is valid. This effect has not been studied. However, Williams (17, cf. p. 24) has mentioned the possibility that deviations from hydrogen-like binding of the electrons in helium might be responsible for a 10 per cent departure in stopping power from the prediction of the hydrogen-like model, the latter yielding too great a stopping power. Such an effect in He would be a close analog to the effect in a valence bond of a molecule. It would be of great interest to compare accurate stopping-power data for He (which are not available at present) with the results of a refined theo- retical calculation based on an accurate dispersion model, such as is provided by the work of Vinti (18) and of Huang (19). (The former has shown, for example, that 2 per cent of the total oscillator strength of the He electrons arises from double excitations, which are automatically ignored in the hydrogen-like approximation.) 154 PENETRATION PHENOMENA IN LIQUID WATER difference between molecular and atomic binding of the electron is cer- tainly insignificant. But of approximately equal importance for the total stopping power are those collisions in which the energy transfer is of the same order of magnitude as the ionization potential. For such col- lisions the penetrating particle has an effect equivalent to electromag- netic radiation, the frequency distribution of which corresponds to a Fourier analysis of the impulsive field of the passing particle. The en- ergy transfers are thus closely related to the optical absorption spectrum, both discrete and continuous. (This relation was encountered, in the discussion above, as the dependence of I on the dispersion.) Now molec- ular binding has a decisive effect on the discrete spectrum and would without doubt be known to have as great an influence on the continuous absorption in the vicinity of the absorption edge, were our knowledge of the continuous absorption in the case of molecules sufficiently advanced. We must thus anticipate a pronounced alteration by molecular binding of the details of energy loss in these "lighter" collisions, a somewhat smaller influence on the over-all energy loss, and a still smaller but never- theless appreciable influence on the stopping power, which, as may be recognized by inspection of formula 4, depends chiefly on 2kNZ and less sensitively on details of the binding of the electrons. Molecular binding will also influence, to some extent, the oscillator strengths, but not appreciably the binding energies, of inner electrons. This is related to the fact that the osciUator strength of an inner electron is smaller than unity, because its transitions to occupied discrete states are not possible. For a molecule there is, in crude terms, a fuller occu- pancy of the lowest discrete states, thus decreasing the total oscillator strength of an inner electron. This decrease must be compensated by an augmentation of oscillator strengths of outer electrons, so that one might expect / in molecules to be decreased and the stopping power therefore increased by this effect. However, the character and positions of the energy levels are so altered in the molecule that it is not certain whether this conclusion has any validity. The second and third factors listed above are simply additional modes of energy loss, and must be added to the energy loss to electrons in order to obtain the total stopping power. Some energy transfer to vibrational modes will in general accompany electronic excitation, of course; this follows from the Franck-Condon principle and the fact that bond dis- tances in excited states usually differ from those in the ground state. However, energy transfer to vibrational and rotational modes can occur independently of electronic excitation, as is seen at once by considering the equivalent radiation field of the particle. This view also shows that, as in the corresponding optical effects, the energy transfer will be neg- ' M ^ fin / 2my2 ^jme. Wvib • M ^ 2mi;^ STOPPING POWER OF A POLYATOMIC GAS 155 ligible unless the molecule possesses a permanent electric dipole mo- ment. * The stopping power arising from these two types of energy loss has not been calculated.! However, we may easily deduce the following approximate expressions which must be correct, at least in order of mag- nitude, and which suffice for the present discussion: -SL-"^fe'lrr;-)in— (5) \ rf.r/rot \me /2h / \h /me/ i«rot Here ifvib and Wtot are the energies of the first molecular vibrational and rotational levels, respectively. Note that this contribution to the stop- ping power is not of order of magnitude kN\ the great magnitude of atomic relative to electronic mass enters through the small value of w (in atomic units). The equations show at once that these contributions are small compared to the electronic stopping power for fast particles, being roughly 10"^ and 10"^ of the total, respectively: the small factor w'/(me^/2h^) dominates the greater logarithmic factor. In the language of the method of impact parameters, the energy loss is smaller because of the greater mass (atomic rather than electronic) which must be set in motion, although this is partly compensated by the greater p„,a,x — greater because the dynamic screening is less stringent and permits en- ergy transfer to greater distances. In the language of the Williams- Weizsacker method, the energy transfer by emission and absorption of virtual, infrared quanta is smaller, although the smaller energy in such quanta is partly compensated by their greater abundance in the Fourier spectrum. The magnitude of these two additional contributions to the stopping power, relative to the total, is not, however, independent of the energy * There is always a small probability of vibrational excitation arising from col- lisions in which the particle actually passes through the molecule. This probability is small compared to that for "indirect" excitation in the case of polar molecules, and for fast particles is always insignificant in its contribution to the total stopping power. For extremely slow particles (for example, electrons of a few-electron-volt energy) it is known to be appreciable, even for homopolar molecules, but this case does not concern us in the present study. t Massey (20) has derived an expression for the cross section for rotational ex- citation which leads at once to our expression (Eq. 6) for the stopping power, but which is valid only if the dipole moment ijl is small compared to one-half of an atomic unit (h^/me). However, Eq. 6 is still approximately correct for greater values of n, a fact worth noting because n is comparable to h^/me for many molecules of practical importance. Cf. also Wu (21). 156 PENETRATION PHENOMENA IN LIQUID WATER of the particle. Both increase, relatively, as the energy declines. The contribution of rotational excitation is, of course, much the smaller of the two. Although for fast particles they are beyond the discrimination of present stopping-power data, it is quite possible that excitation of vibrations may play a detectable role for particles having velocity of the order of magnitude? of, or only a few times greater than, t^o- We shall now examine a specific and most important example, molec- ular hydrogen. The difference in stopping power between the molecule and the extreme of isolated atoms has already been emphasized, for /isolated = 15.00 ev, whcrcas /bound ~ 18 ev, the former being an exact value from theory and the latter a very approximate one from experi- ment. A theoretical treatment of the stopping power of molecular hy- drogen, or, in effect, a theoretical calculation of /bound, has not yet been achieved. It would be a valuable contribution and appears to lie within the realm of possibility. Values of 8„ for the molecule are, of course, known with great accuracy from analysis of the molecular spectrum. They show a crude, but necessarily far from close, similarity to the corresponding values for the hydrogen atom. The disparity, which arises chiefly from the difference in binding energies for the dif- ferent states, is enhanced by the fact that transitions induced by the passing particle may not appreciably alter the internuclear separation — this being the demand of the Franck-Condon principle — and hence, be- cause all excited states have considerably greater equilibrium separa- tions than the ground state, always leave the excited molecule in a high vibrational level. This coupled vibrational excitation is about 3^ - 1 ev for most excited (attractive) states. For all ionization processes it is slightly greater than 1 ev. For "repulsive" excited states, of course, a great additional amount of energy is transferred as momentary potential energy of the molecule. Although a detailed calculation of /bound for molecular hydrogen will not be attempted here, a rough estimate may readily be given. We write Eq. 3, appropriately generalized to the many-electron case, in the form ■n^^iE/.lni" (7) where P is the ionization potential. In the single instance in which this expression can be evaluated exactly, namely atomic hydrogen, it is found that I/P = 1.102, and it has been common practice to assume that this ratio has the same value for certain other related atoms and molecules; thus, for molecular hydrogen (for which the ionization po- tential is 15.43 ev), it is assumed that / = I.IOP = 17 ev. This pre- STOPPING POWER OF A POLYATOMIC GAS 157 scription is not correct, however. Exchange effects in atoms other than hj^drogen, or in molecules, effectively "tighten" the binding, and this may be considered to have two more or less distinguishable results: the ionization potential P, and the various excitation energies e^ relative to P, become greater than the predictions of the hydrogen-like model; and the ratio I/P is increased.* The last-mentioned effect derives in part from the higher values of excitation energies (even when expressed rel- ative to P), and in part from a shifting of oscillator strengths toward higher excitations and especially ionizations. (In general, the total fraction of oscillator strengths residing in the continuum is greater, the more saturated is the character of the valence-shell binding.) It is ob- viously necessary to take both effects into account. Thus, for hydrogen, Pat = 13.60 ev and Pmoi = 15.43 ev. The first excited atomic level (2p) has e/Pat = 0.75, but the corresponding molecular levels (2p, ^2^+ and 2p, ^n„) are located at about e/Pmoi = 0.81. Similarly, higher molecular levels also exceed corresponding atomic levels in their values of En/P. This analysis is impeded, however, by the fact that not much is known, either empirically or from theory, about the detailed dispersive properties of molecular hydrogen. However, a preliminary study has been reported by Mulliken and Rieke (22). Their results give a total/ of 0.31 for the first excited levels (specified above), per atom, compared to 0.42 for the corresponding atomic level. The second group of levels also has lower / for the molecule. Thus both the effects of departure from hydrogen-like binding are apparent in the case of H2. We there- fore conclude that the ratio I/P should be greater for the molecule than for the atom. Unfortunately, in the absence of theoretical information concerning oscillator strengths for transitions to the continuum (and of empirical information on the continuous absorption of H2) this analysis cannot be carried much further with confidence. A crude estimate leads to /bound ~ l-2Pmoi ~ 19 ev. The effects under discussion all tend to make the stopping power of the molecule smaller than that of the separated atoms. Thus, the above estimate suggests a decrease of about 5 per cent for a 5-Mev alpha particle. It would be of great interest to construct an approximate but complete model for the dispersion of molecular hydrogen, carry through the analysis sketched above, and then compare the conclusion with accurate empirical stopping-power data which will eventually become available. (Those now at hand give merely //P « 1.1 ± 0.1.) The rather great molecular effect indicated above will not enter in the application of the Bragg rule to practical cases involving hydrogen, be- * An extreme example is helium, for which the values of S„/P greatbj exceed hydro- gen-like values, and I/P has a value of about 1.8. 158 PENETRATION PHENOMENA IN LIQUID WATER cause atomic hydrogen is not accessible to stopping-power studies, and the binding of hydrogen in different molecules is of a particularly uni- form character. One would anticipate only minor deviations (ordinarily smaller than 1 per cent) in the contribution of hydrogen to the stopping power for fast particles of various gaseous compounds, the mean value probably being close to the value for ^H2. Nevertheless the analysis for H2, the simplest of all molecules, is of interest as a guide to the understanding of more complex molecules. For such molecules the alterations in fn, Zn values arising from molec- ular binding will be vastly more complex, and there is hardly hope that they will yield to detailed theoretical analysis. Of course, there may be discovered some method of deducing the effect of chemical binding on the stopping power in a general way, without elaborate analysis of the /„, e„ values. Some promise is also offered by the possibility of determin- ing and analyzing the ultraviolet absorption spectra (including continua). Only transitions involving a valence electron will be strongly affected by molecular binding. Thus deviations are likely to be suppressed for all but the lightest elements, notably C, N, O, and F. Because the character of the valence bonding in molecules containing these atoms can vary most markedly, significant deviations from the Bragg rule should be anticipated. Double bonds, triple bonds, and resonating- group structures (such as benzene) seem especiallj^ suggestive in this respect. Because of the inert character of the inner electrons, however, deviations exceeding about 5 per cent will be rare. Molecules, such as NO and NO2, having an odd electron, may present unusual features. V. Stopping Power of a Solid or Liquid The Bragg rule of additivity of stopping powers is usually presumed to be applicable to liquids and solids as well as to polyatomic gases. One would anticipate, however, that the rule should in most circumstances be even more in error for condensed media, in so far as the contribution to the stopping power by outer electrons is concerned, because in ad- dition to effects of chemical binding there may enter effects of inter- atomic or intermolecular interactions involving several or many units of the medium. Such "collective" effects have been investigated only * for the special and important case of metallic conductors. Here the valence electrons * In this paper we exclude problems of relativistic velocities, at which there occurs a decrease in stopping power, arising from screening of the field of the moving parti- cle by the excited and ionized atoms in its wake, that may be very important for condensed substances. This effect, which has attracted much attention in recent STOPPING POWER OF A SOLID OR LIQUID 159 are essentially "free," and naive application of the method of impact parameters, and Eq. 1 with Pmax = 2t;/w, would lead to a paradox for these electrons, for which co '^ 0. Resolution of this paradox, and for- mulation of the theory for the contribution to the stopping power by conduction electrons, have been given by Kramers (23) [cf. also A. Bohr (24)]. In essence, it is found that the effective "radius of action" of the field of the moving charged particle is, after the polarization of the medium by the passage of the particle has been taken into account, not simply p^ax = 2y/c \ V \ \ \ \ \ \\ \ A \ > I h\ \ \ \ \ ~^\ \ ^ \ A \\ ■A \ \\\ \\\ V 1 1 1 ^ \ 100 200 Dose, x-rays, kr 300 Fig. 2. Survival curves at 5 min in the case of multiple infection system, with ultra- violet and x-rays. [Latarjet (15).] INFLUENCE OF PLOIDY IN YEAST We have just observed the influence of multiplicity of units on sensi- tivity, taking as a criterion injury of cytoplasmic elements. Let us now consider the influence of a nuclear constituent, comparing haploid and diploid cells of the same species of yeast as to inhibition of their division by x-rays. All the survival curves obtained for yeasts are sigmoid in shape. Especially with x-rays, the most precise determinations give two-hit curves. Using ionizing radiations with increasing ionization density: x-rays of 0.7, 4.15, and 8 A, and alpha rays of polonium, it has been shown (7) that two distinct units are affected, as all these radiations 246 FACTORS INFLUENCING CELL RADIOSENSITIVITY give two-hit curves, independently of the ionization density (Fig. 3). As the yeast used in this experiment was diploid, one could wonder whether the involvement of two units might not be linked to diploidy. To answer this question, irradiations with x-rays were carried out com- paratively on diploid strains and on haploid lines derived from them by LAg 4.15A 20 30 erg/mm^ 5 10 15 a/mm^ x 10"* Fig. 3. Survival curves of diploid yeasts after irradiation with several monochro- matic radiations (KCu, LAg, KAl) and alpha rays from polonium. All are two-hit curves. [Frilley and Latarjet (7).] Ephrussi (16). The lesion used as a criterion was the inability to multiply indefinitely (immediate death + delayed death). The results (Fig. 4) show that each diploid line gives a classical two-hit sigmoid survival curve, whereas the haploid lines give one-hit curves and, at the same time, show much greater sensitivity than the diploids. This phenomenon cannot yet be interpreted in terms of a precise mechanism, but there is an undeniable influence of the number of repUcations of each chromosome.* * Most of our results have recently been confirmed by Magni in Italy and by Tobias and Zirkle in America (cf. Tobias' paper). INFLUENCE OF PLOIDY IN YEAST 247 An analogous situation has recently been observed by Atwood and A. Norman (personal communication) in Neurospora, where mono- nucleate cells give one-hit curves with ultraviolet rays, whereas multi- nucleate cells show greater resistance and give multihit curves whose multiplicity equals that of the nuclei. 100 24 X 1000 r Fig. 4. Survival curves of x-rayed yeasts of the same family. I, Haploids; II, di- ploids. [Latarjet and Ephrussi (16).] Another difference, perhaps more important, appears between diploid and haploid cells. Figures observed under the microscope in irradiated haploids (Fig. 5) are limited to normal colonies arising from uninjured cells; single cells, normal in size or enlarged (immediate death); and pairs of enlarged cells (delayed death after one division). These figures are definitive and do not change in the course of prolonged incubation. Haploid yeasts do not recover; their lesions are irreversible. In the diploids, on the other hand, one sees, besides the preceding figures, numerous recovery figures which develop in the course of incuba-* 248 FACTORS INFLUENCING CELL RADIOSENSITIVITY tion (Fig. 5). On chains of giant cells apparently doomed to die, cells of normal appearance often arise from which normal colonies then develop. In yeasts this ability to recover from radiation injury is thus linked to polyploidy. Haploid Diploid Fig. 5. Aspect of colonies and injured cells of S. cerevisiae after x-irradiation. Action of Temperature When temperature influences the production of a lesion, it is difficult to know whether this influence takes place during the primary effect, or later, during the "dark reactions" (chemical reactions initiated by the primary effect). However, in certain cases, an experiment may answer this question. Radiochemical reactions are usually considered insensitive to tem- perature. Although rather general, this affirmation should not be ac- cepted in all cases. Thus Schreiber (26), in suppressing the motility of Sphaerocarpus donelUi spermatozoa by monochromatic ultraviolet rays (2650 and 3025 A), obtained the following results: with 2650 A, the efficiency of irradiation remains constant between 0 and 15° C, then increases regularly above 15°; with 3025 A, the efficiency increases steadily with temperature from 0° on. The temperature coefficient of the phenomenon therefore depends on the wave length ; this proves (a) that temperature acts on the primary effect rather than on the dark reactions; (6) that this primary effect is a photochemical reaction with- out intermediate; (c) that this photochemical reaction is sensitive to temperature. (In this case, a relatively simple experiment informs us of the nature of the primary effect.) ACTION OF TEMPERATURE 249 Such effect of temperature on photochemical reactions can be inter- preted by a modification of the absorption spectrum of the absorbing molecules. A rise in temperature increases the molecular oscillations and shifts the entire absorption spectrum toward the red. Such radia- tion, which at a low temperature can only excite the molecule, may dissociate it at higher temperature, the molecule then being more prob- ably in a state of predissociation. The temperature coefficient of the reaction then depends on the wave length. In Schreiber's experiment, in the case of 3025 A, which is of low efficiency, this efficiency increases steadily with temperature; in the case of 2560 A, w^hich is more efficient, the energy of oscillation must reach a noticeable value (15°) in order to manifest itself in respect to the actual energy of the photon. If, as we have just seen, the primary effect is sometimes sensitive to temperature, the latter always accelerates the dark reactions and pre- cipitates the appearance of the lesion. But the earlier appearance of the lesion does not mean a change in sensitivity. What is important here are the frequency and the gravity of the lesions, which temperature may also influence. The unknown processes involved in the dark reac- tions interfere and compete with the cellular metabolisms, some of which, although more or less disturbed by irradiation, may secure dif- ferent types of recovery. These recoveries are influenced by temperature in so far as it affects differently the speeds of the chemical reactions put into play, and in the sense of this difference. When there is no such difference, no temperature recovery takes place (6, 24). It may happen, however, that a cooling off during the latent period slows down the process of lesion more strongly, so that it favors recovery and lowers sensitivity. Strangeways and Fell (28) observed that degenerative modifications produced by x-rays in chicken embryos are lessened and sometimes stopped if embryos are kept for 24 hr at 5° C after irradiation. They concluded that slowing the metabolism gave to the tissues the possi- bility of repairing the lesions. Cook (5) confirmed this observation by submitting several groups of Ascaris eggs to an x-ray dose of 5000 r. The controls incubated at 25° immediately after irradiation gave 2 per cent normal embryos. The other groups were kept at 5° for increasing periods, then incubated. This exposure to cold increased progressively the proportion of normals : 4 per cent after a period of 1 week, 15 per cent after 4 weeks, 45 per cent after 8 weeks, after which no more recoveries took place. By irradiating, either with x-rays or with ultraviolet rays, a bacterium {B. dysenteriae) and a yeast (S. ellipoideus), I noticed (14) that a stay at 5° C before incubation favors recoveries among the irradiated yeast 250 FACTORS INFLUENCING CELL RADIOSENSITIVITY cells but not among the bacteria (Table 1). The significance of this difference in behavior is not clear, and it probably will be useful to check what happens in haploid yeasts, which (cf. p. 245) give no spon- taneous recovery, whereas the above-mentioned diploid yeast gives some. TABLE 1 Type of Radiation I. X-ray, X 1.54 A (Ka of the curve), intensity 6250 r/min Yeast A B Dose 12,500 r 0 44.8 2 22.8 5 11.2 Bacterium A B Dose 12,500 r 0 77.7 2 80.5 5 78.6 7 83.4 II. Same radiation Dos( i 13,100 r 0 37.0 3 30.0 4 28.6 6 22.3 10 19.0 Dose 5,000 r 0 55.3 2 7 10 13 61.6 55.4 68.7 60.0 rDose 700 ergs/mm^ Dose 400 ergs/mm^ III. Ultraviolet radiation, X 2537 A, intensity 26 ergs/mm^/sec 60.0 46.4 32.0 32.4 29.0 3 7 10 86.9 91.8 90.2 93.4 A. Time at 5° C (in days). B. Number injured. Some Problems of Radio-Oxidations sensitization by oxygen According to a very early and well-tested observation (cf. 4, 23) microorganisms capable of living either in aerobiosis or in anaerobiosis are appreciably less sensitive to radiation when they live in the absence of oxygen.* The same observation holds for cells of higher organisms (25). This influence of oxygen is probably most evident when mammals completely deprived of oxygen are irradiated in toto. In their experi- ments, Lacassagne and Latarjet (12, 13) have taken advantage of the remarkable capacity of newborn mice to resist for as long as 20 min complete asphyxia with cessation of circulation and respiration and complete anoxia of tissues. The animals can be irradiated during this * See Hollaender's paper. SENSITIZATION BY OXYGEN 251 period, and later most of them can be easily revived. Mice asphyxiated by nitrogen or carbon dioxide received soft x-rays (0.8 A), at a dose of 1500 r, on the whole body. Whereas the normally irradiated controls all died within 12 days, the animals irradiated during anoxia not only all survived, but subsequently grew at the same rate as the non-irradiated Fig. 6. Effects of anoxic irradiation on newborn mice. Right to left: 1, Non-irradiated control; 2, irradiated in anoxia; 3 and 4, normally irradiated. controls. With ultraviolet rays the difference between normal and oxygen-deprived animals was as notable as with x-rays, with reference both to cutaneous lesions (erythema, epidermitis, deep burns) and to general toxic effects (delay in growth, death). Figure 6 shows, starting at the right, a non-irradiated control, an animal irradiated in anoxia (similar in all respects to the control), and two animals normally exposed to the same dose. The last two show severe cutaneous lesions and dis- tortion due to deep edema. The animal at the left died. The same influence of oxygen is found at the other end of the organic scale, in a simple radiochemical reaction such as formation of hydrogen peroxide in water irradiated with x-rays or alpha rays. The primary 252 FACTORS INFLUENCING CELL RADIOSENSITIVITY role played by oxygen in the reaction has been verified (Loiseleur et al.), but has not been found in the case of alpha rays. With x-rays, for example, about 50 times less H2O2 is obtained in the absence of oxygen. It is mainly in the presence of dissolved oxygen that irradiation pro- vides aqueous media with the properties of an active oxidation system. This fact can be demonstrated directly by recording the platinum po- tential of an aqueous redox system during irradiation. This was done by Loiseleur and Latarjet (19) by x-raying an aqueous solution of quin- hydrone either saturated with or completely deprived of oxygen. The 100 E 90 Fig. 7. Conversion of hydroquinone to quinone by irradiation in the presence of oxygen (curve A), but not in the absence of oxygen (curve B). [Loiseleur and Latarjet (19).] platinum potential of the solution permits observation of the trend of the system, either towards quinone (oxidation) or towards hydroquinone (reduction). One observes (Fig. 7) a progressive oxidation of the satu- rated solution, whereas, in the alternate case, no oxidation takes place. This proves that irradiation of water alone does not liberate oxidative radicals in sufficient quantity to displace the equilibrium in the direction of oxidation, whereas dissolved oxygen insures the phenomenon. Although a mechanism involving free oxygen may not explain all radio-oxidations, this constituent is responsible for a high proportion of them. This is proved by a number of experiments carried out on various biological systems. We may cite, as examples, inactivation of auxine by x-rays (27), and the experiments of Thoday and Read (29) on chromosome alterations produced by x-rays and alpha rays in bean roots suspended in water saturated with nitrogen or oxygen : with x-rays the lesions are more severe in samples irradiated in the presence of oxygen, whereas with alpha rays this gas has almost no effect. Giles and Riley (8) observed in Tradescantia that chromosome sensitivity is OXYGEN ACCEPTORS AS RESISTANCE FACTORS 253 markedly decreased in the absence of oxygen, the ratio of sensitivities between O2 and N2 being about 5 to 1.* From the results obtained in these experiments, it is almost certain that this influence of O2, even in complex systems, is expressed at the level of the primary effect rather than during the dark reactions. Weiss's (30) interpretation of this phenomenon during the primary effect in aqueous systems attributes to the O2 molecule the role of a buffer in the course of the reactions initiated in water. This inter- pretation has the advantage of explaining the disappearance of this factor in the case of alpha rays. It is probable, nevertheless, that O2 acts also by other processes. Direct oxidation of a biological molecule bj^ O2, after activation of one of these two molecules, also plays an important part, according to the theory of Loiseleur and Latarjet. This second mechanism leads to a better understanding of the decrease of sensitivity caused by oxygen acceptors, a phenomenon which we will now discuss. OXYGEN ACCEPTORS AS RESISTANCE FACTORS Whatever the nature of the oxidating agents produced in irradiated medium, whether activated oxygen or free radicals are involved, these agents may reach the oxidizable molecules at the mercy of unknown chemical affinities. A true competition is established in any complex medium, and the oxidation of a biological molecule has to contend with the presence of other oxidizable molecules which more or less protect it. These pro- tection phenomena have been thoroughly studied, and we will consider them here only in so far as they concern oxygen acceptors (without underestimating all other types of protection which are pointed out by Dale, Hevesy, and Barron). When desensitization is obtained, by means of prior injection of pro- teins, serum, or hormones (9), in animals irradiated in toto, it is im- possible, in the present state of our knowledge, to decide which sensi- tivity factor is involved. In certain cases, however, the properties of the injected substance may suggest a mechanism. Thus radiologists long ago observed that diabetics are less sensitive than normal subjects. This fact has been experimentally verified in animals (1, 20); in this particular case it was assumed that glucose acted as an oxidation buffer at the level of primary processes. In fact, working with simple systems, it has been observed that glucose acts as a buffer in that it behaves as a hydrogen donor (17). In water * Cf. Giles's paper. 254 FACTORS INFLUENCING CELL RADIOSENSITIVITY irradiated with x-rays, glucose gives up some hydrogen which combines with oxidative agents to form H2O2; so doing, it blocks these agents, which are thus prevented from combining with other solutes. I take this opportunity to approve Dale's statement regarding H2O2. I should like, however, to point out that if the French school has attached great importance to this substance (cf. 3) it is not always in order to endow it with an active role in the production of the lesions. On the contrary, we have emphasized (17) that H2O2 is, in many instances, a subsidiary product whose presence attests that oxidizing agents have been diverted from the lesion process. This is true to such an extent that in certain cases increase in H2O2 means a decrease in the yield of the biological reaction. The decrease in sensitivity of mice injected with a-tocopherol (10) seems also to be connected with an oxidative process since this substance, as well as glucose, is a strong inhibitor of peroxides. The protection afforded by oxygen acceptors is clearly displayed in irradiation of aqueous solutions of strychnine (20). X-rays inactivate this alkaloid by probable oxidation into genostrychnine with almost complete loss of toxicity. Inactivation is not changed by the presence of non-oxygen-accepting solutes such as NaCl, NaNOs, Fe2(S04)3, SnCU, and saccharose. On the other hand, almost complete protection is afforded by oxygen acceptors closely related to these solutes, that is, NaN02, FeS04, SnCl2, and glucose. This experiment gives strong support to the idea of participation of free oxygen in this inactivation and of a very simple mechanism by which oxygen acceptors assure such protection. PEROXIDASES AS FACTORS OF RESISTANCE AND RECOVERY Instead of decreasing sensitivity by diverting oxidating agents toward acceptors, one can obtain the same result by destroying these agents. For this purpose, peroxidases, and in particular catalase, come first in mind. The preliminary results that I am about to describe reveal the great complexity of the primary processes and especially their depend- ence on cell metabolisms. Chemical destruction of the first irradiation products implies either an immediate contact with the chemical, which therefore must be present in the cell at the time of irradiation, or sufficiently stable radioproducts. We do not yet know the magnitude of the life span of oxidative free radicals, but experiments show that catalase may remain active when added after periods of a minute or even an hour. Accordingly it is possible to conceive of a general method for decreasing certain radiation effects which would consist in treating PEROXIDASES AS FACTORS OF RESISTANCE AND RECOVERY 255 with appropriate substances after irradiation. This "recovery method" would differ from the "protection method" previously discussed. It may be illustrated by some of the results recently obtained in my laboratory by treatment of ultraviolet-irradiated bacteria with catalase. This study followed an observation by Monod et al. (22) that bacteria {E. coll, strain K12) irradiated with ultraviolet light can be reactivated by addition of catalase to the culture medium. This phenomenon, which recalls Kelner's photoreactivation (11), seems to depend to a certain extent on the presence of visible light. The preliminary results that we have already obtained with two bacteria {E. coli, strains B and K12) can be summarized as follows: 1. Both bacteria, enriched in catalase content by growing in a medium containing a large amount of this enzyme, show increased resistance to ultraviolet light. Survival rates may be 20 times as high as those of the controls. This effect depends on the dose; weak with low doses, it increases with higher doses. The table shows the results of one experiment : Dose, grgg / Survival Rates mm^ Controls I Catalase II II/I 800 3 X 10"'' 6.7 X 10"* 2 1000 3.3 X 10-^ 3.3 X IQ-'' 10 1200 5 X 10"^ 1 X 10^ 20 2. This effect does not occur with x-rays, either in B or in K12. This negative result recalls an observation by Barron et al. (2) that catalase does not prevent the inhibiting action of x-rayed water on cellular respiration. With ultraviolet rays, since catalase is present in the cell at the time of irradiation, we may be dealing with either a pro- tection or a recovery effect. 3. According to Monod's observation, the recovery effect appears when K12 bacteria grown in synthetic medium and sterilized by ultra- violet radiation are plated with catalase and incubated. The extent of the reactivation varies widely, depending on the dose and on the meta- bolic conditions of the cell. We did not succeed in controlling the metabolic factors, and results varied greatly between experiments carried on under apparently identical conditions. So far reactivation has occurred sometimes when catalase is administered without any addition of visible light. But it has been a constant and striking phe- nomenon when, before incubation, the bacteria undergo an exposure to visible light which in itself would produce only a very slight effect. 256 FACTORS INFLUENCING CELL RADIOSENSITIVITY The following are the results of a typical experiment : Ki2 irradiated with 1200 ergs/mm" (X 2537 A) Number of irradiated bacteria plated per Petri dish: 4 X 10^ Number of colonies after 36-hr incubation: Irradiated control (dark) 22 Irradiated control (+ visible light) 220 Irradiated control (+ catalase dark) 35 Irradiated control ( + catalase, same dose + visible light) 2500 (Sometimes catalase alone would give 1000 colonies.) 4. This phenomenon seems to require much more stringent conditions than photoreactivation. For example, we were not able to reproduce it in bacterium B under the conditions used with K12, although these con- ditions allowed B to undergo strong photoreactivation and although this bacterium had shown increased resistance to ultraviolet radiation when grown in the presence of catalase. 5. This recovery takes place in K12 even when catalase is added some time after irradiation, the length of the time depending on the tem- perature. In cultures kept at 37°, reactivation is still possible when catalase is added 2 hr after irradiation. It is thus evident that the action of the enzyme involves relatively stable compounds. 6. No reactivation was obtained after x-irradiation. Such facts might throw some light upon the origin of the radioresistance displayed as a hereditary character by some mutant strains (31). REFERENCES 1. Baclesse, F., and J. Loiseleur, Compt. rend. soc. bioL, 141: 7-43-745, 1947. 2. Barron, E. S., V. Flood, and B. Gasvoda, Biol. Bull., 97: 51-56, 1949. 3. Bonet-Maury, P., and M. Lefort, Compt. rend., 226: 1363, 1445, 1948. 4. Braun, R., and H. Holthusen, Strahlentherapie, 34: 707-734, 1929. 5. Cook, E. v.. Radiology, 32: 289-293, 1939. 6. Duryee, W. R., J. Natl. Cancer Inst., 10: 735-795, 1949. 7. Frilley, M., and R. Latarjet, Compt. rend., 218: 480-481, 1944. 8. Giles, N. H., and H. P. Riley, Proc. Natl. Acad. Sci., 35: 640-646, 1949. 9. Graham, J. B., and R. M. Graham, Proc. Natl. Acad. Sci., 35: 102 106, 1949. 10. Herve, A., and Z. M. Bacq, Compt. rend. soc. biol, 143: 881, 1158, 1949. 11. Kelner, A., Proc. Natl. Acad. Sci., 35: 73-79, 1949. 12. Lacassagne, A., Compt. rend., 215: 231-232, 1942. 13. Lacassagne, A., and R. Latarjet, Compt. rend. soc. bioL, 137: 413-414, 1943. 14. Latarjet, R., Compt. rend., 217: 186-188, 1943. 15. Latarjet, R., J. Gen. Physiol, 31: 529-546, 1948. 16. Latarjet, R., and B. Ephrussi, Co7npt. rend., 229: 306-308, 1949. DISCUSSION 257 17. Latarjet, R., and J. Loiseleur, Compt. rend. soc. bioL, 136: 60-62, 1942. 18. Latarjet, R., and R. Wahl, Ann. inst. Pasteur, 71: 336-339, 1945. 19. Loiseleur, J., and R. Latarjet, Compt. rend. soc. bioL, 135: 1530-1532, 1941. 20. Loiseleur, J., and G. Velley, Compt. rend., 230: 784-786, 1950. 21. Luria, S. E., and R. Latarjet, J. Bad., 53: 149-163, 1947. 22. Monod, J., A. I\L Torriam, and M. Jolit, Compt. rend., 229: 557-559, 1949. 23. Mottram, J. C, Brit. J. Radiol, 8: 32-39, 1935. 24. Patt, H. M., and M. N. Swift, Am. J. Physiol., 155: 388-393, 1948. 25. Scherk, R., Radiology, 46: 395, 1946. 26. Schreiber, H., Naturwiss., 29: 669, 1941. 27. Skoog, F., /. Cellular Comp. Physiol., 7: 227-270, 1935. 28. Stangeways, T. S., and H. B. Fell, Proc. Roy. Soc., B102: 9-29, 1927. 29. Thoday, J. M., and J. Read, Nature, 160: 608, 1947; 163: 133, 1949. 30. Weiss, J., Nature, 153: 748, 1944. 31. Witkin, E. M., Genetics, 32:221-248, 1947. 32. Wollman, E., and A. Laeassagne, Ann. inst. Pasteur, 64: 5-39, 1940. DISCUSSION Rubin: I should like to discuss briefly the radiation deaths in various strains of E. coli. It has been shown that the B strain, the parent strain, and the B/r, which is derived from the B strain, show different sensitivities to ionizing radiation. It has further been shown that B/r resembles K12 and most other strains of E. coli. In other words, the B strain tends to be the one that stands by itself rather than the B/r strain. If one examines the data, it would appear to be evident that two different mechanisms of killing both apply to the B strain and only one mecha- nism of killing to the B/r strain and to the K12 strain. It may be that the reason that Latarjet has not found reactivation is that in one strain a certain mechanism is operating and in the other strain another mechanism is operating, so one would not expect to find comparable results in his two strains. Latarjet : I am very much interested in this remark. As a matter of fact, K12 is signifi- cantly more resistant to radiation than B, though still more sensitive than B/r. Therefore, B/r should be tested for catalase reactivation. However, there might remain some differences between K12 and B/r, such as recombination, which could account for differences in beha\aor regarding the catalase phenomenon. Dale : I am very interested in the fact that the results varied with the presence or ab- sence of oxygen in these experiments. I should like to ask Latarjet whether in his quinhydrone experiment the control without radiation in the presence of oxygen showed a change to the oxidized form. Latarjet : No. 258 FACTORS INFLUENCING CELL RADIOSENSITIVITY Dale : The recovery of Ki2 in the presence of catalase may possibly indicate that a highly active, organically bound ion causes of itself some inactivity. Lataejet: If catalase is added after the radiation has been done, the factor is controlled. Dale: It may be possible that, even if added later, catalase brings about some re- covery. Barron: Latar jet's experiment on oxygen is confirmed by our experiment with ferrocy- tochrome c, in which the inactivation was due to the hydroxyl and HO2 radicals. Catalase does not protect ferrocytochrome c during exposure to x-rays. Ferrocy- tochrome c irradiated in the absence of oxygen shows 37 per cent protection, as compared with the results of radiation in the presence of oxygen. /4 On the Localization of Radiation Effects in Molecules of Biological Importance MARTIN D. KAMEN Mallinckrodt Institute of Radiology Washington University Medical School St. Louis, Missouri A biological system is a theorist's nightmare. Even in its simplest manifestation it consists of a semiliquid, heterogeneous aggregation of molecules of all sizes and complexities, interacting in a precisely ordered manner by means of various mechanisms largely unknown. Study of these systems underlines the embarrassing gaps in our knowledge of the liquid state and of polymer chemistry at the presumably more amenable level of inorganic physical chemistry. The abstractions derived from the quantum description of molecules like methane or benzene in the gaseous state, or from the radiochemistry of simple molecules in aqueous solutions, are necessary but not sufficient for an understanding of the interaction of radiation with biological systems. At present the only recourse is to grope empirically — at least until we come to grasp an adequate understanding of protein structure, enzyme synthesis, and the mechanism of enzyme action. However, it does not follow that studies of the effect of radiations in complex systems cannot be made and their results correlated with what is already known from the study of simpler systems. Among the questions which can be posed in such a study is whether, in large molecules of biological importance like protein or nucleoprotein, there is varying radiosensitivity in any particular bonds or regions. Ex- periments in which external radiation is used may provide data like ionic and excitation yields and quantum efficiencies. But they give no information on this very fundamental point. It is possible to make an experimental approach to this problem using chemical complexing agents specific for particular atomic groupings, like S — H and free amino groups. A more direct attack, however, would seem to result from placement of unstable isotopes of elements involved in definite bond sites. An example which comes to mind readily is sub- 259 260 LOCALIZATION OF RADIATION EFFECTS stitution of radioactive carbon (C^'*) in the carboxy-peptide linkage of protein. But, if we are to achieve a sufficient number of bond dis- turbances from the disintegration act, it is more practical to use a rel- atively short-lived isotope, like P^^. Such a short-lived isotope may be used to study the effects of disturbing 0 — P bonds in phosphorus-con- taining structures, particularly in nucleoprotein. To exemplify this ap- proach and the manner in which interpretation of results obtained await the solution of purely physical problems, we will consider briefly some of the recent experiments by Hershey, Kennedy, Gest, and the writer.* When bacteria and bacteriophage are grown in a medium containing phosphate with a high P^^ content (0.003-0.03 per cent), the viral progeny are unstable and show a progressive loss of infectivity with time. We find that this inactivation is primarily the result of the radioactive decay of the assimilated P, and not of the ionization resulting from pas- sage of the beta particles through the phage. There is a linear relation between the inverse of the P^^ content for phage and the average sur- vival time. This relation holds over a sufficient range to provide a use- ful technique for examining distribution of P^^ atoms among the phage population. The phage particles studied may be thought of as spherical nucleo- protein macromolecules with a maximal diameter of about 110 milli- microns. Each particle contains '^5 X 10^ P atoms distributed in some unknown manner. Some of these P atoms are radioactive and decay with frequency determined by the disintegration constant, X. The re- sults of this act in any given decay will be excitation and probably rup- ture of the 0 — P bonds, which may initiate a chain of reactions leading to inactivation of the whole molecule. In analyzing the expected dependence of the phage survival on the P^^ content, we begin by assuming (a) that disintegration of a single atom can occasionally inactivate a phage molecule, (h) that all the phage particles are equally radiosensitive, and (c) that the P^^ atoms are dis- tributed at random among the phage particles. From these assump- tions, it follows that the change of phage titer with time, —dS/dt, is proportional to the number of disintegrations occurring in unit time, which can be shown to equal 3.4 X 10^^a\NSAoe'''''K In this expression the known quantities are X, the disintegration constant, or fractional decay per day; N, the total number of P atoms per phage particle; Aq, the * A detailed presentation of the data and methods involved has appeared since preparation of this article; see A. D. Hershey, J. W. Kennedy, H. Gest, and M. D. Kamen, J. Gen. Physiol., 34: 305, 1951. The writer wishes to express his gratitude to Dr. Hershey for his wiUingness to permit this discussion to appear before publi- cation of the formal report. LOCALIZATION OF RADIATION EFFECTS 261 initial specific P^" content (specific radioactivity in curies per gram); and S, the surviving phage titer per miUiUter. Thus, a, the efl^iciency of inactivation, can be determined, knowing the rate of decay of the phage population. Setting S = Sq and t = 0 when the initial specific activity is Aq, it follows that log 5 - log>So = 1.48 X 10-V4oA^(l - e"'') Extensive experimental data have been obtained which in all cases agree with this relation in presenting a straight line when the logarithm of the phage surviving is plotted against the function (1 — e~^'). The intercept at f = 0 agrees with the value log Sq, and the slope is propor- tional to the initial specific radioactivity Aq. The values of aN derived from these plots vary from 41,000 to 58,000, with a mean of 43,000 for the phage tested (phage type T'* and three closely related strains were examined). Since N is known to be 5.0 X 10^ E, where E is the efficiency of the counting method (plaque count), the value for a is 0.086/E' per atomic disintegration. E is believed to be very close to 100 per cent, so that on the average, under the experimental conditions used, a phage particle is inactivated at least once in 11.6 disintegrations. Now what can be done about interpreting this figure? First it is necessary to examine the efficiency of inactivation to be expected, on the basis that the inactivation results from the passage of beta particles resulting from the P^^ decay. The average energy of the P^^ beta par- ticles is 7.0 X 10^ ev. The average initial specific ionization is 7.1 ion pairs per mm air. The air-path equivalent of such beta particles in a phage molecule would average 0.04 mm, so that 0.28 ion pair would be produced per phage particle. For the highly energetic P^^ beta particles this probably represents a maximum, because clustering of ion pairs would be expected to occur rather frequently. Using the value for a derived experimentally, this means 11.6 X 0.28, or 3.3, ionizations pro- duced by the beta particles inside a phage for inactivation. Thus, the efficiency is 0.3 per ionization. Actual measurements of inactivation of unlabeled phage placed in labeled phosphate solutions show an efficiency of only 0.009 per ioniza- tion. Comparable efficiencies found from perusal of x-ray data in the literature are somewhat higher but still far from the expected figure of 0.3. Hence it is highly improbable that we are dealing with inactivation resulting from ionization by beta particles. It can be concluded that the overwhelming majority of inactivations occur as a consequence of the events following transformation of the P atom itself, and that these experiments bear on the question of the relative radiosensitivity of the 0 — P bonds in the nucleoprotein. 262 LOCALIZATION OF RADIATION EFFECTS We must now examine mechanisms available for inactivation follow- ing transformation of P atoms. These may be of two types. One in- volves bond rupture, the other bond excitation. Many uncertainties arise in attempting to assess the fraction of the phosphate bonds ruptured per disintegration. The bond energies prob- ably range between 10 and 20 ev. If one assumes the nuclear recoil to be taken up by the resultant S atom and one or two of the 0 atoms, the mass available for the recoil momentum ranges from ^^30 to ^^60. Still more uncertainty attends this estimate because the angular dis- tribution of the neutrinos emitted in the beta process is not known. If the neutrino and beta particle fly off in the same direction, the residual nucleus can experience its maximum recoil (~100 ev) while showing a continuous spectrum of recoil energies ranging down to some value be- low 10-20 ev. Other distributions are a consequence of assumptions in which the average angle between neutrino and beta particle is taken as 180° or, what appears to be the most popular value, 135°. A very rough calculation indicates that for the most part there is sufficient energy to effect a bond rupture for at least 50 per cent of the disintegrations. Another effect, namely, the replacement of P by S in the nucleoprotein at the site of the disintegration, might be expected to alter radically the functionality of the particular bond involved. This alteration would not be expected to be so much a consequence of the mere replacement of P by S as of the rearrangement of electrons resulting during the trans- formation. Thus, the overall change would involve only departure of one proton along with the nuclear electron. Little strain on the bridging bonds between the phosphate and the nucleotide moieties would re- sult because the S— 0 and P— O bond distances are not very different. It can be appreciated that much uncertainty attaches to the estimate of percentage bond rupture following radioactive decay. It seems rea- sonable, however, to conclude that any of the processes involved would be quite as effective in immobilizing a portion of the nucleoprotein and, taken in the aggregate, would be more than sufficient to explain the high efficiency of assimilated P^^ for inactivation as compared to external p32 or x-rays. We must suspend, for the time being, the interesting query whether bond rupture is more effective than bond excitation in inactivation of nucleoprotein. Incidentally it would be of interest to see whether properties other than mere inactivation are affected by a few radioactive events. Although the experimental exploitation of this direct procedure for probing variation in radiosensitivity is still very much in its infancy, it is possible to distinguish certain features of the data which indicate that LOCALIZATION OF RADIATION EFFECTS 263 specific atom groupings, such as phosphate in nucleoprotein, vary con- siderably in response to radiation. Thus, data on inactivation of phage — whether derived from x-ray experiments or from experiments in which P^^ is incorporated into phage — indicate that, although one "hit" or event may be sufficient to inactivate a whole phage molecule, numerous such events occur before inactivation takes place. In x-ray inactiva- tion, for instance, the survival curve shows the "one-hit" type of process, while dosage measurements indicate approximately 75-100 ionizations occurring inside each phage. Experiments employing incorporation of P^^ directly into the phosphate of phage yield data of a similar nature, but they reveal besides that a few specific bond sites, that is, phosphate groups, cannot be disturbed without inactivation of the molecule as a whole. Only when the structure of nucleoprotein and the disposition of phosphate within the viable molecule are understood will it be pos- sible to propose definite mechanisms for inactivation by radiation inter- action with the P atoms of the nucleoprotein. Some extensions of these procedures to other enzymes and systems come to mind. A few systems which might be examined by these methods include: 1. Inactivation of enzymes containing more or less firmly bound co- factors, that is, triosephosphate oxidase of rabbit muscle with its one molecule of diphosphopyridine nucleotide (1). 2. Changes in physicochemical properties of proteins into which large amounts of phosphate can be incorporated, as in so-called phosphopro- teins of yeast. 3. Use of S^^ in studying radiosensitivity of H — S and S — S bonds in proteins, such as insulin. 4. Possible localization of short-lived C^^ in carboxy-peptide bonds by biosynthesis of protein from labeled CO2 or carboxyl-labeled amino acids. 5. Comparison of products obtained by x-ray bombardment of and by P^^ incorporation into simple molecules like glucose-1-phosphate and adenosinetriphosphate. Ramifications of considerable potential importance may also be ex- pected from exploitation of the suggestion by Hershey that the relation between survival time and P^^ content of phage be utilized for distin- guishing distributions of P^^ in heterogeneous phage populations, and in particular for distinguishing parent from progeny phage in experi- ments designed to establish mechanisms of phage reproduction. The future exploitation of these methods depends primarily on the availability of radioactive elements with high specific activity. In ex- tending researches of the type described, P^^ samples with specific 264 LOCALIZATION OF RADIATION EFFECTS activities greater than 50 curies per gram are required. Such samples can be prepared using the present uranium-pile installation, apparently without marked changes in routine procedures. However, the po- tentialities of these researches warrant increasing efforts to produce large amounts of the various radioactive isotopes needed with specific activities some orders of magnitude greater than are now available routinely. REFERENCE 1. Taylor, J. F., S. F. Velick, G. T. Cori, C. F. Cori, and M. W. Stein, J. Biol. Chem.,' 173:619, 1948. DISCUSSION Rubin: Absorbed P^^ was found to increase significantly the rate of mutation at a specific locus in E. coli (streptomycin resistance). The calculation of the distribution of P^^ decays of such low energy as to in- crease significantly the specific ionization in the cells shows no great difference from Kamen's calculations. Wyss: The decay of radioactive P to S might be of primary importance. The phage containing such a substitute for P might readily invade a bacterial cell but could not there reproduce itself because of the absence of identical building blocks, that is, nucleic acids containing S instead of P. In Kamen's experiments such a phage is recorded as being non-infective, and the impUcation is that it became non-infective during the emanation. Kamen : It is not obvious that a single sulfate radical-containing nucleotide would necessarily be unavailable for synthesis into a nucleic acid. Such a "thionu- cleotide" would be only a slight modification of the natural nucleotide and might be used to synthesize a slightly modified macromolecule in which only 1 in several 100,000 P atoms was replaced by an S atom. When it is possible to prepare sulfur analogs of nucleotides, it will be interesting to see how well the nucleic acid synthesis system is able to incorporate them into the natural nucleoprotein. Failla : It could also be assumed that only the low-energy beta particles produce enough ionization in the phage; that is to say, only the low-energy beta particles will have a high probability of effectiveness and of consequent biologic change. Powers: The experiments at Argonne on the phenomenon reported today have given qualitatively the same result.* In Paramecium aurelia it has been shown that * Powers, E. L., Genetics, 33: 120, 1948. DISCUSSION 265 incorporated P^^ is about 5 times as efficient in inducing death after autogamy as are Sr^^'^^Y^" solutions delivering the same beta-radiation dosage to the cell. In our system the accumulation of phosphorus by the organism is a complicating factor which reduces the calculated efficiency of P^^ to about 4 times that of the Sr solutions. It should be noted that the Paramecium evidence concerns the induction of changes in micronuclei which undergo a series of maturation di- visions followed by the fusion of two gametic nuclei before the expression of the effect. The phenomenon has, then, been detected in a system which is genetic in the "gene-chromosome" sense. The experiments have been extended to radioactive hydrogen. Recent results show that tritium is certainly no more efficient than one would expect on the basis of dosage of ionizing radiation delivered to the cell by the beta particles from the H^ atoms. It appears that the respective positions of P and H in the structure of the nucleus are markedly different in importance. Solomon : In addition to the bond rupture around radioactive sulphur which Kamen pointed out, it might also be possible to have the situation complicated by the reacti\dty of the sulfur which is formed ; perhaps it can combine with the oxygen atoms. In ovir laboratory we have found that sulfur produced from chlorine will exchange with sulfur in the carbon disulfide molecule, which does not in the ordinary way happen. Latarjet : What would be the efficiency of an ionization in Kamen's case as compared to the one I obtained with soft x-ray * irradiation of intracellular T2? I am attempting at present to induce host range mutations in a bacteriophage by ultraviolet irradiation of the intracellular phage during its growth process, f I should like to ask Kamen whether he thinks that his technique would be suitable for the same kind of experiment with ionizing radiation. Kamen : The effect could be studied. It might be possible to take mutants containing P^^, cross them with members of a normal strain, and study the results of hy- bridization. Latarjet : Is a the probabiUty of the efficiency of ionization? Kamen: Yes. Latarjet : It is interesting that studies with x-rays on the same phage give -values of approximately 0.12, which is in fair agreement with Kamen's value of 0.08. * J. Gen. Physiol, 31: 529, 1948. t Compt. rend., 228: 1354, 1949. 266 LOCALIZATION OF RADIATION EFFECTS Muller: Is there any reason to believe that 1 in 11 represents the chance that inacti- vation will result from the conversion of any P atom in the nucleoprotein to S, rather than the proportion of P atoms in the nucleoprotein which are so situated that inactivation will inevitably result from their conversion to S? Kamen: It is not known whether 8 per cent of the phosphorus is in the right portion of the molecule and will have 100 per cent probability of inactivation following disintegration of the P^^, or whether distribution of the energy throughout the molecule will give rupture at the appropriate bond in 12 per cent of the cases. 15 Recent Evidence on the Mechanism of Chromosome Aberration Production by Ionizing Radiations * NORMAN H. GILES, JR. Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee Understanding the mechanism by which ionizing radiations produce chromosome aberrations is one of the fundamental problems in radiation genetics. The present discussion will deal with recent evidence on this subject, obtained for the most part from experiments with plant chro- mosomes, especially those of the spiderwort, Tradescantia. At the out- set it appears desirable to review briefly the interpretation of aberration production in Tradescantia as proposed originally by Sax (24, 25) and developed in quantitative form primarily by Lea (20). After this, more recent results, stemming principally from the observations by Thoday and Read (28, 29) on the effect of oxygen on aberration frequency, will be presented. Finally the implications of the oxygen effect for an interpre- tation of the biochemical mechanism of radiation-induced chromosomal changes will be considered, in particular the extent to which this effect requires a revision of previous views as to the mechanism involved. The Production of Chromosomal Aberrations in Tradescantia BY Ionizing Radiations The general experimental technique for observing the effects of ion- izing radiations on Tradescantia chromosomes as developed by Sax (24) has been described in detail previously by Sax (25), Catcheside, Lea, and Thoday (6, 7), and Lea (20) and will be considered here only briefly. The typical procedure is to expose entire inflorescences, consisting of several buds containing microspores in various stages of development, to penetrating radiations such as x-rays or fast neutrons. Cytological * This work was done under Contract W-7405-Eng-26 for the Atomic Energy Commission, Oak Ridge, Tennessee. 267 268 CHROMOSOME ABERRATION PRODUCTION examinations are then made at appropriate intervals after treatment to detect aberrations at the first postmeiotic mitosis in the microspore. Two general categories of aberration types are noted: (a) chromatid types, resulting from irradiation of chromosomes which are effectively double in prophase, and (6) chromosome types, resulting from irradiation of chromosomes which are effectively single in resting stage. In both in- stances, radiation produces breaks in one or more of the six chromosomes in the nucleus of a microspore, and the resulting broken ends may re- main as such, undergo restitution, or rejoin with other broken ends to produce aberrant configurations which are cytologically detectable. The principal configuration types with which we shall be concerned in the experimental results to be discussed in this paper may be designated as chromosome interchanges. These rearrangements, observed in cells examined 4-5 days after irradiation, are either dicentric or ring chromo- somes and result from breaks in two separate, undivided chromosomes, or chromosome arms, followed by the reunion of broken ends to give one chromosome with two centromeres, or one continuous ring chromo- some, plus an accompanying acentric fragment in each case. The essential features of the hypothesis developed by Sax, Lea, and others to explain the production of chromosome aberrations in Trades- cantia may be outlined as follows. Electromagnetic or particulate radi- ations produce their effects as a consequence of the formation of ion pairs within a chromosome during the passage through the chromosome of either primary or secondary charged particles. Chemical changes re- sulting from this direct ionization of the molecules composing the chromo- some lead to the production of chromosome breaks. The resulting broken ends may remain as such, yielding terminal deletions, or undergo restitution, giving rise to apparently normal chromosomes again, or re- join with other broken ends, producing cytologically visible aberrations. The restitution and reunion processes are competitive and both space and time factors are involved. Several ionizations (betw^een fifteen and twenty) must occur within the chromosome to produce a break, and the factor of major consequence in distinguishing the quantitative effects of various radiations is the difference in ionization distribution along par- ticle tracks. For certain radiations (for example, gamma rays and x- rays) ionization distribution along the tracks (of secondary electrons) is such that the probability of breakage of a chromosome by a traversing particle is considerably less than 1 for most of the length of the track except near the end. As a consequence, such radiations are relatively inefficient in producing breaks, on the basis of total ionization produced per track. Furthermore, breaks in two separate chromosomes (or chro- mosome arms) are almost always produced by two separate particle RADIATION— INDUCED ABERRATIONS 269 tracks, with the result that the frequency of interchange aberrations (for example, dicentrics and rings) increases as the square of the dose (a two-hit curve) when the time of irradiation is kept constant. Because such aberrations are two-hit phenomena, there is also an intensity ef- fect— the yield of interchanges decreasing when a constant dosage of radiation is administered over periods of increasing duration. The average time during which a break may remain "open" before restitu- tion or reunion occurs (that is, its half life) is at least 4 min. For other radiations (for example, recoil protons from fast neutrons), ionization distribution along tracks is such that the probability of breakage of a chromosome by a traversing particle is close to 1, and as a consequence the efficiency of such radiations in producing chromosomal changes is very high compared to those of gamma or x-rays. In addition, both the breaks in the two separate chromosomes (or chromosome arms) taking part in an interchange are usually produced by a single particle track, resulting in a linear relationship between interchange frequency and dose (a one-hit curve) and the absence of an intensity effect. In addition to aberrations resulting from breaks in two separate chro- mosomes, certain types are produced oy breaks in a single chromosome arm (either divided or single). The majority of these aberrations are one-hit types resulting from the passage of a single ionizing particle, and exhibit no intensity effect with any type of radiation. The interpretation of chromosome-aberration production as just out- lined has been quite generally successful in accounting for most of the quantitative results of radiation experiments w^ith Tradescantia. How- ever, experimental data of two sorts have been obtained which indicate that this hypothesis in its simplest form is not entirely adequate. The first evidence was that obtained by Kotval and Gray (17) in their studies with alpha particles. On the basis of comparative ionization distribu- tion and particle numbers, the hypothesis predicts that a given amount of ionization produced by alpha particles should be considerably less efficient in producing chromosome breaks than an equal ionization dose produced by fast neutrons, whereas the experimental results indicate that for equal ionization doses alpha particles are somewhat more efficient. It was concluded that a proportion of the breaks produced by alpha particles arises from ionization produced in the immediate vicinity of, but not within, a chromosome, thus suggesting the involvement of an indirect as well as a direct mechanism. The second, and even more striking, evidence was that obtained by Thoday and Read (28), who noted a pronounced effect of oxygen on the frequency of x-ray-induced aberrations in the root-tip mitoses of the broad bean, Vicia faha. Their experiments indicated that the absence of oxygen during irradiation re- 270 CHROMOSOME ABERRATION PRODUCTION suited in a marked decrease in aberration frequency. Similar results were obtained by Hay den and Smith (15) in experiments with barley seeds. Experiments on the effect of oxygen have also been performed with Tradescantia, and the results of some of these will now be discussed. The Effect of Oxygen on X-Ray-Induced Chromosomal Rearrangements in Tradescantia The experimental methods used to expose Tradescantia inflorescences to x-radiation while in atmospheres containing various percentages of oxygen have been described in some detail by Giles and Riley (11, 12) and will be outlined only briefly here. Inflorescences were placed in an appropriate holder inside an airtight Lucite exposure chamber. This chamber was placed inside the x-ray machine and connected through ports by pressure tubing and appropriate valves to a vacuum pump, a gas pressure cylinder, and a mercury manometer. Air in the chamber could be evacuated and replaced by the appropriate gas or gas mixture from the cylinder. The chamber could also be maintained under vac- uum, or under pressures up to 3 atm above normal atmospheric. Rapid introduction or removal of gas could be effected with the apparatus. All these manipulations could be carried out before, during, or after ir- radiation, depending on the experimental conditions desired. The x-ray intensity for each exposure was determined by means of a Victoreen thimble ionization chamber which could be inserted into the box in the same position as that normally occupied by the inflorescences. A series of dosage curves was obtained for inflorescences exposed in air, in oxygen, and in nitrogen. The yield of both interchanges (di- centrics and rings) and interstitial deletions was markedly reduced when nitrogen replaced air in the chamber, and increased somewhat when oxygen replaced air. Additional comparative exposures were made in other gases, such as helium and argon, and also under vacuum. In all instances, reduced aberration frequencies similar to those obtained in nitrogen resulted, indicating that the absence of oxygen was responsible for the decrease in radiosensitivity (11). No chromosomal effects were noted in control experiments in which similar exposures to nitrogen and oxygen were made without irradiation. It was clear from these resuhs that the presence of oxygen resulted in a marked increase in aberration frequency. The next problem was to determine the reason for this effect of oxygen. If it is assumed that all breakage is the result of direct-hit effects, the same number of breaks should be produced in the presence or absence of oxygen by a given x-ray dose. Thus the increased aberration fre- OXYGEN EFFECT WITH X-RAYS 271 quency obtained in oxygen might result from an effect of oxygen itself on the recovery process, such that when oxygen is present new reunions of broken ends are favored as opposed to restitution. It seemed possible, for example, that such an effect could result from the stimulation of chromosome movement by oxygen. Another and perhaps more likely possibility appeared to be that the effect of oxygen itself is an indirect one, such that in the presence of dissolved oxygen x-rays produce in cells a certain substance or substances which increase the yield of aberra- tions. In this event, such an intermediate substance could produce an effect by way of either the recovery or the breakage mechanism. In the former instance, the x-ray breakage of chromosomes would still be con- sidered a direct effect; in the latter, however, the breakage would have to be considered an indirect effect, at least in part. It appeared feasible to attack some of these problems experimentally in Tradescantia, since in this organism the recovery process extends over a considerable period of time (the average time between production of a break and restitution or reunion being at least 4 min). Thus it is pos- sible to separate to a considerable degree the two processes of breakage and recovery and to test the effect of oxygen on each. To do this, in- florescences were exposed to a single dose of 300 r of x-rays in 1 min, either in the presence of pure oxygen or in the absence of oxygen (in vacuo). Immediately after the irradiation oxygen was either removed (by evacuation) or introduced (to a positive pressure of 1500 mm of Hg). The exchange of gases, as recorded by the manometer, could be effected quite rapidly, and in this fashion it was possible to have the breakage process occurring in oxygen and recovery largely in its absence, or the reverse. In addition, other experiments were performed in which oxygen was either introduced or removed during part of the x-ray exposure. The results of such comparative exposure are reported in the paper of Giles and Riley (12). Certain other experiments of a similar type have been performed, and these results will be presented here. In the original experiment in which a single exposure of 1 min to 300 r was made in vacuum followed by the immediate introduction of oxygen, no effect on the recovery process was noted. It was decided to increase the possi- bility of detecting such an effect by fractionating the dose so that a rel- atively larger portion of the recovery period would take place in oxygen. The following procedure was used. A set of inflorescences was exposed in vacuum for 20 sec at 300 r per min; after the irradiation, oxygen was immediately introduced into the chamber to a positive pressure of 1500 mm of Hg and allowed to remain for 8 min; the chamber was then re- evacuated and another 20-sec exposure made, followed by the reintroduc- tion of oxygen as above; this procedure was repeated 5 times to give a 272 CHROMOSOME ABERRATION PRODUCTION total dose of 500 r. Two control series were run, in one of which the procedure just described was followed except that helium rather than oxygen was introduced. In the other control a total dose of 500 r was administered in 100 sec of continuous exposure in vacuum; helium was then introduced and remained in the chamber for 8 niin after the x-ray exposure. The data from this experiment are presented in Table 1. It TABLE 1 Test of the Effect of Oxygen on the Recovery Mechanism, Using Fractionated Doses All series received a total dose of 500 r at 300 r per min. In series A this dose was delivered in five equal fractions, with irradiation occurring in vacuum, and each intervening recovery period of 8 min occurring in oxygen at a positive pressure of 1500 mm of Hg. Series B was similar, except that the recovery periods occurred in helium. Series C received one continuous exposure in vacuum, with recovery occurring in helium. Series Conditions A Fractioned dose; irradiation in vacuum; recovery in oxygen B Fractioned dose; irradiation in vacuum; recovery in helium C Continuous exposure; irradiation in vacuum; recovery in helium is evident that there is no increase in aberration frequency in series A, in which oxygen was present during recovery, as compared with B, in which helium was present. The somewhat higher values for C are to be expected, since this was a continuous exposure with no intervening re- covery periods. A second experiment has been performed (in cooperation with A. V. Beatty) to retest the observation of Giles and Riley (12) that the ad- dition of oxygen during irradiation results in an immediate increase in aberration frequency. These data are presented in Table 2. In this in- stance, essentially the same experimental conditions were utilized as previously, except that a second exposure (series C) was made in which oxygen was present during only the last 15 sec of the total x-ray exposure of 1 min. The results of this experiment are in agreement \\\ih the earlier one in indicating that the introduction of oxygen during irradia- tion results in an immediate increase in aberration frequency. It is clear from these comparisons (and those reported earlier) that the addition or removal of oxygen immediately after irradiation does not No. of Interstitial Cells Interchanges Deletions Examined per Cell per Cell 323 0.23 ±0.027 0.20 ±0.025 450 0.28 ±0.025 0.31 ±0.026 582 0.31 ±0.023 0.35 ±0.024 OXYGEN EFFECT WITH X-RAYS 273 modify the aberration frequency, thus indicating that oxygen itself has no effect on the recovery process. There seems to be Uttle question that, under the experimental conditions utilized, oxygen diffuses very rapidly into the cells and is present during the recovery process. This is shown by the fact that the introduction of oxygen during irradiation causes a TABLE 2 Experiments on the Introduction of Oxygen during X-Irradiation OF Tradescantia Inflorescences (All exposures of 300 r at 300 r per min) Series Pretreatment Conditions Exposure Conditions Post-Treatment Conditions No. of CeUs Inter- changes per CeU Interstitial Deletions per Cell A Buds in vacuum Vacuum Vacuum — 10 min 850 0.11 ±0.01 0.08 ± 0.01 B Buds in vacuum 1st 30 sec; vacuum 2nd 30 sec ; oxygen intro- duced (within 3 sec) to 1500 mm of Hg Evacuation (within 25 sec) Vacuum — 10 min 900 0.32 ± 0.02 0.36 ± 0.02 C Buds in vacuum 1st 45 sec; vacuum Last 15 sec; oxygen intro- duced (within 3 sec) to 1500 mm of Hg Evacuation (within 25 sec) Vacuum — 10 min 600 0.22 ± 0.02 0.26 ± 0.02 D Buds in oxygen Oxygen at 1500 mm of Hg Evacuation (within 25 sec) Vacuum — 10 min 900 0.61 ±0.03 0.67 ± 0.03 pronounced increase in aberration frequency. This latter result is also important in providing additional evidence that oxygen, to be effective, must be present during the actual irradiation and that there is little or no latent period between the introduction of the gas and its effect in terms of increased aberration production. Other experiments have also demonstrated that a pre-exposure of buds in pure oxygen before they are irradiated in helium has no effect. From all these observations, it seems clear that the effect of oxygen itself is an indirect one, presumably arising from the production by x- 274 CHROMOSOME ABERRATION PRODUCTION rays, when oxygen is present, of some substance which increases the frequency of aberrations. As indicated previously, this effect of such a substance might operate by way of either the breakage or the recovery mechanism. The most Hkely hypothesis seems to be that such a sub- stance would cause an increased production of chromosome breaks. However, the alternative possibility, that the recovery process is mod- ified, cannot be immediately excluded on the basis of the data just pre- sented. This problem must be attacked by other methods. Experi- ments designed to determine whether the recovery time is different for breaks produced in the presence and absence of oxygen are under way (Giles and Riley, unpublished). The results to date are compatible with the view that the recovery mechanism is essentially the same under these two conditions. The previously reported differences in the slopes of dosage curves obtained at a constant intensity of 45 r per min in air, in oxygen, and in nitrogen, which at first appeared to be due to an effect on restitution time, can be explained on the basis of an intensity effect resulting from a lesser production of breaks per unit time in nitrogen. It has been shown by Sax (26) that the exponents of dosage curves from chromosome interchanges decrease with decreasing radiation intensity. Evidence has also been obtained (Giles and Beatty, unpublished) that the effect of temperature, which has been previously interpreted as in- fluencing the recovery process (25), is actually, at least to a considerable extent, an indirect effect on oxygen availability. The experiments of Baker and Sgourakis (4) with Drosophila have demonstrated that oxygen has a marked effect in increasing the frequency of other types of x-ray- induced genetic changes, sex-linked lethal mutations, where there is no evidence that a recovery process is involved. All these results suggest very strongly that the oxygen effect is on the breakage and not on the recovery mechanism in Tradescantia. In order to provide additional evidence concerning the mechanism of the oxygen effect, it appeared desirable to determine the relationship between the amount of oxygen present during irradiation at a constant dosage and aberration frequency. Consequently, a series of exposures has been made of inflorescences in atmospheres containing different per- centages of oxygen (mixed with helium) at a single x-ray dose of 400 r. Some data on this point have already been published; see Giles and Riley (12). Another experiment (Giles and Beatty, unpublished) has been carried out to determine this relationship more precisely. In this instance special attempts were made to free the helium used of any residual oxygen. The percentages of oxygen (2, 5, and 10 per cent) in the other gas mixtures used were accurate to within ±0.2 per cent, and a larger number of cells was scored to increase the statistical reliability OXYGEN EFFECT WITH X-RAYS 275 of the determinations. A graphical summary of the results is presented in Fig. 1, together with the averages of points obtained at higher per- centages of oxygen in previous experiments. It is clear that there is still a substantial yield of aberrations even in the complete (or nearly com- plete) absence of oxygen. When oxygen is present in irradiated cells, there is a rapid rise in aberration frequency above this base level. This 0 10 20 30 40 50 60 70 80 90 Percentage of oxygen in exposure chamber (normal atmospheric pressure) Fig. 1. Reproduced by permission from Science, 112: 643, 1950. increase is linear between 0 and 10 per cent oxygen, after which the rise is apparently more gradual. Certain additional experiments (Giles and Beatty, unpublished) pro- vide further evidence that the amount of dissolved oxygen present in the cells is an important factor in determining aberration frequency. In these experiments, a constant percentage of oxygen was used in the ex- posure chamber, but irradiations were carried out with the inflorescences under pressures up to 3 atm above normal. The data obtained for ex- posures at 0, 1, 2, and 3 atm above normal pressure (approximately 740 mm of Hg) in 10 per cent oxygen (plus 90 per cent helium) have been in- dicated in Fig. 2, on the assumption that the amount of effective dis- solved oxygen in the cells is directly proportional to the pressure. Con- trol experiments in which comparable exposures were made in helium under pressure indicated that pressure alone did not change the aberra- tion frequency. As can be seen from the graph (Fig. 2) there is good agreement with previous exposures in different percentages of oxygen 276 CHROMOSOME ABERRATION PRODUCTION I 1 1 1 1 - - ] f ^: - - 4 / r - - / 400r at 50r/min. - o = 5%0, (subscripts) pressu es above normal - I = 10% O 1 2 (subscripts) press 1 I J res above 1 norma - at normal atmospheric pressure. As mentioned previously, preliminary experiments have also been completed which strongly suggest that the effect of temperature on aberration frequency is, at least in large part, actually an oxygen effect (Giles and Beatty, unpubhshed). Similar evidence concerning the temperature effect on sex-linked lethals in Drosophila has already been obtained by Baker and Sgourakis (4). 1.1 -55 0.9 - 2P0.7 •E 0.5 - 0.3 - 0.1 - 0 20 40 60 80 100 Percentage of oxygen in exposure chamber Fig. 2. Reproduced by permission from Science 112 : 643, 1950. The Biochemical Mechanism of the Oxygen Effect It is clear from the results which have just been presented that oxygen has a marked effect in increasing the radiosensitivity of Tradescantia chromosomes, as measured by the frequency of x-ray-induced inter- changes and deletions. We shall assume, as most of the evidence seems to indicate, that this effect arises only if oxygen is present in cells during the actual period of irradiation, and results from the production by x- rays of more chromosome breaks under these circumstances. The sim- plest explanation for this situation would appear to be that, in the presence of oxygen, irradiation results in the production within the nucleus of some substance (or substances) which causes an increase in chromosome breakage and that the amount of this substance produced is positively correlated with the amount of oxygen present. It thus be- comes of interest to determine, if possible, what this substance is. Since these cells are composed largely of water, it seems very probable that the substance is a product of irradiated water, more particularly, a product characteristically formed when oxygen is present in irradiated BIOCHEMICAL MECHANISM OF OXYGEN EFFECT 277 water. It has already been suggested by Thoday and Read (28, 29) that this product may be hydrogen peroxide. Additional evidence is now available which supports this conclusion. The results of four entirely unrelated types of experiments furnish evidence favoring or compatible with the H2O2 hypothesis. Bonet-Maury and Lefort (5) have in- vestigated the production of H2O2 in water irradiated with x-rays and with alpha particles under various conditions, including the effect of oxygen concentration and of temperature. In addition, data are avail- able on the effect of pH on peroxide yield; see Loiseleur (21). At least four striking parallels exist between H2O2 production and chromosome-aberration production under varying conditions of irradia- tion, (a) It is found that with x-rays H2O2 is not produced in oxygen- free water [or is produced in very small amounts, as shown by Allen (1)], but that when oxygen is present the amount of H2O2 produced depends markedly on the concentration of oxygen. The type of curve obtained for the increased yield of H2O2 with increasing oxygen concentration at a constant x-ray dose is generally similar to that obtained in the present studies for the relation between aberration frequency and the percentage of oxygen present during x-radiation. (6) In oxygen- saturated w^ater, H2O2 production by x-rays decreases regularly with decreasing temperature, a definite discontinuity marking the pas- sage from water to ice. Below — 116°C no H2O2 can be detected. Faberge (10) has shown that, when Tradescantia pollen grains are x- rayed at various temperatures, the general character of the sensitivity curve (as measured by the number of chromosome breaks observed in pollen tube divisions) resembles that for H2O2 production. There is a dip in the region of 0° C and a gradual decline thereafter. However, al- though H2O2 production stops at — 116° C, chromosome breaks are still produced at — 192°C, their frequency being almost one-fifth that at +25° C. (c) The pH of the solution exerts an effect on the yield of H2O2 with x-rays, an alkaline pH favoring a lowxr H2O2 concentration. The experiments of Marshak (22) have shown that the frequency of chromosome aberrations observed at anaphase in root tips of Vicia faba and Allium cepa is markedly reduced when x-radiation is carried out in the presence of penetrating bases, such as ammonium hydroxide, {d) Undoubtedly the most cogent evidence obtained to date in favor of the H2O2 hypothesis is that derived from a comparison of x-ray and alpha- particle effects in the presence and absence of oxygen. With alpha par- ticles, H2O2 production occurs even in oxygen-free water and the ad- dition of oxygen does not increase the yield. Thoday and Read (29) have shown that, for aberrations induced in the root tips of Vicia faba, the removal of oxj^gen results in little if any decrease in aberration fre- 278 CHROMOSOME ABERRATION PRODUCTION quency when the cells are irradiated with alpha particles, as compared with a very marked decrease when x-rays are used. These observations have been confirmed in preliminary results obtained by Conger (un- published) in experiments on the irradiation of mature pollen grains of Tradescantia with alpha particles. Although these comparisons suggest very strongly that H2O2 may be the product involved in the oxygen effect, they do not establish this point unequivocally. The possibility exists that other products of the radiodecomposition of water may be concerned. The observations of Krenz and Dewhurst (18) on the effect of dissolved oxygen on the oxida- tion of ferrous sulfate in aqueous solution by gamma rays can apparently best be explained by a mechanism involving the HO2 radical. The marked similarity between the magnitude of the decreased oxidation of ferrous sulfate in the absence of oxygen (to about one-fourth) and the decrease in aberration frequency obtained in the early experiments with Tradescantia is noteworthy. However, the absolute magnitude of the decrease in aberration frequency occurring in the absence of oxygen apparently depends on the dose and the intensity in a particular experi- ment. Further, the experimental results with alpha particles in the presence and absence of oxygen appear to make it unlikely that HO2 is the intermediate involved. These results are in agreement with the view, on radiochemical grounds, that H2O2 is formed directly in large amounts by alpha particles because of the very close proximity of the OH radicals produced in the center of the particle track [Gray (13)]. The failure of O2 to increase the yield of H2O2 apparently indicates that the usual reaction for peroxide formation by way of the intermediate HO2 radical is relatively unimportant [Bonet-Maury and LeFort (5)]. It thus appears that H2O2, rather than HO2, may be responsible for chromosome breakage. There are, however, additional mechanisms by which HO2 may be produced from peroxide, and the possibility cannot be entirely eliminated that this radical may also be to some extent an effective agent in chromosome breakage. Additional evidence on the presumptive role of H2O2 should be ob- tained from experiments with enzyme inhibitors such as cyanide which should block the action of catalase and permit H2O2 accumulation. A suggestive slight mutagenic effect of cyanide alone, which has been in- terpreted on this basis, has already been reported by Wagner et al. (30) in Neurospora. The experiments of Mottram (23), which show that cyanide increases the sensitivity of roots of Vicia faha to x-rays, as judged by inhibition of growth, also lend support to this possibility. In the Neurospora experiments a direct mutagenic effect of H2O2 was also noted. In experiments utihzing enzyme poisons to elucidate the mecha- OXYGEN EFFECT AND PREVIOUS HYPOTHESES 279 nisms of the oxygen effect, however, the specificity of the poison would appear to be exceedingly important; otherwise an unequivocal interpre- tation of the results is not possible. Effects due to cyanide might result from an inhibition of the cytochrome system, thus preventing the uti- lization of oxygen by the respiratory enzyme systems of the cell. If such a utilization of oxygen is necessary to bring about its effect on radiosensitivity, then it might be expected that cyanide would decrease, rather than increase, radiosensitivity. There is, in fact, one report, that of Bacq et al. (3), that cyanide exerts a protective action against the killing of mice by x-rays, but these results are not confirmed in similar experiments by Dowdy et al. (9) with rats, in which a clear effect of anoxic anoxia was demonstrated. With respect to the production of chromosomal aberrations, it appears probable that the oxygen effect is produced by oxygen dissolved in the aqueous medium of a cell, but ad- ditional experimental evidence on this point is being sought. There is also the possibility that, even though H2O2 may be one of the essentially primary radiation products associated with the oxygen effect, it still may not be the actual mutagen directly responsible for chromosome breakage. It may be only an intermediate in the formation of other substances such as organic peroxides, some of which have been shown by Dickey et al. (8) to have marked mutagenic effects in Neuro- spora. It appears likely that the effect of organic peroxides may result from free-radical formation; and, indeed, it is possible that most, if not all, chemical mutagenic effects may be explicable on this basis and thus turn out to be fundamentally related to radiation-induced mutations [Auerbach (2), Dickey et al. (8), Jensen et al. (16)]. The Relation of the Oxygen Effect to Previous Views on THE Mechanism of Chromosome Breakage in Tradescantia BY Ionizing Radiations The preceding discussion has indicated the remarkable effect of oxy- gen in increasing the radiosensitivity of Tradescantia chromosomes. This effect can be most easily interpreted as resulting from an increased production of chromosome breaks by x-radiation, the amount of increase being positively correlated with the amount of oxygen present in cells. On the basis of such results, it would appear that the previous hypothe- sis utilized to explain the production of aberrations in Tradescantia must be modified. On this hypothesis, as outlined earlier, chromosome break- age has been considered to result from the direct action of the radiation in ionizing the molecules actually composing a chromosome, as a con- sequence of the passage through the chromosome of an ionizing particle 280 CHROMOSOME ABERRATION PRODUCTION [Lea and Catcheside (19)]. It now appears most likely that an indirect mechanism is involved, in which irradiation of the oxygen-containing aqueous medium in the cell leads to the production of some substance which in turn produces chromosome breaks. It should be recalled, however, that a substantial aberration frequency is still produced by irradiation in the absence of oxygen (at least in so far as oxygen can be removed from these cells). The question thus arises whether there are two mechanisms for chromosome-break produc- tion, one involving direct ionization of the chromosome molecules and the other an indirect effect from the irradiated aqueous medium, and further whether the relative importance of the two mechanisms may be judged by the degree of the oxygen effect. That such is the situation is by no means clear. It seems possible, in fact, that at least some of the aberrations induced in the absence of oxygen may also be the result of an indirect effect, being produced by substances other than H2O2 or HO2, such as OH radicals, resulting from the radiodecomposition of es- sentially oxygen-free water. Attempts have been made to test this point by experiments (Giles and Beatty, unpublished) designed to minimize the effectiveness of the OH radical by promoting, during irradiation, the back reaction to form H2O [Allen (1)]. To do this, inflorescences were exposed to 400 r of x-rays in atmospheres of hydrogen, both at normal pressure and at 3 atm above normal. Interchange frequencies were essentially the same for the tw^o exposures, and although both values were somewhat lower than those obtained in comparable exposures in helium or nitrogen, the difference is not significant. If it is valid to assume that hydrogen would in fact react to remove OH radicals formed during irradiation, the failure to detect a reduced aberration yield in these experiments supports the view that chromosome breakage produced by x-rays in the absence of oxygen may all result from direct ionization of the chromosome mole- cules. It should be pointed out, however, that this conclusion is based on the assumption that reactions leading to H2O2 production or suppres- sion in cells from which oxygen has been removed as completely as pos- sible are similar to those occurring in oxygen-free pure water. There is as yet little experimental evidence on this point, and it is quite pos- sible that the complexity of the cellular environment may cause very different reactions to occur. Unfortunately it does not appear to be experimentally feasible to as- sess the relative importance of the indirect and direct effects on chromo- somes by the method normally employed for enzymes and viruses— that of determining the effect of a constant radiation dose w^hen the solute concentration is varied over a considerable range. The nearest approach SUMMARY 281 to this situation would appear to be sensitivity tests of cells that vary in water content. This is not possible with developing microspores of the type used in these experiments. It might be feasible with mature pollen grains, however. There are in fact many data which indicate that dry seeds of plants show increased radioresistance, both with re- spect to killing and to induced genetic changes, genie as well as chro- mosomal, as compared with hydrated seeds [Gustafsson (14)]. However, it is not always possible from such comparisons to conclude that the sensitivity changes are directly correlated with water content, and do not result from changes in the mitotic state of the nucleus, Avhich are known to affect radiosensitivity. Regardless of the degree of importance of the direct effect, it appears probable that in Tradescantia, at least, the magnitude of the indirect effect is considerably greater. However, it is still possible to interpret the results in terms of target theory, as indicated by Thoday (27). The essential requirement is that the action of ionizing particles, whether direct or indirect, be relatively localized. If the effect is principally in- direct, it appears that a substance, such as H2O2, must be produced along the track of an ionizing particle and must have a relatively limited effective diffusibility (or short half life). In fact it seems necessary that its effective distribution within the nucleus must correspond in pattern rather closely to that of ionization distribution along particle tracks. Such a localized distribution would appear to be essential, as has been pointed out by Zirkle (31), in order to explain the striking quantitative differences among various radiations, as, for example, the shapes of the dosage curves for interchanges induced by x-rays and alpha particles. Summary Recent experiments have demonstrated that oxygen has a marked ef- fect in increasing the sensitivity of chromosomes in Tradescantia and other plants to x-rays as measured by the frequency of cytologically detected aberrations. It has been shown that this is not an effect of oxygen itself on the behavior of broken ends of chromosomes. The ef- fect apparently arises from the production by x-rays, as a result of the radiodecomposition of water in cells containing oxygen, of some sub- stance which causes an increase in aberration frequency. Several inde- pendent lines of evidence indicate that this substance may be H2O2. It appears likely that the increased frequency of aberrations arises from an increased production of chromosome breaks when oxygen is present dur- ing irradiation, rather than from a modification of the recovery process. Thus a major fraction of the radiation effect on Tradescantia chromo- 282 CHROMOSOME ABERRATION PRODUCTION somes is to be considered indirect rather than direct. In the absence of oxygen, however, there is still an appreciable aberration frequency and some evidence indicates that this entire fraction may be the result of direct radiation action. Since the previous hypothesis explaining chromosome breakage in Tradescantia assumed that all the effect of the radiation resulted from the direct ionization of the molecules of the chromosome, the demonstration of an indirect effect necessitates a revision of this interpretation. How- ever, it is still possible to interpret the results in terms of target theory. The essential requirement is that the action of ionizing particles, whether direct or indirect, be relatively localized. If the effect is principally in- direct, it appears that a substance, such as II2O2, must be produced along the track of an ionizing particle and must have a relatively limited effective diffusibility (or short half life). In fact, it seems necessary that its effective distribution within the nucleus must correspond in pattern rather closely to that of ionization distribution along particle tracks. REFERENCES 1. Allen, A. O., Radiation chemistry of aqueous solutions, /. Phys. 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H., G. A. Cleland, and C. Lotz, The role of organic peroxides in the induction of mutations, Proc. Natl. Acad. Sci., 35: 581-586, 1949. 9. Dowdy, A. H., L. R. Bennett, and S. M. Chastain, Study of the effect of varying oxygen tensions on the response of mammalian tissues to roentgen irradiation. Atomic Energy Comm. Document UCLA-55 (unclassified), 1950. 10. Faberge, A. C, Chromosome breakage by X rays at low temperature and the radiodecomposition of water. Genetics, 35: 104, 1950. 11. Giles, N. H., Jr., and H. P. Riley, The effect of oxygen on the frequency of X-ray-induced chromosomal rearrangements in Tradescantia microspores, Proc. Natl. Acad. Sci., 35: 640-646, 1949. DISCUSSION 283 12. Giles, N. H., Jr., and H. P. Riley, Studies on the mechanism of the oxygen effect on the radiosensitivity of Tradescantia chromosomes, Proc. Natl. Acad. Sci., 36:337-344, 1950. 13. Gray, L. H., Electrons, neutrons, and alpha particles. Chap. XV in Biophysical Research Methods, F. M. Uber (Ed.), Interscience, New York. 14. Gustafsson, A., Mutations in agriculture plants, Hereditas, 33: 1-100, 1947. 15. Hayden, B., and L. Smith, The relation of atmosphere to biological effects of X rays, Genetics, 34: 26-43, 1949. 16. Jensen, K. A., G. Kolmark, and M. Westergaard, Back mutations in Neurospora crassa induced by diazomethane, Hereditas, 35: 521-525, 1949. 17. Kotval, J. P., and L. H. Gray, Structural changes produced in microspores of Tradescantia by a-radiation, J. Genetics, 48: 135-154, 1947. 18. Krenz, F. H., and H. A. Dewhurst, Mechanism of oxidation of ferrous sulfate by 7-rays in aerated water, J. Chem. Phys., 17: 1337, 1949. 19. Lea, D. E., and D. G. Catcheside, Induction by radiation of chromosome aber- rations in Tradescantia, J . Genetics, 44: 216-245, 1942. 20. Lea, D. E., Actions of Ionizing Radiations on Living Cells, Cambridge, 1946. 21. Loiseleur, J., Sur les echanges electroniques dans I'eau soumise a Taction du rayons X, Compt. rend., 224: 76, 1942. 22. Marshak, A., Alteration of chromosome sensitivity to X rays with NH4OH, Proc. Soc. Exptl. Biol. Med., 38: 705-713, 1938. 23. Mottram, J. C, Variations in the sensitivity of the cell to radiation in relation to mitosis, Brit. J. Radiol, 8: 643-651, 1935. 24. Sax, K., Induction by X rays of chromosome aberrations in Tradescantia micro- spores, Genetics, 23: 494-516, 1938. 25. Sax, K., An analysis of X-ray-induced chromosomal aberrations in Tradescantia, Genetics, 25: 41-68, 1940. 26. Sax, K., Types and frequencies of chromosomal aberrations induced by X rays. Cold Spring Harbor Symposia Quant. Biol., IX: 93-103, 1941. 27. Thoday, J. M., Oxygen and chromosome mutation in plants, Brit. Sci. News, 3:66-69, 1950. 28. Thoday, J. M., and J. Read, Effect of oxygen on the frequency of chromosome aberrations produced by X rays. Nature, 160: 608-610, 1947. 29. Thoday, J. M., and J. Read, EfTect of oxygen on the frequency of chromosome aberrations produced by alpha rays, Nature, 163: 133-135, 1949. 30. Wagner, R. P., C. H. Haddox, R. Fuerst, and W. S. Stone, The effect of irradiated medium, cyanide, and peroxide on the mutation rate in Neurospora, Genetics, 35 : 237-248, 1950. 31. Zirkle, R. E., Relationships between chemical and biological effects of ionizing radiations. Radiology, 52: 846-855, 1949. DISCUSSION Barron : I had no intention of taking part in this discussion. However, at the sugges- tion of Eyring, I will re-emphasize the role of oxygen in ionizing radiations, inasmuch as under normal conditions biological fluids are constantly saturated with oxygen. It is well known that the formation of H2O2 on irradiation of water with x-rays occurs only when oxygen is present. Furthermore, the atomic hy- drogen, formed on the primary ionization of water, may reduce molecular oxygen 284 CHROMOSOME ABERRATION PRODUCTION to form the powerful radical O2H: O2 + H -» O2H. The presence of oxygen, therefore, increases considerably the oxidizing power of ionizing radiations. Molecular oxygen can be used, for these reasons, as a test for the mechanism of action of ionizing radiations. If the effects obtained on irradiation are greater in the presence of oxygen, there will be no doubt that the effect was due to prod- ucts of the irradiation of water. The radical OH and jitomic hydrogen are formed regardless of the nature of gas dissolved in water ; O2H and H2O2, how- ever, are formed only in the presence of oxygen. Thiol compounds, such as glutathione, — SH enzymes, — SH proteins, are oxidized by all three agents, and irradiation will be more effective in the presence of oxygen. Ferrocyto- chrome c is not oxidized by H2O2; in this case the presence of oxygen would increase only oxidation due to the radical O2H. Finally, there is the possibility, which must be investigated, that the H2O2 produced on irradiation may act as an oxidizing agent when combined with catalase. In summary, the presence of oxygen introduces two more oxidizing agents. It is obvious, therefore, that the absence of oxygen will decrease the toxic effects of ionizing radiations, a decrease which reaches 70 per cent in the case of the oxidation of thiol compounds. If ionizing radiations have the same effect in the presence as well as in the absence of oxygen, oxidation processes cannot be dis- missed because the powerful OH radicals are still formed. It would be inter- esting to study the effect of low oxygen tensions on irradiation of tissues with small doses of radiation. It is quite possible that x-irradiation of bacteria at low oxygen tensions, such as those prevailing in the high plateaus of the Andes and the Himalayas, would produce fewer mutations than irradiation at sea level. /6- Physical and Chemical Factors Modifying the Sensitivity of Cells to High-Energy and Ultraviolet Radiation * ALEXANDER HOLLAENDER Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee Microorganisms are in some ways ideal materials for obtaining imme- diate quantitative information on some of the basic aspects of radiation effects. It is well known that they can often be handled in very large 100 Aspergillus terrcus Normal spores Irradiated dry ■ Proton or alphas • X-rays 0 20 40 120 60 Dose, kilorep Fig. 1. Comparative lethal action of x-rays (250 kev), of protons, and of alpha particles on dry spores of Aspergillus terreus. [From Stapleton and Martin (18).] numbers, thus giving statistical results on effects which are hardly rec- ognizable on higher organisms. They are, in general, not suitable for * This work was done under Contract No. W-7405-Eng-26 for the Atomic Energy Commission. 285 286 FACTORS MODIFYING THE SENSITIVITY OF CELLS studies of radiation effects on chromosomes, which have been so well demonstrated by Giles, and for most genetical studies, which will be discussed by Muller. Typical killing curves which have been obtained with fungi in our laboratory are given in Fig. 1. They refer to a comparative study of x-rays, and alpha particles and protons, on Aspergillus terreus [Stapleton and Martin (18)]. We are, however, not too enthusiastic about the use of these curves as the only basis for interpretation of the mechanism of lethal action of radiation, since "death" could be caused by a variety of mechanisms which are difficult to untangle. There are many treatments which in themselves have little or no ef- fect on the radiation sensitivity but, if given in supplement to ionizing radiation, produce striking effects. In general, these supplementary treatments have increased the sensitivity of cells to radiation rather than protected them; however, there are a few exceptions to this. Heat and Visible Light The first two factors to be discussed are the effect of heat and light in the treatment after irradiation. Cells are more sensitive to heat after irradiation; this applies to x-rays as well as ultraviolet [Gaulden (8)]. I should like to refer you also to the work of Curran and Evans (5) and of Anderson and Duggar (2). E. H. Anderson (1), in our laboratory, found that a certain strain of Escherichia coli can be reactivated after exposure to ultraviolet several hundred-fold, if incubated at 40° C, as compared with incubation at 30° C. This effect can be found to a shght degree after x-raying (about five-fold). However, this heat recovery has been recognized wdth only strain B of E. coli. In this connection it should be mentioned that Hayden and Smith (9), in their work with maize, found heat reactivation of seeds after x-ray exposure. Unfortu- nately, no repetition of this work has been reported. A careful test in our laboratory has given negative results [Suskind (20)]. Photoreactivation In 1949 Kelner (14) and Dulbecco (7) first reported "photoreactiva- tion" after exposure to short ultraviolet. Most of the work reported until now has dealt with primary irradiation of 2537 A. Some work by Carlson and McMaster (4) in our laboratory has shown that, in the nucleolus of the grasshopper neuroblast, the photoreactivation declines after exposure to wave lengths shorter than 2537 A. The photoreactiva- tion itself is limited to wave-length range of 3650-4500 A, given im- INFRARED 287 mediately after "short ultraviolet" exposure. There is some difference of opinion in regard to the most effective wave length for different or- ganisms or even different strains in one species of organism [Kelner (15), Knowles and Taylor (16)]. No significant photoreactivation has been found after exposure to x-rays. It appears from the present informa- tion that photoreactivation either destroys a toxic substance which is formed by irradiation with 2537 A or reverses the destructive action oi an essential compound needed for the survival of the cell. The fact that photoreactivation is found after exposure to short ulti-a- violet fits into the general picture of the behavior of cells after irradi- ation with ultraviolet as compared with x-rays. Bacteria or fungi sur- viving ultraviolet have a much extended lag phase [Hollaender and Duggar (10)]. This extended lag phase has been determined very care- fully, but it can also be recognized by observing plate cultures which have been incubated about 12 hr. There is very little of this extension after x-ray exposure. Our interpretation at present is that the extension of the lag phase is a non-chromosomal effect and that photoreactivation works mostly through the cytoplasm. Such an interpretation seems reasonable at first but would have to be checked experimentally. Infrared We reported several years ago, first in cooperation with Kaufmann (12) and later with Swanson (19), that infrared around 10,000 A given before {Drosophila, Tradescantia, and Aspergillus terreus) or after {Trades- cantia) x-radiation will increase the effectiveness of x-radiation in pro- ducing chromosomal rearrangements and chromatid breaks and muta- tions (Aspergillus terreus). The effect is somewhat more pronounced in regard to chromatid breaks, as Giles and Beatty (unpublished) have found in our laboratory. The infrared alone, given under carefully con- trolled conditions, will produce no recognizable chromosome changes. It is important to point out that a carefully designed experimental tech- nique must be used so as not to raise the temperature in these biological materials to a level in which heat damage could appear. This infrared work indicates that, in addition to the usually recognized damage, some "potential" damage must be produced in the chromosomes by x-radia- tion which is not obvious under normal conditions and which can be re- paired if x-radiation is given alone. These additional effects of infrared radiations have also been observed in regard to nitrogen mustards by Swanson and by Kaufmann. These experiments indicate that x-radia- tion causes an unstable condition in chromosomes which can be made obvious by infrared treatment. 288 FACTORS MODIFYING THE SENSITIVITY OF CELLS Water Content I am sure that you are acquainted with the work on the sensitivity of plant viruses to x-rays, depending on the water content of the virus crystals. Similar work has been done by Stapleton. in our laboratory in regard to irradiated Aspergillus terreus spores, (a) suspended in water, (b) freshly removed from an agar slant culture containing about 40-50 per cent water, and (c) dried and desiccated for 3 days, and containing only about 20-25 per cent water. There is a striking increase of resist- ance to x-rays of very dry spores. Water apparently sensitizes the spores to x-rays by bringing in direct contact some substances formed in the water by x-rays. However, what we call "dry" spores in our experiments still have a water content which should be an important factor in the sensitivity. In connection with the irradiation in water, we should discuss the work of the Te cas group [Wyss, Stone, and Clark (21)], who found that, when the medium was irradiated with wave lengths shorter than 2537 A, bacteria and fungi grown in this irradiated medium showed a some- what increased mutation rate. Hydrogen peroxide produced the same effect. Certain organic peroxides have been found to increase mutation rates, as reported by a California Institute of Technology group [Dickey, Cleland, and Lotz (6)]. These findings point to the possibility that hydrogen peroxide, or certain organic peroxides which may be produced by radiation in the medium or inside living cells, may be an important factor in regard to radiation sensitivity. Further evidence is also brought out in the following discussion. Oxygen Tension The effect of oxygen tension on the sensitivity of chromosomes to x-radiation has been reported by Giles. R. S. Anderson reported in 1941 that yeast irradiated in the presence of oxygen was much more sensitive to x-rays than yeast irradiated in the absence of oxygen. We have carried out similar studies in regard to Escherichia coli, the experimental details of which, although important, will be omitted here for the sake of brevity. All data presented are based on plate counts of bacteria grown aerobically after irradiation. Figure 2 illustrates results obtained with aerobically grown bacteria irradiated in oxygen, air, and nitrogen. This graph shows that the ratio of survivors at 60,000 r under two different gases is nitrogen/oxygen = > 1000 OXYGEN TENSION 289 for aerobically grown E. coli (B/r), whereas the relative sensitivity for oxygen-treated samples, as compared with nitrogen-treated ones, is ap- proximately three-fold. The same modification of sensitivity has been demonstrated on E. coli, strain B, which has been reported to be more sensitive to radiation Anaerobic 40 60 X-ray dose, kiloroentgens Fig. 2. "Anaerobic" and "aerobic" refer to the mode of growing tbe organisms. "Oxygen" and "nitrogen" refer to type of suspension in which bacteria were kept during irradiation. than B/r. Since strain B is more sensitive to x-rays, the rate of killing is greater under both conditions studied; nevertheless the ratio of sensi- tivity of oxygen-treated cells to nitrogen-treated cells is approximately equal to that found for B/r. Nitrogen can be replaced with helium, hydrogen, or carbon dioxide without significantly changing sensitivity. 290 FACTORS MODIFYING THE SENSITIVITY OF CELLS SENSITIVITY OF AEKOBic VERSUS ANAEROBIC Escherichia coli (fi/r) Since it is well established that E. coli is a facultative anaerobe, it was decided to compare the relative sensitivity of this organism grown an- aerobically before irradiation with that of the same strain grown under strictly aerobic conditions. The bacteria grown anaerobically before ir- radiation and irradiated anaerobicallj^ were found to be extremely radio- resistant. The ratio of survival for the cells grown anaerobically before radiation is nitrogen/oxygen =10^ The ratio of sensitivity between oxygen- and nitrogen-treated anaero- bic cells is again, perhaps fortuitously, very close to 3. If the sensitivity of the extreme cases is compared, that is, cells grown aerobically before irradiation and irradiated in the presence of oxygen, and those grown anaerobically before irradiation and irradiated in the absence of oxygen, a factor of 10 is found. USE OF ORGANIC COMPOUNDS (aMINO ACIDs) AS PROTECTIVE AGENTS It was noticed that bacteria {E. coli, B/r) were more sensitive to x-rays when exposed in phosphate buffer than in nutrient broth (Difco), 8 gm per liter. Since broth contains a wdde variety of amino acids, it was decided to test the protective action of amino acids, first in groups and then individually, if the group tests appeared promising. The effect of amino acid solutions as protective agents was studied from two points of view: (a) the effect of amino acid concentration on bacterial survival; (h) the protective action of optimum concentration of amino acids as obtained from survival (a) as a function of x-ray dose. The results indicate that only glutamic acid and cysteine afford in- creased protection over the range of concentrations used in these ex- periments (Fig. 3). It appears from the data presented here that there are two different effects: (1) radiation produces changes in the medium which can be reduced by lowering the oxygen tension of the medium; (2) the studies with anaerobic cells, on the other hand, indicate that essentially com- plete removal of oxygen from the cells also results in lowering their x-ray sensitivity. The combination of two protective systems results in extreme resistance of the organisms to radiation. The possibility that respiration is tied up with radiation sensitivity seems to be indicated by these tests. DISCUSSION 291 100 80 g 60 S-. 40 20 ^ 1 1 \l 1 1 1 A = Aerobic broth culture \ exposed in oxygen \ B = Aerobic or anaerobic _ \ glucose broth culture \ exposed in oxygen \ C = Anaerobic glucose \ broth culture exposed _ y \ in nitrogen \a \s V W=i Vat = 4 \Ar=24 n V , 1 1 1 20 100 120 40 60 80 X-ray dose, kiloroentgens Fig. 3. Data from Fig. 2 recalculated for "target" determination. See discussion in text. Discussion The data from Fig. 2 were recalculated on the basis of the target theory [see Lea (17)] and are given in Fig. 4. A purely physical interpre- tation on the basis of the target theory of the effects of x-rays on E. colt leads one to the conclusion that it is possible to vary the number of "targets" at will, by means of adjustment of growth conditions or conditions during irradiation. Whereas this type of physical interpre- tation may be a helpful tool in the study of radiation effects, the chemi- cal approach appears more promising at the present time. The hypothe- sis that hydrogen peroxide formation induced by radiation is responsible for radiation effects on chromosomes is in many ways attractive, as Giles has so well shown. However, this hypothesis is less attractive in the interpretation of lethal effects on bacteria and fungi for the following reasons, (o) Bacteria suspended in a medium which has been irradiated with x-rays (60,000-80,000 r) will not be killed by this medium. The half life of hydrogen peroxide in buffer solution is of sufficient length that it should show a residual effect. (6) Hydrogen peroxide added to the suspension medium becomes toxic to bacteria only if concentrations are used which are far in excess of the ones produced by the usual amounts of radiation (> 200,000 r). (c) Organic peroxides, as well as hydrogen peroxide, should be produced by irradiation in broth suspen- 292 FACTORS MODIFYING THE SENSITIVITY OF CELLS sion and amino acid solutions. However, bacteria become more resistant if irradiated in the presence of these compounds. There is a possibility that the hydrogen peroxide attaches itself to the organic compounds, especially amino acids, and thus makes itself less available to the bac- teria. 100 0.001 0.005 0.01 0.02 Molar concentration of amino acids Fig. 4. Effects of amino acids, prepared immediately before irradiation and added to the suspension to be irradiated, on the survival of B/r at 60,000 r. [Hollaender, Stapleton, and Martin (11).] It appears that radiation probably produces a radical Avhich has a relatively short life but exists long enough to diffuse through the medium and into the bacteria or is produced in the cells themselves and dis- tributed by diffusion. At present this radical could be HO2. However, there may be other compounds formed by irradiation, the existence of which is not yet established. REFERENCES 293 We mentioned in the introduction that radiation death in organisms could be effected by many factors. Hydrogen peroxide may be one of the contributing factors, but other radicals found in the decomposition of water by radiation may play a special role in contributing to the killing of the organisms. The effect of oxygen in regard to the sensi- tivity could be largely explained by such a hypothesis. I feel that we are basing our speculation too much on studies which have been con- ducted with pure water, whereas in living cells, where really "pure" water does not exist, there must be a loose association of molecules which could be affected by these radiation-produced radicals. A typical example would be a nucleoprotein built into chromatin material. It appears somewhat promising that untangling metabolism mechanisms effected by oxygen under the influence of radiation will give us a clue to this problem. Finally, the inactivation of the sulfhydryl-requiring enzyme systems discussed by Barron may also play an important role. REFERENCES 1. Anderson, E. H., Heat reactivation of ultraviolet-inactivated bacteria, J. Bad., 61: 389, 1951. 2. Anderson, T. F., and B. M. Duggar, The effects of heat and ultraviolet light on certain physiological properties of yeast, Proc. Avi. Phil. Soc, 84: 661, 1941. 3. Anderson, R. S., and H. Turkowitz, The experimental modification of the sensi- tivity of yeast to roentgen rays. Am. J. Roentgenol. Radium Therapy, 44: 537, 1941. 4. Carlson, J. G., and Rachel D. McMaster, Nucleolar changes induced in the grasshopper neuroblast by different wave lengths of ultraviolet radiation, ahd their capacity for photorecovery, Exp. Cell Research, 2: No. 3, 1951. 5. Curran, H. C, and F. R. Evans, Sensitizing bacterial spores to heat by exposing them to ultraviolet light, J. Bad., 36: 455, 1938. 6. Dickey, F. H., G. H. Cleland, and C. Lotz, The role of organic peroxides in the induction of mutations, Proc. Natl. Acad. Sci., 35: 581, 1949. 7. Dulbecco, R., Reactivation of ultraviolet-inactivated bacteriophage by visible light. Nature, 163:949, 1949. 8. Gaulden, M. E., Effects of pretreatment and posttreatment with heat on the frequency of X-ray-induced chromosomal breaks in the grasshopper neuroblast, Hereditas, supplementary vol., p. 579, 1949. 9. Hayden, B., and L. Smith, The relation of atmosphere to biological effects of X rays. Genetics, 34: 26, 1949. 10. HoUaender, A., and B. M. Duggar, The effects of .sublethal doses of monochro- matic ultraviolet radiation on the growth properties of bacteria, /. Bad., 36: 17, 1938. 11. HoUaender, A., G. E. Stapleton, and F. L. Martin, X-ray sensitivity of E. coli as modified by oxygen tension. Nature, 167: 103, 1951. 12. Kaufmann, B. P., A. HoUaender, and H. Gay, Modification of the frequency of chromosomal rearrangements induced by X rays in Drosophila. I. Use of near infrared radiation. Genetics, 31 : 349, 1946. 294 FACTORS MODIFYING THE SENSITIVITY OF CELLS 13. Kelner, A., Effect of visible light on the recovery of Strepiomyces griseus conidia from ultraviolet radiation, Proc. Natl. Acad. Sci., 35: 73, 1949. 14. Kelner, A., Photoreactivation of ultraviolet-irradiated E. coli, with special refer- ence to the dose reduction principles and to ultraviolet induced mutation, J. Bad., 58:511, 1949. 15. Kelner, A., Action spectra for photoreactivation, Bact. Proc, 1950: 53, 1950. 16. Knowles, T., and A. H. Taylor, Spectral radiation involved in photoreactivation of ultraviolet-irradiated cultures of microorganisms, Bact. Proc, 1950: 49, 1950. 17. Lea, D. E., Actions of Radiations on Living Cells, Cambridge, 1946. 18. Stapleton, G. E., and F. L. Martin, Comparative lethal and mutagenic effects of ionizing radiations on Aspergillus terreus (abstract). Am. J. Botany 36: 816, 1949. 19. Swanson, C. P., and A. Hollaender, The frequency of X-ray-induced chromatid breaks in Tradescantia as modified by near infrared radiation, Proc Natl. Acad. Sci., 32:295, 1946. 20. Suskind, S. R., Resuscitation of heat-inactivated seeds by X radiation, /. He- redity, 41:97, 1950. 21. Wyss, O., W. S. Stone, and J. B. Clark, Production of mutations in Staphylococ- cus aureus by chemical treatment of substrate, J. Bad., 54: 767, 1947. DISCUSSION Taylor : From the standpoint of radiation chemistry, an interesting feature of HoUaen- der's and Giles' papers is that there is no mention of a marked effect of hydrogen. This would seem to indicate a small importance for H2O2, since its concentration is repressed in irradiated aqueous systems by the presence of hydrogen. Definite, quantitative conclusions are made difficult by the relatively small concentration of hydrogen (less than 10"^ molar at 1 atm) relative to other solutes, and by the laclc of detailed knowledge of radiation chemical effects in solutions (that is, individual radical concentrations, individual reaction rates). One gets, however, the qualitative impression that much of the biological effect occurs through immediate coUision of freshly formed oxidizing radicals (for example, OH) with the biological material. Sparrow : In cooperation with M. J. Moses we have obtained some information con- cerning the effect of x-radiation on chemical changes in fixed nuclei. The pre- liminary results indicate that the desoxypentose nucleic acid of the fixed cells is more susceptible to mild acid hydrolysis after x-irradiation than before. The interpretation is that modifications induced by the irradiation permit hydrolysis to proceed at a faster rate. It is not known whether the changes induced are chemical or physical or a combination of both. I should also like to mention that we now have data indicating that sensitivity of chromosomes to x-ray breakage may be related to the synthesis of desoxy- pentose nucleic acid (DNA). Sensitivity increases as DNA synthesis progresses, reaching a maximum where synthesis stops and decreasing to a minimum to the point where synthesis again begins. DISCUSSION 295 Plough : I should like to comment briefly on HoUaender's discussion of the relation of oxygen tension to the production of mutations by radiation. He and others have said that many other chemical processes must be taken into account in connection with the problem, and I should hke to call attention to certain rele- vant facts from our studies of radiation-induced auxotrophic mutations in Salmonella. Just as in E. coli, we now have strains of S. typhimurium which differ markedly in resistance to radiation. In strain 533 (sensitive) we can isolate after exposure to a dosage of about 50,000 r about 70 per cent of bio- chemical mutants — after penicillin screening — whereas with strain 519 (resist- ant) only about 20 per cent of similar mutants appear after the same treatment. Thus the frequency of mutations parallels the resistance to radiation. Yet the optimal oxidation-reduction potential for each of these strains is the same. On the other hand, preliminary tests indicate that *S. neicyort grows best at a much lower oxidation potential than does S. typhimurium (strain 533), and yet the frequency of radiation-induced mutations in relation to dosage is approximately the same through the range of dosages tried. It would certainly appear from this finding that other factors than available oxygen influence the frequency of radiation-induced mutation. Hollaender: I agree with Plough that the need of the organism for oxygen is not the de- ciding factor as to whether it will be radiation resistant or not. It appears from our findings that oxygen is an important factor in regard to x-ray sensitivity because of (a) its prevalence in the medium in which the organisms are suspended during irradiation; and (b) the presence of oxygen inside the organism. Absolute resistance of the organism is, of course, also influenced by many factors such as inherited resistance, nutritional background, and growth phase during which the organism is irradiated. /7 Gene Mutations Caused by Radiation H. J. MULLER Indiana University Bloomington, Indiana The Relation of Radiation Mutations to Those of Spontaneous Occurrence Perhaps the most striking thing about the gene mutations induced by radiation, in organisms in which they have been intensively studied, is the fact that they have thus far been in no wise distinguishable from the so-called spontaneous mutations. Pick out any locus in Drosophila in which a spontaneous mutation is known, and the production of a similar-appearing mutation in this locus by means of ionizing radiation may be guaranteed. Moreover, if multiple alleles of the locus are known to have occurred spontaneously, a similar series of multiple alleles can in time be obtained by radiation also. We do not mean to imply here that mutant genes whose effects look alike are necessarily the same in their inner genie structure but only that no consistent differences have been found in the alleles, or series of alleles, arising with and without radiation. It is further to be observed that, if mutations in the reverse direction have been obtained spontaneously, they too can be produced by exposure to radiation. And it is probably true, conversely, that any gene mutation arising as a result of irradiation could be found in un- treated material, if a prolonged and intensive search were made. It must be admitted that it has not yet proved possible to put this comparison into quantitative form. That is, although spontaneous gene mutations in Drosophila have been known for over 40 years and those induced by radiation for 24 years, the per-locus rate of mutation, es- pecially that occurring spontaneously, is so low as to have prevented the making of a comparison of the per-locus distribution of mutation rates, or what has been termed the mutational spectrum, in spontaneous and radiation samples. That would require a long-term project, the funds of which were not subject to year-by-year uncertainties, because the so-called "personal equation" is so strong a factor in the detection of visible mutations. Thus in a given series of radiation experiments 296 COMPARISON WITH "SPONTANEOUS" MUTATIONS 297 the present author found a rate of visible mutations 5 times that found by a group of specially trained graduate students. In view of this, it is evident that unless the same person, who must be one with special apti- tude for such work, has had a chance to accumulate data of this kind on spontaneous mutations at specified loci over a period of years, and to obtain corresponding data on mutations induced by radiation, the com- parison will be dubious, either on quantitative or on qualitative grounds or both. This then poses a condition which present Droso-phila projects, with their short terms, can hardly meet. The same stricture applies also to the determination of the spectra of both spontaneous and induced mutations to be obtained under given conditions, as in different cell stages, on application of chosen chemicals, in various physiological states, and in the presence of particular genes. But, although no such exact comparison of spectra has been possible, nevertheless there is at least rough agreement between spontaneous and irradiated samples in regard to their ratios of visible to lethal gene muta- tions. Indeed, the data of Timofeeff-Ressovsky (72, 73) on this point appeared to show a really good agreement, but since in this work the results for spontaneous mutations had to be obtained over a consider- able period, as is usually the case, the errors due to probable personality differences, referred to above, must make us hesitate to place reliance on the exact figures obtained. To turn back to a consideration of in- dividual loci, although again really quantitative conclusions are impos- sible, it is in general to be noted that gene mutations which are obtained relatively readily without treatment, such as white eye, garnet eye, and cut wing, are comparatively frequent after irradiation also, whereas cer- tain others, such as scute and achaete, which have seldom been found spontaneously, are similarly rare (although they were carefully looked for) in the irradiated material. At the same time, we must acknowl- edge that even in untreated material, as in the comparisons of "high- mutation-rate lines" with other lines reported by Neel (60) and by Ives (29), the rates for one or two loci (especially that of yellow body) are sometimes found to have been disproportionately changed. If this can be true even without artificial treatment, and if the result was not caused by unstable alleles of the particular genes in question having been present in some lines (a very likely possibility in these cases), then it would hardly seem likely that irradiation, a much stronger influence, would raise the mutation rates of all loci to just the same extent. This is a matter that merits further analysis, if similar cases of high mutation rate are found in the future, by transference of genes at the loci in ques- tion to different genetic backgrounds. Yet, whatever the answer may be to the question last raised, it is 298 GENE MUTATIONS CAUSED BY RADIATION evident that the gene mutations induced by radiation form no distinc- tive category. Moreover, the results show that the likelihood of occur- rence of mutations at different loci, and of different mutations at a given locus, if not raised quite equally by the application of radiation, must usually be raised at least sub-equally. This is a quite remarkable rela- tionship in view of the fact that the absorbed energy of the radiation "hit" is so inordinately higher — in a considerable proportion of the hits by some two orders of magnitude — than the energy likely to be involved in the course of most spontaneous mutations. Evidence was presented by Muller and Altenburg (55) in 1919, and confirmed almost a decade after that [Muller (40, 41)], that temperature influences the production of spontaneous mutations in Drosophila and acts in fact as a limiting factor in controlling their frequency. The warning was given (1928) that the effect of thermal agitation here might have been indirect, as, for instance, by having caused some special kind of chemical change in the food. For this reason it has been especially desirable to have the necessary large-scale work of this kind repeated with other organisms, and also with other stages of the same organism. Yet the decades have passed without this having been done by anyone. However, as the present writer also pointed out in 1928, the agreement in sign and in general magnitude of the effect with what was to be ex- pected of a simple intervention of thermal agitation in the mutation process itself makes it seem likely that this temperature effect is a direct one. The above conclusion seemed to be strengthened, after Timofeeff-Res- sovsky (71) had obtained similar data on Drosophila, by considerations presented by Delbriick (11) concerning the relatively large amount of temperature influence, having a Qio of about 5, to be reckoned for re- actions with rates as slow (molecular changes as infrequent) as those here dealt with. The molecular changes in question, here represented by mutations in individual loci, are so infrequent as to result in a half life of some thousands of years for the individual gene, as first shown by Muller and Altenburg (55). It was noteworthy that, in agreement with the calculated Qio of 5 for rates as slow as this, there is in fact an approximately five-fold increase in mutation frequency with a 10° C rise in temperature. The basic assumption in this calculation was that the mutation occurs whenever a certain energy level, which is inferred to be about 1.5 ev, happens to be attained by the mutable material. This supposedly requisite energy level is so high in comparison with the average kinetic energy of the particles in the protoplasmic medium (some 50-70 times as high) that it would be attained very infrequently, giving the reaction the low rate found, yet this rate would be raised by a 10° C COMPARISON WITH "SPONTANEOUS" MUTATIONS 299 temperature increase by about 5 times instead of by only the 2 or 3 times usual for chemical reactions. To this latter line of reasoning, however, we must interpose several words of warning. According to one possible criticism, low reaction rates are not always or entirely caused, as assumed above, by the fact that sufficiently high energy levels are so seldom attained by the reac- tive particles. They may equally well be caused, especially in a complex medium like protoplasm, by infrequency of encounters of just the right structural (that is, "qualitative") types for the production of the reac- tion, despite the fact that the energy level remains low. This infre- quency of effective encounters may simply be due to rarity (low con- centration) of the reactive substances or, what amounts to nearly the same thing, to a highly specific and unusual type of encounter being necessary. However, the high temperature coefficient would not be ex- plained in this way without bringing in some accessory assumption, such as that the reactive (mutagenic) substances were present in higher con- centration at a higher temperature. As another alternative, a low reac- tion rate for mutation (long half life) may be due to a coincidence of two or more unusual events being required for its occurrence, even though both these events took place at a relatively low energy level. In that case the temperature coefficient could be as high as the product of the coefficients of the two or more participating events, and so it would simulate the coefficient to be expected for a high-energy-level re- action. Thus w^e see that, although a high energy level, of some 1.5 ev, seems to be the simplest way of interpreting the finding of a low rate of mutation combined with a high temperature coefficient, it is by no means the only plausible possibility. That the occurrence of mutation does not depend merely upon a given energy level being reached at a given point, but also upon the confor- mation of the material, is indicated by a comparison of the energy levels for spontaneous and ultraviolet-induced gene mutations. Even the high energy level of some 1.5 ev proposed by Delbriick for spontaneous mu- tations falls far short of the 10 ev or more (accumulated in units of about 5 ev each) which, as we shall see in the next section, is probably necessary for mutation by ultraviolet. This seems to mean that, from an energetics standpoint, the chemical pathway to mutation by ultra- violet activation of purines or pyrimidines is far less efficient than the spontaneous pathway or pathways. However, we cannot yet exclude the opposite possibility that, instead of involving lower energy, the spontaneous mutation process may in- volve even higher energy than that of Delbriick's hypothesis. Living matter can on occasion attain energy levels higher than those found in 300 GENE MUTATIONS CAUSED BY RADIATION the plus "tail" of the statistically random energy distribution which re- sults from the operation of ordinary thermodynamic principles. These high levels are due to mechanisms, utilizing the energy-transferring prop- erties of some nucleotides, which cause an accumulation of the potential energy from multiple quanta, absorbed at different times, and which can then suddenly release this energy at a high level, as occurs, for in- stance, on a molar scale in electric organs. At present, however, there seems no need to postulate an energy level above 1.5 ev for spontaneous mutations merely because of its being needed for ultraviolet mutations. It seems more plausible to refer the difference to the nature of the chemi- cal pathways and/or to a multiplicity of key events being necessary to effect the occurrence of mutation. The above diversity of possibilities should show what a high degree of caution is necessary when the attempt is made to interpret biological events on the basis of simple physical principles, without regard to the chemical complexities that may be involved. Nevertheless, despite our uncertainty regarding the energy level necessary for spontaneous muta- tion and the nature of the chemical steps involved, we can be sure that the random encounters of thermal agitation play an important and neces- sary part in the process of spontaneous mutation. Moreover, this being the case, it is probable that the increase in spontaneous mutation fre- quency that accompanies a rise in temperature within the range normal to the organism is, in part at least, caused by the increase in thermal agitation which the higher temperature entails. That the spontaneous gene-mutation process must usually depend upon thermally activated reactions follows, for one thing, from the cal- culations and data presented by Muller and Mott-Smith (56), later con- firmed by others, which showed that natural radiation is entirely inade- quate in amount to be an important cause of spontaneous mutations in Drosophila at the rate at which they occur in that material. That these thermally activated reactions which lead to gene mutations are random events beyond individual control by cellular regulative processes and that they are therefore subject to the statistical principles of ordinary thermodynamics is attested to by their sporadic, pointwise distribution in space and time. For one thing, this randomness is of such scope that, as yet, it has not been found possible (at any rate in experiments that have been confirmed) to bias the occurrence of ordinary spontaneous mutations by applying special conditions that would favor one type against another. But, more telling still, the randomness can be shown to exist even in the range of microscopic or submicroscopic dimensions. For, as the present writer pointed out in an early discussion of this question (38, p. 470): "Mutation is due to an event of such minute COMPARISON WITH "SPONTANEOUS" MUTATIONS 301 proportions, so circumscribed, that it strikes only a single one of two near-by, similar loci in the same nucleus"; that is, when one gene mu- tates, its allele of identical composition, usually only a small fraction of a micron away, remains unaffected. Thus the determination of just which gene mutates in a given case, and to which allele, must be a consequence of the ultramicroscopic ac- cidents of thermal agitation rather than of the chemical nature of the material reacting with the gene. And it may be concluded [Muller (39), p. 43] that "mutations are not caused by some general pervasive influ- ence, but are due to 'accidents' occurring on a molecular scale. When the molecular or atomic motions chance to take a particular form, to which the gene is vulnerable, then the mutation occurs." Since the time when this statement was made, it has become possible to add that the similarity of radiation mutations to spontaneous mutations, in regard to the kinds of effects produced, their relative numbers, and their random distribution in space and time, lends strong support to this viewpoint. In this connection it is especially noteworthy that in the genesis of the radiation mutations, unlike that of the spontaneous ones, the acci- dents are initiated by a fast particle the path of which can have had no re- lation to cellular needs or metabolic processes, and that nevertheless the spontaneous mutations appear as random as those produced by radiation, and essentially similar to them. If in the natural accidents that cause spontaneous gene mutations different kinds of protoplasmic substances differed much from one another in regard to the types of mutations they favored, then we should hardly expect spontaneous gene mutations as a group to agree as much as they do with the group of gene mutations produced by the absorption of high-energy radiation. Hence it seems likely that a thermally occasioned encounter of the right kind to produce one mutation w^ould also, according to w'hich gene and gene-part hap- pened to be involved, have sufficed to produce practically any other mu- tation. Thus there seems to be very little difference in the type of process, or amount of energy necessary, for the occurrence of different kinds of gene mutations, and the same general sort of chemical substitu- tion may well be involved in all cases. If we had more data on muta- tional spectra w^e might make this inference more secure. Our inference that the mutations of different genes can be occasioned by chemical encounters of the same type by no means implies, con- versely, that the type of chemical encounter to which a gene is exposed is of no account in the determination of whether or not a mutation will be produced in it. That is, the occurrence of spontaneous mutations does not depend solel3^ on the energy level reached, as has sometimes been assumed, but also on the energy being conveyed in an appropriate 302 GENE MUTATIONS CAUSED BY RADIATION manner, that is, by given substances and/or with attendant conditions of a suitable kind. This is shown clearly by the great variations in the overall frequency of spontaneous gene mutations found in different ex- periments, amounting to 10 times or more [Muller (40, 41)]. Not only can genetic differences have effects as great as this [Demerec (12), Neel (60), Ives and Andrews (30)], but also differences in cellular conditions, depending on age and stage of development [Muller (48)]. The action of ''temperature shocks" appears to come in the same category. Finally, as has been known since the work of Auerbach and Robson [see sum- mary by Auerbach (2)], chemical influences of specific types are potent causes of mutation, a finding long to have been expected in view of the above facts and considerations. The question, w'hich the present author raised in 1928 (41, p. 345), as to whether gene mutation occurs by the chemical change of a pre-existing gene or by a misstep in the synthesis of the daughter gene by its mother gene, is one which, for spontaneous mutations, seldom admits of an answer in any given case. However, the intense concentration of spon- taneous gene-mutational occurrences in Drosophila into those stages of the germ cycle in w^hich gene duplication (as evidenced by mitosis) is going on more actively [Muller (48)], and earlier findings of a similar nature in bacteria by Zamenhof (80) and later by others, strongly sug- gest that in these cases spontaneous mutations usually take place by misconstruction of the daughter gene. On the other hand, the accumula- tion of spontaneous mutations in mature, dormant spermatozoa [Muller (48)] indicates that in them it is the old gene which is being transformed. The proof of this will not be complete, however, until it is shown that the mutant offspring from the aged sperm are mutant throughout their bodies. To obtain evidence on this matter for spontaneous mutations, very large-scale work involving the study of certian particular types of mutations, which affect the whole external surface of the body visibly, is required. Yet, however that may be, the answer to the question is already clear so far as the great majority of gene mutations induced by radiation of spermatozoa is concerned: that is, in this case, the pre- existing gene undoubtedly becomes altered in its composition. For, wherever evidence concerning the point at issue is available, for radi- ation mutations induced in spermatozoa, it is found that the whole body is usually involved. In view of this apparent difference in the usual method of their origi- nation, the spontaneous mutations studied having more often arisen through the misconstruction of the daughter gene and the radiation mu- tations studied having more frequently involved changes in the com- position of the already finished gene, the previously noted similarity in type of product between the spontaneous and the radiation mutations THE GENE MUTATION RATE— DOSAGE RELATION 303 is the more interesting. It would indicate that those spots which are more vulnerable in the finished gene are also more prone to be effectively disturbed during the process of gene construction. It is not surprising, however, that the unfinished gene should be more labile in general than the finished one; that is, that it should be more susceptible to having mutational disturbances caused in its synthesis by the relatively mild influences that operate in non-radiated material.* If the above general view of the spontaneous gene-mutation process has validity, it is not at all strange that high-energy radiation also in- duces the occurrence of gene mutation, for it not only releases energy at a far higher level than necessary for such a result but in virtually as many different forms as it would naturally be encountered in, and more besides. Indeed, such a chaos of different, molecularly more or less localized reactions must arise in irradiated protoplasm, both as direct results of the ionizations and activations on the molecules hit, and as secondary, tertiary, etc., consequences of the varied combinations into which these products later enter, that it would be strange if those reac- tions which, in non-radiated material, result in spontaneous mutations were not included among those here arising. In addition, the gene itself could be struck directly by a fast particle, with results that might resem- ble those brought about by the intermediation of mutagenic substances. In all this welter of effects and of possibilities, the tracing of the more usual trains of mutational processes, in physicochemical terms, is a mat- ter of the greatest difficulty. This field is only now opening up, as a re- sult of studies of chemical influences upon the occurrence of gene mu- tation, both with and without radiation. On the Proportionality between Induced Gene Mutations and Ionizations Before referring to some of the specific findings along these lines, let us consider the question of how the production of mutations is affected by changes in the dose of radiation. Since the doctoral thesis presented * While this manusciipt was in press, results were reported by Novick and Szilard {Proc. Natl. Acad. ScL, 36: 708-719, 1950), showing that in Escherichia coli growing at a given temperature spontaneous mutations continue at the same rate regardless of the rate of growth and multiplication (varied by controlling the amount of some minimal nutrient available for them), provided they are able to multiply at all. Here, then, the changes must be in the "old" gene; however, we do not know how much turnover of material it is undergoing. A paper by Maale and Watson {Proc. Natl. Acad. Sci., 37: 507-513, 1951) reports results on phage "tagged" with P^- which may be interpreted by assuming that at least the phosphorus in the genetic material of the phage does undergo exchange apart from reproduction, but other interpreta- tions of these results are possible. 304 GENE MUTATIONS CAUSED BY RADIATION to the present author by OHver 20 years ago, in which he reported a simple proportionaHty of the frequency of induced gene mutations to the dose of x-rays apphed, this finding has been ever further substanti- ated and extended. There have been only such discrepancies as are to be expected in consideration of the nature of the difficulties involved, and of human frailty, and these have in general been rather quickly corrected. The same statement applies to the independence of the fre- quency of the induced gene mutations from the time distribution or intensity of the dose of radiation. Thus, despite a number of unfortu- nate publicized statements by non-geneticists, casting doubt on the muta- genic effectiveness of small doses of radiation, the work has gone so far as to make it inconceivable, on physicochemical grounds, for a single ionization track traversing a nucleus to be without its proportionate probabihty of inducing a gene mutation. Of course, this does not directly prove that one ionization or activa- tion can cause a mutation, since the ionizations come grouped within tracks, and a good share of them (for the most frequently employed wave lengths about the same share) in tight clusters. This may be one of the bases of Opatowski's (63) mathematically expressed objection to the conclusion that a single ion is effective. It is hardly sufficient to answer that soft x-rays have been found to have the same effectiveness as ordinary x-rays, since the wave lengths tried have seldom been quite long enough to meet possible objections, and the measurement of the amount of penetration of those which are long enough presents great uncertainties. A more telling argument might seem to be the fact that, despite their greater ionization density, neutrons are not more effective, but are in fact somewhat less so, than ordinary x-rays, in inducing sepa- rately recordable gene mutations. This, however, proves somewhat too much, for here the ion density is obviously beyond the optimal for in- dividual effects. It might be thought that, if two or more ionizations near together were ordinarily needed for producing a gene mutation, then this would cause the mutation rate to rise more rapidly than the dose. For, al- though the frequency of clusters located at the ends of tracks and of their branches, as in the course of delta rays, would all increase linearly with the dose, there would in addition be some independently produced ionizations, lying in different tracks, which happened to be as near to- gether as those in the natural clusters, and the frequency of such ac- cidental juxtapositions would rise with the square of the dose for two ionizations, with its cube for three, etc. If, however, we calculate the frequency of such groupings in comparison with that of the natural clusters, for any doses of the size actually used for Drosophila, we find IMPLICATIONS OF THE RESULTS WITH ULTRAVIOLET 305 accidental groups to be so rare, even at best, that they could not exert a perceptible influence on the results found. This is the more true the higher the number in the group, but it holds even for groups of two. Thus, even if a gene mutation did require two or more ionizations, such a large proportion of the clusters that produced them would at all doses used be "natural" clusters, the frequency of which varied linearly with the dose, just as that of single ionizations does, that we should obtain no evidence on the matter through mere dosage studies. Iaiplications of the Results W'ITh Ultraviolet Nevertheless it seems unlikely that more than one ionization should be necessary to produce a gene mutation, in view of the mutagenic action which even ultraviolet exerts, and the fact that this compara- tively low-energy agent works through activations which, within any given substance, are distributed at random with regard to one another. Yet it is true that the total amount of energ}^ which must be absorbed for the production of a gene mutation is far higher for ultraviolet than for x-rays or gamma rays. Some work on Drosophila by Meyer, E. and L. Altenburg, Edmondson, and Muller shows that, for the most efficient ultraviolet doses which we have thus far worked with, between 100 and 1000 times as much energy must be absorbed by the chromosomes them- selves as is absorbed by them when x-rays enough to give the same mu- tation rate are applied.* The precise interpretation that should be placed on the mutation fre- quency-dosage relation found for ultraviolet is subject to much uncer- tainty because of the complexity of that relation. In both fungi [Hol- laender (24, 25), Hollaender, Sansome, Zimmer, and Demerec (27)] and Drosophila [Sell-Beleites and Catsch (65), L. and E. Altenburg, Meyer, and Muller (1)] the curve expressing this relation, with mutation fre- quency as ordinate, after rising for a short space in a more or less linear * In the paper as presented at the meeting it was stated that approximately equal amounts of energy were absorbed for the production of a gene mutation whether by ultraviolet or by x-rays. This is, however, true only of the energy absorbed by the cells in question (primordial germ cells) as wholes. As the ultraviolet, unlike the x-radiation, is absorbed selectively by the chromatin, which constitutes less than one-hundredth of the cell material, we must reckon the ultraviolet as correspond- ingly less efficient. (See later discussion on the limitation in the spatial range of mutagenic effectiveness of the energy derived from x-ray absorption.) The treat- ment of the relation of mutation frequency to dosage of ultraviolet as the latter is varied has also been radically revised in the article as now presented. The author wishes to acknowledge his indebtedness to Dr. C. P. Swanson and Dr. N. H. Giles for having called his attention to some facts in this connection, although these in- vestigators are in no way re-sponsible for the interpretations here presented. 306 GENE MUTATIONS CAUSED BY RADIATION manner (see below), tends gradually to level off and then even to sink at very high dosage levels, levels resulting in a great mortality of the germ cells or offspring. It seems reasonable to infer, as Hollaender has done, that this flagging of the curve is caused, in part at least, by the mortality being selective, for the distribution of ultraviolet can seldom be made very even, and cell susceptibility also may vary. It is to be ex- pected that the deleterious action of the radiation on viability would in general be exerted more heavily against the same cells as have more mutations induced in them, sometimes because these cells have received more radiation and sometimes because they are in a more susceptible condition. Moreover, in some material (that in which the effect of the mutations can show relatively early, before the physiological effect of the ultraviolet on viability has faded away) the mutations themselves would give the cells, in their further development, a greater mortality as compared with non-mutated specimens when they had at the same time received a physiologically more effective dose of ultraviolet; this too would tend to lower the apparent mutation rate more at higher doses. There may in addition have been appreciably more "light re- activation" at the higher doses in some of the experiments, and this would have weakened the effective doses more just when they were in- tended to be stronger. It is also conceivable, as an explanation of why the curve becomes convex at higher doses, that ultraviolet of mutagenic wave length exerts a second effect on the mutation process, similar perhaps to that of very long ultraviolet and short visible light, so as to interfere with the pro- duction (or to cause the reversal before their completion) of the very mutations which this same mutagenic ultraviolet, presumably through separate quanta, induces. In that case we should have to assume further that this counteracting effect rises more steeply with the dose than the mutagenic primary effect does. This would, for instance, be true if the frequency of mutations primarily induced varied as (P, that is, as the nth power of the dose, because of n hits being required for a mutation, whereas the final or net frequency of mutations, that remaining after the counteracting quanta had taken effect, varied as the product of (P times e''^ (where A; is a constant) . This formula would follow the principle which appears to operate in "reactivation" by visible and long ultra- violet light, as judged, for instance, by Novick and Szilard's results (see below), although of course in the case of visible and long ultra- violet light reactivation it would be necessary in this formula to sub- stitute a different letter for the second d, representing the amount of that radiation, since the first d would represent only the amount of mutagenic radiation. It is true that this interpretation has the objec- IMPLICATIONS OF THE RESULTS WITH ULTRAVIOLET 307 tion of requiring a bimodal curve for the spectral distribution of effec- tiveness of the interfering action, since radiation in the region of 3100 A has very little such effect, at least in bacteriophage reactivation [Dul- becco (14)]. However, it is difficult to explain on other grounds why the falling off in net mutagenic effectiveness at higher doses should be so widespread and so extreme as it has been observed to be. But no matter which of the above interpretations, or which combina- tion of them, be adopted, the influence of selective mortality or inter- fering radiation should be negligible at low doses, and at these doses (that is, nearer the orgin of the frequency-dosage curve) the mode -of operation of the positively acting factors should become clear, as is usual for sigmoid curves in general. Moreover, it would become very prob- able that we had reached low enough doses for judging the action of the positive factors by themselves if we found a portion of the curve (to the left of its convex region, and including the lowest doses for which effects were measured) in which, over a considerable range of dosage, involving, for instance, a dosage variation by a factor of 4, the amount of effect (mutation frequency) was found to be proportional to a constant power of the dose. That is, we could assume that at these dosage levels the interfering action had not yet come into play appreciably and that the observed curve of results reflected the influence of only the factor or factors which induced the mutations, in the virtual absence of the coun- teracting influences. Unfortunately, it is extremely difficult to get data of sufficient statisti- cal reliability for such low doses. However, if we examine the most detailed published data approaching these conditions, such as those ob- tained by Hollaender and Emmons (25) on Trichophyton, and by Hol- laender, Sansome, Zimmer, and Demerec (27) on Neurospora, we find distinct indications that at low doses the mutation frequency rises faster than a simple linear relation to dose would allow. On the other hand, so far as one can judge, the frequency hardly seems to rise faster than the square of the dose, as it would if two activations were required for a mutation. Yet the data are too meager for a decision either on this question or on that of whether low enough doses to rule out the inter- fering factors have been reached. Novick and Szilard (61) have reported measurements of the frequency of three specific types of mutation (to phage resistance) in E. coli on application of varied doses of ultraviolet. In this work the amount of effect caused by "reactivation" by light of longer than mutagenic wave length w^as determined at the same time, for each of the types of mu- tation. It seems unlikely that the viability of mutants of these kinds w^ould have been reduced by the ultraviolet much more than that of 308 GENE MUTATIONS CAUSED BY RADIATION the non-mutants would have been. Although it is hard to rule out dif- ferences in susceptibility, there were probably no important differences in the amounts of exposure of the genetic material of the different organ- isms, since they are so minute and were agitated during treatment. It is true that the individual mutation-frequency determinations were sub- ject to a high statistical error, yet the effect of this is minimized by the fact that the results from all the series studied agree very closely in principle. For, when the mutation frequency is represented on a loga- rithmic scale as ordinate and the dose on a logarithmic scale of the same magnitude as abscissa, the lines connecting the datum points are found to have the same slope in the case of all six series (a light-treated and a dark series for each of the three mutant types). Moreover, since the effect of the light follows the same formula throughout (see p. 306), it is possible to allow for it and thus to combine the results of the light and dark treatments into one curve for each of the three types of mutations. When this conversion is carried out it is found that the effective dose (that after correction for light treatment) varied, for two of the mutant types, by a factor of about 3, and for the other by a factor of about 5. There are several scattered datum points along each of the three curves, and each curve is found to form, so far as can be judged, a straight line, on the log-log scheme of representation used. This means that the mu- tation frequency, over the whole range of doses employed, rises as a sensibly constant power of the dose. The slope of the straight lines, on this log-log curve, having a tangent of about 2.3, is such as to show that for each small increment of dose there was about 2.3 times as great a relative increment of mutation fre- quency. This relation, showing that the mutation frequency rises as the 2.3 power of the dose, would ordinarily be interpreted (although the authors do not discuss this matter) as meaning that in the production of each mutation 2-3 independently produced unit elements of the dose, that is, in this case 2-3 activations (sometimes 2 and sometimes 3), usu- ally take part. Indeed it does not seem possible to avoid this conclusion, except by gratuitously assuming still greater complications, with effects canceling one another sufficiently to return the final, net effect to the 2.3-power relationship to dose. In other words, there is evidence for an only slightly higher than two-hit curve. And, since the same curve (al- though with different absolute frequency of effect) was found in the case of each of the three mutants for four or more points over a three- to five-fold dose range, it does not seem likely that it has at these doses been modified appreciably b}^ those influences above discussed, which in other material tend to cause an apparent leveling off or drop at very high doses. A curve of 2-1- "hits" thus seems to represent the process IMPLICATIONS OF THE RESULTS WITH ULTRAVIOLET 309 of ultraviolet mutagenesis here without those complications which so often distort its expression at the higher doses. That not only ultraviolet but also mustard works through a multihit process is indicated by the synergism between the mutagenic effects of these two agents reported by Swanson et at. (68). This makes it the more likely that spontaneous mutations too, since they probably result from changes of still lower energy level, require a concatenation of rare events. The ionizations induced by high-energy radiation rise above this need, perhaps because their higher energy, in its degradation, has the necessary multiple effects, or because it can accomplish, more di- rectly, what the multiple effects in the other cases converge to do. In the work on ultraviolet mutations in Drosophila it has not been feasible to get mutation-frequency data for a number of widely differing doses at levels low enough to make the probable role of differential via- bility unimportant. Hence we have not been able to determine the prob- able number of hits participating in a positive way in ultraviolet muta- genesis in this material. Nevertheless we do have, in the data of the group of Drosophila workers previously mentioned, a very suggestive point of correspondence with the E. coli results, which raises the pre- sumption that essentially the same mechanism may be at work in both. The point in question concerns itself with the amount of absorbed ultra- violet energy necessary to produce a specified type of mutation. For this purpose we must take into consideration the data for the lowest dose for which significant results were obtained in Drosophila, for that is the dose at which the complications of differential viability, etc., which reduce the apparent effectiveness of the dose, are at a minimum. It happens that, judging by the factors of the type of lamp, the distance, and the time, this dose (involving an exposure to a G.E. "germicidal" lamp at 200 cm for 3 min) came within the range used by Novick and Szilard with E. coli. Although the intensity of our treatment appeared some 4 times lower than theirs, we have not found such a difference to play a very large role in our own work when the factors of the dose are of the order of magnitude that obtained here. Thus we can arrive at an approximate comparison of the probability of a mutation of a given type (say, to resistance to phage T4) being produced in E. coli at a given dose with the probability of a mutation being produced at an individual locus in Drosophila by the same dose. It is true that in this particular Drosophila work we have not dealt with individual loci, but the relation between the overall lethal frequency (the modulus used in the present work) and the average per-locus frequency of mutation has been approxi- mately determined in other studies. Thus our present results can be translated into these terms. 310 GENE MUTATIONS CAUSED BY RADIATION Reference to Novick and Szilard's graphs shows that, at the dose in question (without light treatment), E. coli has a frequency of about 1 induced mutation in 5000 to T4 resistance and 1 in 20,000 to Tq resist- ance or Ti resistance, respectively. At this same dose, Drosophila gives visible mutations in the specified individual loci studied at the rate of some 1 in 5000, although actually the rate varies from about twice to half this value, according to the locus.* There are too many sources of error in both the above sets of values for the comparison to be valid except as regards the orders of magnitude involved. Moreover, the assumption has not been proved that the same locus is always involved in the independently arising bacterial mutations that give resistance to the same type of phage. Nevertheless, it is of interest that the results on tw^o such different organisms should agree as closely as those here found seem to do. We are reminded at this point of some similar apparent agreements in per-locus rates of spontaneous and of x-ray mutations that have been reported in other comparisons of widely different organisms. At any rate, in view of the wide range of conceivable values of mutation rate, and of values found under other circumstances, it does not appear very plausible to regard the present agreement as a complete coincidence. If it is not, however, it would indicate a similarity in the quantitative features of the mechanism of mutagenesis, as well as in the nature of the genetic material, in the two cases. The above considerations appear to favor the conclusion that more often only two and seldom more than three quanta of 2537 A ultraviolet are involved in the production of a gene mutation. At the same time, however, it can be calculated that there is probably somewhat less than one chance in a thousand of actually getting a mutation, even when as many as three of these ultraviolet quanta have been absorbed by one and the same purine or pyrimidine group of the desox^Tibonucleic acid por- tion of a chromosome. Other "accidental" circumstances, therefore, must determine whether the absorbed energy results in a genetically re- producible change. It is to be noted that the chance is also not much over one in a thousand (about }