The Initial Effects of Ionizing Radiations on Cells The Initial Effects of Ionizing Radiations on Cells Edited by R. J. C. HARRIS Head oj the Division of Exjjerimenfal Biology and Virology Imperial Cancer Research Fund, London A Symposium held in Moscow, October, lOGO, supported by UNESCO and the IAEA and sponsored by the Academy of Sciences of the U.S.S.R. 1961 ACADEMIC PRESS LONDON and NEW YORK ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House Berkeley Square London, W.l U.S. Edition published by ACADEMIC PRESS INC. 1 1 1 Fifth Avenue New York 3, New York COPYRIGHT © 1961 BY UNESCO Library of Congress Catalog Card Number: 61-lSSSl PRINTED IN GREAT BRITAIN BY J. W. ARROWSMITH LTD. BRISTOL LIST OF CONTRIBUTORS P. Alexander, Chester Beatty Research Institute, Royal Cancer Hospit ah London, England. Z. M. Bacq, Lahoratoire de PathoJogie GeneraU, Universite de Liege, Liege. Belgium. N. F. Barakina, a. N. Severtzov Institute of Animal Morphology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. G. W. Barendsen, Radiobiological Institute. The Organization for Health Research TNO, Rijswijk {Z.H.), The Netherlands. I. J. Barsky. Institute of Biophysics. Institute of Radiation and Physico-chemical Biology. Academy of Sciences of the U.S.S.R., 3I0SC01V, and Central Scientific Research Institute of Medical Radio- logy, Leningrad, U.S.S.R. L. Benes, Institute of Biophysics, Czechoslovak Academy of Sciences, Brno, Czechoslovakia. y. C. Blokhina, Institute of Biophysics, U.S.S.R. Academy of Sciences. Moscow, U.S.S.R. L. A. Blumenfeld, Institute of Chejnical Physics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. E. M. Brumberg, Institute of Biophysics, Institute of Radiation and Physico-chemical Biology, Academy of Sciences of the U.S.S.R., Moscow, and Central Scientific Research Institute of Medical Radiology. Leningrad, U.S.S.R. Tor Brustad, Donner Laboratory, University of California, Berkeley, California, U.S.A. N. N. DoEMix, Institute of Biojjhysics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. V. Drasil, Institute of Biophysics, Czecholovak Academy of Sciences, Brno, Czechoslovakia. M. Errera, Lahoratoire de Biophysique et de Radiobiologie, Universite Libre de Bruxelles, Bruxelles, Belgium. G. M. Frank, Institute of Biophysics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. L. H. Gray, British Empire Cancer Campaign Research Unit in Radio- biology, Mount Vernon Hospital, Northwood, Middlesex. England. E. Y. Grayevsky, A.N. Severtzov Institute of Animal Morphology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. Vi LIST OF CONTRIBUTORS F. Hercik, Institute of Biophysics, Czechoslovak Acadetny of Sciences, Brno. Czechoslovakia. Alexander Hollaender, Biology Division, Oak Ridge National Laboratory. Oak Ridge. Tennessee. U.S.A. Barbara E. Holmes, Department of Radiofherapeutics, University of Cambridge, Cambridge. England. O. Hug, Strahlenbiologisches Institut der Universitdt MiXnchen und Institutfur StraMenschutzforschung der Gesellschaft fur Kernforschung , Neuherberg bei Miinchen. Germany. A. E. Kalmanson, Institute of Chemical Physics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. Z. Karpfel, Institute of Biophysics, Czechoslovak Acadeyny of Sciences, Brno. Czechoslovakia T. M. Kondratjeva, Institute of Biophysics, Institute of Radiation and Physico-chemical Biology, Academy of Sciences of the U.S.S.R., Moscow, and Central Scientific Research Institute of Medical Radiology, Leningrad, U.S.S.R. M. M. KoNSTANTiNOVA, A.N . Sevcrtzov Institute of Animal Morphology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. A. M. KuziN, Institute of Biophysics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. A. V. Lebedinsky, Academy of Medical Sciences of the U.S.S.R. Moscow, U.S.S.R. Thomas Manney, I)o7iner Laboratory, University of California. Berkeley, California, U.S.A. H. Marcovich, Service de Radiobiologie et de Cancerologie, Institut Pasteur, Paris, France V. M. Mastryukova, Academy of Medical Sciences of the U.S.S.R., Moscow, U.S.S.R. M. N. Metssel, Institute of Biophysics, Institute of Radiation a,nd Physico-chemical Biology. Academy of Sciences of the U.S.S.R., Moscow, and Central Scientijic Research histitute of Medical Radiology, Leningrad, U.S.S.R. E. Palecek, Institute of Biophysics, Czechoslovak Academy of Sciences, Brno, Czechoslovakia A. G. Passynsky, A.N. Bach Institute of Biochemistry, U.S.S.R- Academy of Sciences, Moscoiv, U.S.S.R. Ernest Pollard, Biophysics DejKirtment, Yale University, New Haven, Connecticut, U.S.A. LIST OF CONTRIBUTORS vii E. L. Tow i; us. J)iri.sion of Jiiological and Medical Rtatarch, Anjonnc National Laboratory. Argotme, Illinois, U.S.A. H. J. ScHLiEP, Sfrahlenbiologisches Institut der Universiiat MunrhcM, und Institut fur Sfrahlenschutzforscfmny der Gesellschaft fiir Kernfor- schung, Neuherberg bei Milnchen, Germany I. M. Shapiro, A.N . Severtzov Institute of Animal Morphology , U.S.S.R., Academy of Sciences, Moscoiv, U.S.S.R. N. M. SissAKiAN, Department of Biological Sciences, U.S.S.B. Academy of Sciences, Moscow, U.S.S.R. M. Skalka, Institute of Biophysics, Czechoslovak Academy of Sciences, Brno, Czechoslovakia A. D. Snezhko, Institute of Biophysics, U.S.S.R. Academy of Sciences, Moscoiv, U.S.S.R. J. SosKA, Institute of Biophysics, Czechoslovak Academy of Sciences, Brno, Czechoslovakia L. A. Stocken, Department of Biochemistry, University of Oxford, O.vford, England A. D. Strzhizhovsky, Academy of Medical Sciences of the U.S.S.R., Moscoiv, U.S.S.R. V. N. Tarusov, Biological Department, Moscow State University, Moscow, U.S.S.R. Cornelius A. Tobias, Doniier Laboratory, University of California, Berkeley, California, U.S.A. [libraiiyI^ ^ 4fN4^2V^>y CONTENTS List of Contributors v Introduction xi Opening Address. By N. M. Sissaktan 1 The Nature^ oftlie Initial Radiation Damage at tlie Sub-Celhilar Level. By P. Alexander and Z. M. Bacv 3 Mechanisms Involved in the Initiation of Radiobiological Damage in Aerol)ic and Anaerobic Systems. By L. H. Gray 21 Action of Radiation on Proteins and Nucleic Acids in Solution and at Interx)hases. By A. G. Passynsky 45 Electron S])in Resonance (ESR) Investigations on Radiation-Induced Chemical Effects in Biological Species. By L. A. Blumenfeld and A. E. Kalmanson 59 The Action of X-Rays on Intracellular Bacterio])hage Formation. Bv F. Hercik '. 09 The Action of Ionizing Radiation on the Cellular Synthesis of Protein. By Ernest Pollard 75 Chemical Species Induced by X-Rays in Cells and their Role in Radia- tion Injury. By E. L. Powers 91 Fluorescence Studies of the Changes Undergone by Nuckoproteins and their Derivatives in Irradiated Cells. By M. N. Meissel, E. M. Brumberg, T. M. Kondratjeva and I. J. Barsky 107 A Discussion of Some Immediate Effects of X-Irradiation on Living Cells. By Barbara E. Holmes 131 Radiation-Induced Disturbances of the Lipids of Cellular Micio- structures. By N. N. Doemin and V. D. Blokhina 141 The Significance of Free Deox;>T:ibonucleotides in Radiation Damage. By J SosKA, L. Benes, V. Drasil, Z. Karpfel, E. Palecek and M. Skalka 153 Biochimie et Radiol)iologie du Noyau Cellulaire. By M. Errera 165 On the Mechanism of Lethal Action of X-Rays in Escherichia coli K12. By H. Marcovich 173 A* i^ 80163 2C CONTENTS Damage to the Reproductive Capacity of Human Cells in Tissue / Ciilture by Ionizing Radiations of Different Linear Energy Transfer. By G. W. Barendsen \ 183 Phosphate Metabolism in the Nucleus. By L. A. Stocken 195 Initial Steps in Radiation Damage to Chromosomes and Means of Preventing this Effect. By Alexander Hollaender 201 On the Mechanism of Inliiliition of Cell Division Induced by Ionizing Radiation. By A. V. Lebedinsky, V. M. Mastryukova AND A. D. Strzhizhovsky 211 The Biochemical Mechanism of the Disturbance of Cell Division by Radiation. By A. M. Kuzin 223 The Role of Cellular Damage in the Mammalian Radiation Syndrome. By E. Y. Grayevsky, I. M. Shapiro, M. M. Konstantinova and N. F. Barakina 237 Studies on Enzymes and Yeast Cells with Accelerated Heavy Ions. By Cornelius A. Tobias, Tor Brustad and Thomas Manney. . . . 257 The Rhythm of Oxidative Processes and its Disturbance under the Action of Radiation. By G. M. Frank and A. D. Snezhko 269 Immediate Reactions of Nerves and Muscles to Ionizing Radiation. O. Hug and H. J. Schliep 287 Mechanisms of Chemical Radiation Protection. By Z. M. Bacq and P. Alexander 301 Kinetics of Primary Reactions and Chemical Protection. By V. N. Tarusov 315 General Discussion 327 INTRODUCTION In this \()liiiiie are published the papers and discussion of the Inter- national Symposium on Primary and Initial Effects of Ionizing Radia- tions on Living Cells — the effects that nndei-lie the biological effects of radiation. The Symposium was held in Moscow in Octol)er 1960 and was organized by the Academy of Sciences of the U.S.S.R., under the auspices of UNESCO, and co sponsored by IAEA. The President of the Symposium and the organizer of the very interesting general discussion was an oustanding Belgian scientist, Professor Z. M. Bacq, who is a foreign member of the Academy of Sciences of the U.S.S.R. The symposium was of round table type. The scientists from different countries, invited personally by the Academy of Sciences jointly with the Department of Natural Sciences of UNESCO, are very well known for their important achievements in the field of radiobiology at the molecular, subcellular and cellular level. In the papers presented at the symposium were given not only their latest results but also some general points of view on the state of the problem as a whole. The scientists discussed the most urgent and yet unsolved problems so, besides the reports and discussions after each report, a certain period of time was allotted to general discussion. Paying attention to this fact it was decided to shorten the pro- lixities and repetitions in the publication of the Proceedhigs of the Symposium and to publish not only the texts of the reports but also the discussion referring to separate reports, as well as the whole of the general discussion. In publishing the papers a number of comments, that were not originally made (for lack of time), but were presented by the participants in written form, are included in that general discussion. This seemed expedient since the material supplemented the interesting discussion. In order to make the discussion more wide and lively not only the participants but also the guests at the Sym- posium took part. The "round table" type of symposium has certain advantages. It provides close contact and a free and easy conversation among the limited numbers of participants. It goes without saying that, as well as the official discussion, the unofficial discussion, during the intervals and after the sessions were formally closed, much more often compared with that of more crowded conferences. The res\ilts of such meetings invisiblv, in, as it were, a "latent^" fashion, affected the points of view XI XU INTRODUCTION which were expressed by the participants during the final general discussion. By the extremely wide contemporary development of each field of knowledge, by the application of the most diverse techniques, and by study of the processes with different objects, we must not be dis- appointed when there seems at first sight to be nothing in a report or discussion that would appear to solve the jaroblem, or give the ini- j^ression of a major qualitative leap forward. Each important scientific problem is being solved step by step through the laborious investiga- tion of many tens and hundreds of scientists, when the data are accumulated and concepts develop. Therefore we think that the summing-up of the modern points of view and of the state of the jjroblem, with the simultaneous distinct demonstration of new ten- dencies and ways of approach, is the most important and principal result of such a symposium; these can be found in the published material. Undoubtedly, too. the published material will, in many respects, be a "stimulus" for a further development of science in the field of primary processes produced by radiation, and by the cells- reactions to irradiation. I should like to express my heartfelt gratitude to all the participants for the efforts they made. I also consider it my duty to express my cordial thanks to all who took part in preparing the materials for publication, and esi^ecially, for their hard work in preparing the debate and general discussion. Simultaneously with the present edition the same material is being published in the Russian language by the U.S.S.R. Academy of Sciences. G. M. Frank ()im^:nin(j addi^vF.ss Delivered ov behalf of the Aaidoiiii of Sciences of the U.S.S.R. by Academician-Secretary of the Department of Biological Sciences, U.S.S.R. Academy of Sciences N. M. SISSAKIAN Ladies and Oentlenien. Dear C'oUeagiies, I welcome you on behalf of the U.S.S.R. Academy of Sciences and the U.S.S.R. UNESCO Committee. We are happy to see our guests, eminent scientists from many countries, who have come to discuss interesting and outstanding problems. In you I also greet the important international organizations, UNESCO and the International Atomic Energy Agency which, in co- operation with the U.S.S.R. Academy of Sciences, have sponsored the present symposium. We are glad to see here the representatives of these organizations. In UNESCO and the International Atomic Energy Agency are com- bined the efforts of many nations in working out important problems of science, in spreading culture and knowledge and in the use of atomic energy for the good of mankind. Soviet scientists are eager to establish international co-operation with the aim of mutual understanding, peace and friendship among nations. They are willing to use all their resources and energy to organ- ize international meetings and contacts, which are so important for the progress of science. The rapid development of science in our age, the interconnection of a number of disciplines and the vast date accunudated require that science should be organized on quite new lines. Individual scientists cannot achieve spectacular results as they used to in the last century. Large teams of research workers in scientific institutions and univer- sities cannot exist in isolation. We require constant exchange of opm- ions, information, data and techniques: during these contacts and discussions new ideas are born and new ways of investigation are discovered. The level of modern science and the rate of its development make international meetings especially necessary. The present twentieth century is justifiably called the age of atomic energy. A new', exceptionally po\\erful agent has entered the life of men and advanced the solution of a number of most important prob- lems. At the same time mankind is facing a number of highly important 1 2 N. M. SISSAKIAN theoretical and practical problems. The use of atomic energy has stimnlated the rapid development of a new biological discipline radio- biology. This science aims to establish the laws of the action of radiation on living organisms. It is connected with the solution of essential tasks in most complex fields of human activity, medicine, agriculture and in- dustry. The use of this energy is connected Avith the discovery of the most effective methods of radiobiology, the breeding of new species of plants and strains of useful micro-organisms. One of the most important tasks of this new science is, no doubt, that of protecting those who come into direct contact with this agent, and their j^rogeny, from the damage of ionizing radiation. To solve these problems, it is first of all necessary to know the initial stages of the reaction developing from the interaction of a living organism with ionizing radiation. A deep scientific knowledge of the processes initiated by every kind of radiation is indispensable. These problems require the close co-operation of experts in various branches of science: physicists, biologists and physicians. Radiobiology is a border discipline: formed on the cross-roads of jjhysics, chemistry, biology and medicine, it makes wide use of approaches and techniques jieculiar to these sciences. The present symposium is devoted to the important problems of the primary action of ionizing radiations on biological substrata and on the cell. I am deeply convinced that as a result of the work of the present meeting of eminent scientists, at which so many outstanding radio- biologists are present, we shall be able to sum up year-long researches on these problems and to map out new paths along which radiobiology will develop. The development of radiobiology is most closely connected with the very important problems of our time, the protection of man- kind from the damaging effect of the production and testing of nuclear weapons. Radiobiologists are aware, more than anybody else of the damage caused by even a minimal dose of radiation not only to the present generation, but to the future ones as well. Therefore, the voice of radiobiologists, warning of the danger of nuclear tests is specially important in the strengthening of peace on earth and in the establishment and development of friendship among nations. I should like to wish success to the work of the members of the present symposium and to express the hope that it Axill enhance friendshij) and creative co-operation between the scientists of our various countries. THE NATURE OF THK IXFIIAL RADLVIMON DAMAGE AT THE SUB-CELLULAR LEVEL i'. ALEXANDER Chester Beafty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, England AND Z. M. BACQ Laboratoire de Pathologic Generale, Universite de Liege, 32 Bd. Constitution, Liege, Belgium SUMMARY A detailed investigation of the changes produced in DNA, when this is exposed to ionizing radiations under a variety of conditions, indicates that radiochemical damage to DNA is unlikely to be the primary event which initiates the processes which eventually result in the death of irradiated cells. Further support for this view is derived from the fact that the sensitivity of the nucleoprotein when irradiated within the cells is approximately the same for cells of widely different radiosensitivities. Attention to the non-genetic components of the cell is indicated by the observation that treatment with iodoacetate before irradiation can in- crease the radiosensitivity of cells seven-fold. Studies of mouse leukaemia cells in tissue culture indicate that chromosome breakage is not an important anatomi- cal lesion for the death of these cells. The hypothesis that interference with the sub -cellular fine structure of the cell consitvites an important primary lesion is discussed. As the radiation passes through the cell it deposits energy, part of which is used up to produce ionizations which initiate chemical reactions that cause some of the cell constituents to be chemically altered. There are several steps between the ionization of a molecule and its final chemical state (see "repair" of molecule in this symposium, p. 301) but the time taken for this process is, in vivo, a small fraction of a second, t t In model experiments, chemical changes have been observed hours after the irradi- ation is complete. Usually this is due to the fact that solids are treated in which the movement of molecules is limited and radical coml)ination is slowed down. This situation may be encountered in dry biological preparations such as spores or seeds, but does not occur in wet cells. This explains why radicals can be detected by electron spin resonance in dry systems (including seeds, etc.) but no such signal can be observed in irradiated wet cells. 4 p. ALEXANDER AND Z. M. BACQ Most of these chemical reactions are biologically trivial and do not harm the cell. But some of these occur at vital points and act as a focus for the development of damage by subsequent cell processes. These we have called the Initial (Chemical) Lesions, and their nature forms the subject of this paper. We have very little definite information about the early chemical events. The U.N. Scientific Committee on the effects of atomic radiation (UNSCEAR) concluded in its report (1958): "The nature of the initial step of radiation damage remains to be deter- mined."' After a certain period, the duration of which depends on the intensity of the metabolism, biochemical lesions can be observed and these lead to anatomical lesions (i.e. biological end effects). At the cellular level the immediate biological effects can be conveniently classified into (i) physiological ; (ii) cell lysis or interphase death ; (iii) delayed death often requiring mitosis before becoming manifest. It is improbable that the same radiochemical reactions initiate all these different biological effects. Also, a number of these may act in conjunction to produce one end effect, such as mitotic death, and their relative contribution may vary from one type of cell to another. Numerous methods are suitable for approaching the problem of the nature of the initial lesions, such as various biophysical techniques, biochemical techniques, comparison between the effects of in vivo and in vitro irradiation of key molecules (enzymes, proteins, RNA, DNA, etc.) and the study of the mechanisms of action of chemical protectors. A study of the physiological effects j of radiations may be particularly useful in this connection since they occur almost immediately after irradiation and the initial lesion must therefore be more closely related to the biological effect observed than in the case of such phenomena as cell death, chromosome abnormalities, etc., where many hours of active metabolism intervene. I It has ber-ome clear within the last few years that numerous disturbances induced by ionizing radiations occur almost immediately after irradiation (i.e. seconds to minutes) and must be attributed to an interference with the ph>-siological function of nerve fibres and cells, and to changes in membranes. Particularly striking demonstration of svich immediate effects have been provided by Brinkman and Lamberts (1060) and by Hug (1960) with snails, echinoderms and isolated mammalian organs. Very rapid effects on the retina of vertebrates have also been reported at the earlier UNESCO symposium at Venice (1959) the proceedings of which have been published as Supplement No. 1 (1960) to the InterndtionalJournal of Radiation Biology. All these changes are physiological in the sense that they are characterized by extremely rapid repair and are therefore very dose- rate dependent and may have to be studied while the irradiation is going on. Very rapid changes in the transjjort of the ions have been encountered following irradiation of plant roots (see Baccj and Alexander, 1961) but we know of some negative unpublished experiments with animal membranes. It is not a paradox to say that for certain biological systems, the ionizing radiation at small doses or dose-rates may be a stimulus comparable to an electric current or visible light and that no permanent lesion is inflicted on the organism. INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 5 DOKS DAMACK OF A XISIliLI-: STlirCTlTHK 1{K( ilTLAI{L^■ I'K IICIIDH CKIJ. I)1-:ATH? The i)r()l)lein ot tiiuliiiu the nature of the initial eheiiiical lesion in cell death would be siin])lilied if this effect could be associated with damage of a particular organelle, such as the chromosome. No generali- zation is, however, possible and not even the relative iniiK)rtance of the cytoplasm and the nucleus has been resolved. In certain cells (insect e(f(rs for instance) the lesion of the nucleus seems to be all important but in other cells (amoebae, amphibian ovarian eggs) the contribution of the cyto])lasm is certainly very great (for ref. see Bac(( and Alex- ander, 1901). \Vhile some cells such as lymphocytes, spermatogonia and oocytes are killed outright (i.e. interphase death) by a few hundred rads, most manmialian cells need rather large doses for this to occur and, in general, they are more sensitive to mitotic death : that is, they will divide once or twice before division stops though the cells continue to grow in size. Fig. 1. Effec-t of X-rays (220kV at 300+ inin) on growth of mouse leukaomia colls in tissue culture (Alexander and Mikulski. 1!»»)0) (The cells were in suspension and fully oxygenated when irradiated). Figure 1 illustrates this effect for mouse leukaemia cells in tissue culture. Until the cell number has doubled, no effect of radiation with doses of 300r or less was seen, then cell division ceases (with 30()r in case of 90 per cent of the cells) and the damaged cells increass in size. (Alex- ander and Mikulski, l!»t)()). The majority grow to about double the normal volume but about a quarter of them continue to gro\\' and form 6 p. ALEXANDER AND Z. M. BACQ i^ 'm- «i.^^^ # Fig. 2.— Electron ijhotoinicrograph of mouse leukaemia c-ell grown in tissue culture. INITIAL RADIATION DAMAGE AT SITU-CELLULAR LEVEL Fig. 3. — Similar cells 42 hr after 300 rads of X-rays. About 10 per cent of the cells turn into giants and an example of these is seen in the centre. The majority of the cells only double in volume (i.e. diameter increased by less than 30 per cent) and two of these are shown at the top. 8 p. ALEXANDER AND Z. M. BACQ giants with up to thirty times the normal vohnne (Fig. 3). Even after cell division has stopped, DNA and protein synthesis proceed normally for some twelve to fifteen hours. Interference with protein and DNA synthesis only becomes apparent in these cultures some 36 hr after irradiation when the cells can be seen microscopically to be degenerat- ing. Cell death under these conditions has frequently been attributed to chromosome damage and undoubtedly the loss of large amounts of genetic material must render a cell non-viable. But this cannot be the only mechanism, for radiosensitivity and chromosome damage do not go hand-in-hand (Oakberg and Minno, 1960; Bender, 1960; Sharman, 1959). The possibility must be envisaged that the time interval between irradiation and cell death is needed for the metabolic development of the injury as is the case for all radiation effects. That in rapidly dividing cells mitoses occur during this essential time interval need not imply that mitosis itself is a necessary step for the process of mitotic death ; the mitosis may be coincidental. Evidence for this view is derived from experiments in which cells are prevented from division after irradiation by a treatment that does not suppress metabolism. If the leukaemia cells are kept after irradiation at 22°C for 18 hr and then returned to their normal temperature of 37°C, cell death takes place without intervening mitosis. At the lower temperatures the cells still metabolize although no division occurs (95 per cent of the unirradiated cells survive 18 hr at 25°C). In this connection it is worth emphasizing that there is no evidence that allows us to link chromosome abnormalities with radiochemical damage to nucleoprotein. The concept that the ionizing particle severs the chromosome or chromotid thread on passing through it finds no support from iw ?;zY/o studies of DNA (Lett et al., 1961a, b) nor does it explain the biological data (Revell, 1959). A time interval is always necessary between irradiation and the appearance of the chromo- some "break". To see a break the cell has to be studied in metaphase or anaphase, yet the irradiation has to l)e carried out hours earlier while the cell is in the resting stage or in early prophase. The explana- tion that the interphase chromosomes are more slender structures that can be severed more easily by an ionizing particle than the visible chromosomes of mitosis is invalid since cells irradiated during mitosis show chromosome "breaks" at a high frequency in the next mitosis. RADIOSENSITIVITY, RADIOSENSITIZERS AND INTRACELLULAR PROTECTORS Many factors are likely to play a part in determining the great INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL !l variation in tlie radiosensitivity of cells, in ])articiilar tliosr of" micro- organisms. One as])ect which we have been investigating is the |)resence Avithin the cell of some substances, e.g. snlphydryl componmls or bacterial pigments, which can act as jirotective agents and lednce the magnitude of the initial chemical lesion by one of the energy transfer or re])air processes discussed in our other pa])er in this symposium. We must stress that the jn'esence of intracellular agents can at best be only one of the factors making for high i-adio-resistance. We have tested this hypothesis by comi)aring the dose needed to damage the same "marker" molecule Avithin different cells having varying radiosensitivities. In these ex])eriments large doses have to be used since a radiochemical reaction unmagnified by metabolism is being studied. The cells are irradiated at 0*^0 and then immediately broken u]) in a Hughes press at — 25°C. The homogenate is kept frozen until the actual assay occurs. The dose needed to inactivate the enzymes of the Krebs cycle in Micro- coccus sodensis with an LD37 of 33,000 rads (220 kV X-rays) was approximately five times that necessary for the much more radio- sensitive Psewc^omo^irts yZworescews (LD37, 1,800 rads). The protection of the enzymes in the cytoplasm of the micrococcus may be related to the presence of a carotenoid pigment since this is much more sensitive to irradiation within the cell than in isolation. Snlphydryl protection does not appear to be operative since the concentration of SH groups is less in the micrococcus than in the pseudomonas. The DNx4 of the cells does not appear to be protected since approximately similar doses of radia- tion (100,000 to 200,000 rads)^ suffice to reduce the molecular weight to one half (i.e. to jiroduce one "double" break, see p. 14) in pseudo- monas, micrococcus, rat thymocytes and mouse leukaemia cells. These measurements were made on DNA extracted immediately after irradia- tion with X-rays at lO^r/min at 0°C. The amount of DNA that could be extracted from the irradiated cells was, however, less than that obtained from non -irradiated controls and this introduces an ambiguity in the interpretation. Removal of the intracellular protective agents should lead to radio- sensitization. lodoacetate, which has been shown to enhance radiation damage in mice, readily combines with suljohydryl compounds that could be physiological protectors. Leukaemia cells in tissue culture exposed to iodoacetate at a concentration of 10-5 m for 30 min prior to irradiation (but not when given after irradiation) are 30 per cent more sensitive to X-rays than those that have not been treated : 300 r produce an effect for which 375r would otherwise be necessary. To make this experiment easily interpretable the treatment with iodoacetate was sufficiently mild so as not to interfere with the growth of the cells in any 10 p. ALEXANDER AND Z. M. BACQ way, and it only blocked 30 per cent of the total SH groups of the cell. Treatments that lead to the loss of more SH groups were toxic. It is not surprising that the extent of sensitization was so small under these conditions. Pre-treatment of bacteria with SH reactors leads to very much greater sensitization. Exposure to 10"4 M iodoacetamide doubles the radiosensitivity oi Pseudomoyias flnorescens and it increases that of Micrococcus soclensis seven times. Post -irradiation exposure to iodo- acetamide is without effect on the survivors. These very great changes in radiosensitivity may not be due solely to the removal of protective SH-compounds but may be due to a change in the physiological state of the cell due to the blocking of SH-enzymes (Alexander and Mikulski, 1961). IS THERE A MOLECULE OR A SERIES OF MOLECULES WHICH MAY BE FOUND ALTERED IMMEDIATELY AFTER IRRADIATION IN VI VO WITH DOSES GIVING IMPORTANT ANATOMICAL LESIONS AFTER A LONG LATENCY? All organic molecules are susceptible to damage by radiation and in the irradiated cell almost every constituent is liable to be altered chemically by the direct or indirect action of radiation. A consequence of this almost complete absence of selectivity is that a high proportion of the few reactions that occur will be harmless as they do not involve a molecule (or structure) of which nearly every one is essential to the cell. Two kinds of molecules — enzymes and nucleoproteins — are at first sight possible candidates for the essential lesion. Eyizymes When purified enzymes are irradiated in vitro — with large doses — a decreased activity is regularly observed, f When enzymatic activities are tested very soon after moderate irradiation in homogenates, tissue slices or whole organisms inhibition — even slight inhibition — (cf. DNA and protein synthesis in cells irradiated in tissue culture is rarely noted. On the contrary, anabolic as well as catabolic enzymatic actions are generally found increased, sometimes as much as ten times (for t The chemical mechanism by which ionizing radiations inactivate enz^aiies deiiends on whether the action is direct or indirect. If direct, a single primary ionization is often sufficient to disorganize the whole of the secondary structure of a protein by causing the breakage of liydrogen bonds (Alexander ct al., 1959). The radicals from water (i.e. in- direct action) react chemically with the protein, but the majority of the reactions occur on groups that are not essential for biological activity and these reactions do not lead to inactivation. Only occasionally (ranging on average from one in ten to one in one hvmdred) does a radical inactivate by reacting with an essential site. Consequently, naany ionizations have to occur in water before a protein is inactivated by "indirect action" and this therefore is much less efficient than direct action. INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 11 ref. Bacq and Alexander, 1961). We shall come back to this ])oint later. Another and perhaps more serious objection to an enzyme theory is that almost all the biological effects and, in particular, cell killing are brought about more effectively by densely ionizing radiations such as a -particles and fast neutrons than by sparsely ionizing radiations such as hard X-rays or y-rays. For a radiochemical reaction, shown to occur in the cell, to be a candidate for the role of primary lesion, it must, at least qualitatively, show the same relative efficiency for radiations of different ionizing densities. This requirement effectively eliminates most radiochemical reactions studied so far since in almost every case densely ionizing radiations were shown to be less efficient. This is the case for the inactivation of enzymes where a-particles are many times less effective than sparsely ionizing radiations. Gordy and Shields (1958) have claimed that when proteins are irradiated in the dry state, the energy is funnelled into the disulphide bonds and Ehrenberg and Zimmer (1959) have ascribed great bio- logical significance to this reaction. The experimental evidence for this hypothesis was the observation that the electron spin resonance patterns after irradiation of protein and of the sulphur-containing amino acid cystine were similar. We have investigated this claim (Libby et al., 1961) and Fig. 4 shows that the pattern of protein and cystine are quite different and that there can be no question that the same radicals are produced. The patterns only resemble one another COMPARISON OF IRRADIATED ALBUMIN WITH CYSTINE TREATMENT 5«IOrAT-l95C TREATMENT S.IOr AT 20°C TREATMENT OPENED TO AIR Fig. 4. — Comparison of the electron spin resonance pattern obtained by irradiating bovine serum albumin and cystine with ^^Co y-rays in vacuo. The irradiations and measui-ements were carried out at — 195°C and at 20°C. After measurement, the samples were opened to the air and their ESR remeasured (for experimental details see Libbj' et al., 1961). 12 V. ALEXANDER AND Z. M. BACQ when oxygen is admitted and this is due to the fact tliat the radicals become pei'oxidized in both snljstances. Chemical analysis of irradiated protein confirms that cystine is not preferentially destroyed (Alex- ander and Hamilton, 1960). Deoxyribonucleic Acid {DNA) On quantitative grounds, DNA is a more likely candidate for the primary lesion than enzymes, since it is probable that every molecule is unique. It is possible therefore that damage to only a few molecules per cell could affect survival. There are however a number of difficulties with this interpretation (Alexander, 1900; Lett et a!., 1961a, b) and before considering these we shall summarize the available information about the chemical and physical changes that DNA undergoes on irradiation. Indirect action. This results in far-reaching chemical changes such as the opening up of the imidazole ring of the purines (Hems, 1960) and the formation of jjeroxide groups on the pyrimidines (Ekert and Monier, 1959). From the point of view of cell death (though not necessarily for mutations) reactions that alter tlie macro-molecular properties of DNA (such as main-chain scission or cross-linking) are probably more important since every one of these is liable to destroy the biological integrity of the molecule while isolated chemical changes in one of the bases need not invariably be damaging. In dilute aqueous solution X-rays reduce the viscosity of DNA (Taylor et al., 1948). This reaction is now known to be the result of rupture of the molecule which occurs whenever a break has been j)ro- duced in both of the constituent chains within a distance of some five nucleotide units. A detailed investigation by Moroson and Alexander (1960) has shown that for a DNA molecule of 5 x 10^ molecular weight (number average) 65 OH radicals are necessary before a break becomes apparent. The majority of the OH radicals are used up in producing isolated (non-coincident) breaks that remain hidden. There has been a great deal of contradictory data concerning the post-irradiation fall in viscosity of DNA following irradiation in dilute solution, and complex mechanisms involving unstable peroxides and phosphate esters have been proj)osed (see review given in Bacq and Alex- ander, 1961). Although these reactions may occur to a limited extent, two main causes for this in vitro post-effect have been found (Alexander, 1959). 1. DNA is known to be unstable in solutions of low ionic strength. If less than 10"^^ ]\([ of salt is present the molecule denatures on INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 13 standing and takes up a more coiled configuration. This change is accompanied by a fall in viscosity. This type of denaturation occurs more readily if there are "hidden breaks". Hence if DNA is dissolved at ionic strengths of less than 10-2 M the molecule coils up, denatures slowly and this coiling leads to a reduction in vis- cosity after irradiation. This type of after-effect is completely pre- vented if DNA is irradiated at ionic strength approaching physio- logical. 2. DNA is very susceptible to oxidation by dissolved chlorine which produces breaks in the main-chain in the same way as OH radicals do (Moroson and Alexander, 1960). If DNA is irradiated in solutions of sodium chloride, some of the OH radicals react to give dissolved chlorine. This will then react slowly with DNA to produce breaks (Alexander, 1959). The addition of sodium thio- sulphate immediately after reaction prevents this type of post- effect by removing the dissolved chlorine. If steps are taken to pre- vent these two reactions the fall in viscosity following irradiation is small and represents less than 20 per cent of the total change. Direct action by sjxirsely ionizing radiations. The changes produced wdien DNA is irradiated in the dry state or as a concentrated gel are extremely complex and the observed effects depend critically on the experimental conditions (Alexander etal, 1960; Lett et al, 1961a, b). DNA containing 20 per cent of moisture or less is almost unaffected by irradiation and the presence of oxygen has relatively little effect. DNA fibres containing an equal weight of water are readily degraded if irradiated in the presence of oxygen but become cross-linked when irradiated in its absence. Cross-linking is recognized by an increase in molecular weight and at higher doses the formation of an insoluble gel. We have explained these findings by postulating that the result of an ionization is to produce a break of one of the chains in such a way that one of the ends is active (e.g. a free radical) so that it can combine with another end to produce a cross-link. The active ends combine readily with oxygen after which they are no longer capable of cross- linking. In the dry DNA, diffusion of oxygen is slow but molecular movement is also limited and few of the active ends have an oppor- tunity to form cross-links and the majority are wasted. When the DNA is swollen with water the opportunity for interaction betw^een active ends is greater, but there is now a competing process due to the rapid penetration of oxygen. This complex set of reactions is summarized iji Fig. 5, (a) Cross-linking X or ,i irradiation DNA ; twin molecule maintained by stereo - specific hydrogen bonds. (a) "Active" end of break capable of combining with another active end. A proportion of the ionizations that occur in the DNA produce a break in one of the chains. .1--^ ^^ ^ \<> ^ \^ Two molecules join if the active ends can ineet. In DNA fibres swollen with water the cross- linking efficiency is higher because the inole- cules are more mobile. \ *o, "Active" end becomes peroxidized and is no longer capable of form- ing a cross-link. (Rate of diffusion of oxygen into dry DNA is slow and "active" ends persist for many days because lack of movement prevents cross-linking.) break in each of the ad- nucleotide units ajjart. an (1)) Main-chain Scission This occurs when there is a jacent chains less than about 5 This is produced by radiation : 1. Every time a DNA molecule is traversed by a-particle* (600 eV/double break). 2. When a cluster of ionizations (or other high en- ergy event) is formed by sparsely ionizing radiations (S.'ib eV; double break). " 3. When by chance two isolated breaks come into juxtaposition. Form statistical calculation one "double break" will occur for every 70 random single breaks. This mechanism is responsible for main-chain scission by the indirect action of H and OH radicals formed in the water. * Some cross-links are produced at the same time as main-chain scission by a-rays due to the relatively sparsely ionizing 8-rays. Fig. 5. — Changes in the macromolecular properties of DNA brought about by ionizing radiations. INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 15 Both cross-linking and degradation have been observed in the DNA in heavily irradiated cells. The DNA in thymocytes and in mouse leukaemia cells suffers only degradation and no evidence for cross- linking, while cross-linking predominates when the sperm of fish are exposed to sparsely ionizing radiations (Alexander and Stacey, 1959). Irradiation ivith y.-raijs. The effect of densely ionizing radiations such as a-rays from polonium is initially less complex, f since only degrada- tion is observed, the efficiency of which is independent of conditions. Quantitatively, a double strand of DNA is severed every time it is traversed by an a-particle. This corresponds to one "double break" for every 650 eV of energy deposited (Alexander et al., 1961). Biological implications. These studies show that DNA is drastically altered by radiation. Some of the effects produced are qualitatively different in the presence of oxygen, but there is no evidence to suggest that oxygen influences the total number of molecules aff"ected. a-rays are slightly more effective than sparsely ionizing radiation (650 eV as opposed to 800 eV) in producing main-cham scission (double breaks) and this is therefore a radiochemical reaction in which the relative efficiencies of different radiations are at least qualitatively the same as for biological effects. It is tempting therefore to speculate that double breaks may constitute an important primary lesion. But this hypothesis fails to provide an explanation for the enhancement of radiation of sparsely ionizing radiations by oxygen which does not interfere with main-chain scission, but only prevents cross-linking. Another difficulty is that one would expect cross-linking to be at least as effective as main-chain scission in rendering DNA biologically useless. If damage to DNA were an important primary lesion then the combined effect of cross-linking and main-chain degradation must be considered. On this basis, electrons are more efficient in inactivating DNA molecules than a-rays. This is confirmed by irradiation of transforming principle for which the inactivation dose increases with increasing LET (Fluke etal, 1952). DAMAGE TO MEMBRANES AND INTRACELLULAR BARRIERS It seems that on the basis of radiochemical studies, we have to reject the view that damage to DNA or DNA-protein constitutes the primary f a-rays are always accompanied by less densely ionizing S-rays which comprise some 20 per cent of the total energy deposited by a-rays. These 8-rays give rise, in the absence of oxygen, to cross-linking and this complicates the results obtained with a-rays. / 16 p. ALEXANDER AKD Z. M. BACQ lesion at least for processes leading to cell death. This is in agreement witli the observation that cells without DNA (red cells or reticulocytes) or anucleate fragments of cells (amoebae, Acetabularia 7nediterranea) show tyjDical radio-lesions. Having eliminated enzymes and DNA, the whole concept that a biologically active entity is involved in the primary jjrocess may have to be abandoned. We suggested in 1955 (cf. Bacq and Alexander, 1961) that the increase in enzymatic activity wdiich is so frequently seen in irradiated organisms soon after irradiation f could be explained if radiations broke down internal barriers within the cell — the enzyme release theory. Not only is there an intracellular increased enzymatic activity but also a leakage of many enzymes (for instance, DNase, peptidase, two transaminases and two dehydrogenases — for ref. Bacq and Alexander, 1961) in the plasma and the urine. Such leakage of enzymes is now well understood as a good but unspecific sign of cellular damage; it can be elicited by anoxia or by drug-poisoning. Increased enzymatic activity in cell homogenates or tissue slices of whole organisms can be theoretically explained in three ways (see ref. in Bacq and Alexander, 1961). (i) Increased synthesis of the enzyme ; this is the case after irradiation for tryptophane peroxidase in the rat, an adaptative enzyme depending for its regulation on the adrenal cortex. (ii) Destruction of an inhibitor. Many enzymes (the DNases and RNases) are known to be inhibited by substances that occur naturally in the cell or in circulating fluids. Feinstein and Ballin (1953) believe that this is the case for a cathepsin. (iii) Increased contact lietween enzymes and substrates. De Duve (1958) has conclusively demonstrated that certain granules (called lysosomes) contain a series of enzymes (acid DNase, acid RNase, acid phosphatase, cathepsin, /S-glucuronidase and aryl-sulphatase) which in vitro react poorly or not at all with their specific substrate unless their "membrane" has been damaged by freezing, osmotic imbalance or by mechanical means. One should never forget that the DNase which is in these lysosomes (and also in the mitochondria) has no substrate available on the spot ; it must travel a long way (if we think in molecular distance) and pass through many cytoplasmic barriers and through the nuclear membrane before reaching some DNA. & t Kecent observations by Libinzon (1959) have shown that in the bone-marrow of rabbits the activity of acid and alkahne rii)onucleases is increased three to five times about one hour after tlie beginning of irradiation (1000 r, y-rays ''^'Co, 15r/min.) The activity of the DNases is increased 50 to 100 per cent one hour after irradiation. INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 17 In a certain way our theory is so obvious that it appears to us as a "lapalissade"t. The cell is a network of phospholipid membranes and one of their chief functions is to keep certain substrates and enzymes apart. If these barriers are disrupted, either by forming holes or altering their selective permeability in some way, serious biochemical disturbances will result. The release of degradative enzymes, for example, could give rise to damage to nucleic acids and proteins. But this is only one of the possible mechanisms by which an interference with internal cell structures could lead to cellular lesions, and reactions such as coagulation of nucleo- protein by calcium ions may have to be considered. Possibly the actual l)iochemical process that is initiated by membrane damage may vary for different cells and for different types of lesions. To break down fine intracellular structures, several ionizations acting in conjunction may be necessary and this could explain the superiority of densely ionizing over sparsely ionizing radiations in initiating cellular lesions (i.e. a given amount of explosive is much more effective in knocking down a wall in the form of one cannon shell than in the form of many rifle bullets none of which can penetrate). A mechanism by which oxygen enhances the radiation lesions of sparsely ionizing radiations can be envisaged for membrane damage. The phospholipid membrane may undergo a chain reaction with oxygen which is initiated by an ionization for they contain unsaturated fats and in this way the effect of a single ionization may be greatly multi- plied. With densely ionizing radiations this multiplication effect may not be necessary as the damage produced by several ionizations close together is already sufficient. The possibility that damage to membranes contributes to the killing of lymphocytes by radiation is indicated by electron microscopic studies. Four hours after 1,000 rads are given to the thymus of rats all the cells and, in particular, the nuclei show gross morphological abnormali- ties (i.e. pyknosis) when fixed with heavy-metal-containing fixatives and then stained by the methods developed by Trowell (1952). Yet when these same cells are examined in the electron microscope (with osmium fixation), the shape of the nucleus is quite unaltered and no morphological abnormalities can be seen (Alexander, 1961). The heavy metal fixative used to show up pyknosis causes clumping of isolated nucleoprotein whether this has been irradiated or not. We believe that t The early release of enzymes may be the reason for the iDrimary step of their inaetiva- tion which may be observed several hours or days after irradiation. Enzj-mes when linked to mitociiondria or microsomes are inactive but protected against destruction; when liberated, they may become a substrate for proteases similarly released. 18 P, ALEXANDER AND Z. M. BACQ irradiation makes it possible for the fixative to penetrate into the cells and that the pyknotic changes are not caused by the irradiation, but are due to access of the fixative. REFERENCES Alexander, P. (1959) Ann. Rep. Brit. Empire Cancer Campaign, p. 63. Alexander, P. (I960). Proc. 8th. Int. Congr. of Radiology, Munich, 1959. G. Thieme. Alexander, P. (1961). Proceedings of 3rd Australasian Radiation Biology Gonf. But- terworth, London. (In press). Alexander, P., and Hamilton, L. D. G. (1960). Radn JRes. 13, 214. Alexander, P., and Lett, J. T. (1960). Nature, Land. 187, 933. Alexander, P., and Mikulski, Z. B. (1960). Biochem. Pliarmacol. 5, Alexander, P., and Mikitlski, Z. B. (1961). Brit. J. Radiol. 34, 363. Alexander, P., and Stagey, K. A. (1959). Nature, Lond. 184, 958. Alexander, P., Hamilton, L. D. G., and Stacey, K. A. (1959). Nature, Lond. 184, 226; (\^m) Radn Res. 12,510. Alexander, P., Goldberg, R., Kopp, P., and Lett, J. T. (1961). Radn Res. 14, 363. Alexander, P., Lett, J. T., Moro.son, H., and Stacey, K. A. (1960). //*/. J . radn Biol., Suppl. 1, p. 47. Bacq, Z. M., and Alexander, P. (1961). "Fundamentals of Radiobiology". 2nd edition. Pergamon Press, London. Bender, M. A. (1960). Int. J. radn Biol., Suppl. 1, p. 103. Brinkman, R., and Lambert.s, H. B. (1960). Int. J. radn Biol., Suppl. 1, p. 167. de Duve, C. (1958). Bidl. Acad. Med. Belg. Vlth series, vol. 23, p. 608. Ehrenberg, L., and Zimmer, K. G. (1956). Hereditas. Lond. 42, 575. Ekert, B., and Monier, R. (1959). Nature, Lond. 184, 58. Feinstein, R. X., and Ballin, J. C. (1953). Proc. Soc. exp. Biol., N.Y. 83, 6 and 10. FluiCe, D., Drew, R., and Pollard, E. (1952). Proc. nat. Acad. Sci., Wash. 38, 180. GoRDY, W.. and Shield.s, H. (1958). Radn Res. 9, 611. Hems, G. (1960). Nature, Lojid. 186, 710. Hug, O. (1960). Int. J. radn Biol., Suppl. 1, p. 215. Lett, J. T., and Alexander, P. (1961). Radn Res. 15, 1.59. Lett, J. T., Stacey, K. A., and Alexander, P. 1961). Radn Res. 14, 349. LiBBY, D., Ormerod, M. J., and Alexander, P. (1961). Int. J. radn Biol. (In press). LiBiNZON, R. E. (1959). Biokhimiya, 24, 679; Biochemistry, 24, 625. MoROSON, H. and Alexander, P. (1961). Radn Res. 14, 29. OaKberg, E. F., and Minno, R. L. (1960). Int. J. radn Biol. 2, 196. UXSCEAR Report of the L^.N. Scientific Committee on the effects of atomic radiations. General Assembly, 13th Session. Suppl. no. 17 (A/3838), New York, 1958. Revell, S. H. (1959). Proc. roy. Soc. 150 B, 563. Sharman, G. B. (1959). Int. J. radn Biol. 1, 115. Taylor. B., Greenstein, J. P., and Hollaender, A. E. (1948). Arch. Biochem. Biophys. 16, 19. Trowell, O. a. (1952). Exp. Cell. Res. 3, 79. DISCUSSION POWERS : What was the effect of heating in your experiments on paramagnetic resonance? In other words do you admit the possibility of changes in your material due to heating? ALEXANDER: Thcse experiments were usually carried out at a temperature of — 195°C. Then the sample was warmed up to room temperature. A change in the spectrum resulted. The sample was kept at room temperature for a day, and showed further changes of the spectrum. We did not cool the sample again, since it is quite evident that at room temperature irreversible changes could have occurred as radicals acquired the possibility of meeting and recombining, which does not occur at a temperature of — 195°C. POWERS: How do you explain these changes? INITIAL RADIATION DAMAGE AT SUB-CELLULAR LEVEL 19 ALEXANDER: Particularly larp;e changos ocfur when air is admitted. On heating the changes are less consitlerable. When irradiation was performed at room tem- perature different changes of the signals were also provided. I cannot go into the details but would like to emphasize the importance of our results in determining the role of sulphiu*. POWERS: T do not think that these data support yovir surmises on the role of sulphur. ALEXANDER : When ii-radiation was carried out in the presence of oxygen, ESR signals of the irradiated cysteine and proteins were similar, but if during irradia- tion oxygen was lacking, the cysteine and protein signals differed. We believe that the similarity of these signals is due to the oxygen, in the presence of which peroxide radicals are formed. LEBEDiNSKY : What experimental data confirm the very valuable, in my oj)inion, sviggestion about the importance of the membrane permeability disturbances? ALEXANDER : I hope to demonstrate this in the future by excluding other possible causes, since it would hardly be possible to obtain direct proof. Besides this, it is probably possible to get physiological and biochemical data confirming this view- point in an indirect way. But I have no direct evidence except the fact that following irradiation of erythrocytes with several hundred roentgens a leakage of the potassium is observed while at the same time we have shown that there is a change of the surface potential following such a dose. However, this is only a suggestion, not a proof. BLUMENFELD : What could you say about the form, width and position of the ESR signals which you obtain from protein at liquid nitrogen temperature? ALEXANDER: We did not obtain ESR signals with unirradiated protein whether this was denatiu'ed or native. MECHANISMS INVOLVED IN THK IXITIATTON OF RADIOBIOLOGICAL DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS L. H. GRAY Briti.'^h Empire Cancer Campaign Research Unit in Radiobiology, Mount Vernon Hospital, Northivood, Middlesex, England. SUMMARY Loss of reproductive integrity in spores^ vegetative bacteria and cells of higher plants and animals is in general a result of damage to a very small number of macromolecules which are important foi- proliferation. In dry spores a good deal is now known about the nature and lifetime of intermediates in the chemical reaction chains leading up to the damage to these macromolecules. Water, when present in small amounts, profoundly modifies the reaction chains and shortens the lifetime of many intermediates. In cells of high water content a considerable fraction of the damage may jjroceed from the radiolysis of the water. The conse- quence of a particular kind of macromolecular damage, which has arisen along a particular chemical pathway, may be greatly iafiuenced by nutrional factors, both before and after irradiation. The influence of the chemical and nuti'itional factors is interrelated. THE NATURE OF THE PROBLEM The aim of radiobiologists is to trace the course of events forward as far as possible, from the initial interaction between the ionizing par- ticles and the molecules present in the living cell, through the formation of excited states, unstable intermediates of finite life, and on to chemical changes which would be stable in a non-living system but which repre- sent functional defects when they occur in certain organelles of the living cell. These functional defects become in turn the starting point for a further sequence of chemical changes, and may be regarded as primary lesions at the biochemical level. The ionizing particles are moving too fast to excite molecular vibra- tion levels directly. The energy is transferred first to the electronic system of the recipient molecule and, almost always, in amounts which are large compared with the activation energies for the ordinary chemical reactions which the molecules are known to undergo. Thus in the irradiated cell there arise, almost at random, centres of energy absorption of such magnitude that many alternative pathways of chemical reaction become possible simultaneously at each site. As B 21 22 L. H. GRAY Platzman and Franck (1958) have pointed out. there also arises simul- taneously in a polar medium around the molecules, which are ionized, electric fields of sufticient intensity to break many — perhaps 20 — hydrogen l)onds in the immediate neighbourhood. It is clear, therefore, that from the instant of irradiation thousands of different reaction chains are proceeding simultaneously. Our concern is to associate ])articidar reaction chains with particular forms of radiation response in the cell or in the organism. It is unlikely that any generalization will be found to cover all forms of response, as two examples wall suffice to show. When ionizuig radiation provokes the sensation of vision (Lipetz, 1960) the chain is evidently a short one. The response occurs in a small fraction of a second, and the reaction chain is probably confined to those changes initiated in the immediate vicinity of the light-sensitive elements of the retina, or to the direct excitation of these elements. Similarly, changes in the swimming habits of DapJmia magna, resulting from exposure to a brief pulse of X-radiation, are traceable to dis- turbances in the nauplius eye (Baylor and Smith, 1958), which in turn may consist of a radiation-induced reduction of certain pigment mole- cules, since the effects of the radiation are mimicked Ijy reducing dyes and opposite to those produced by dyes having a redox potential greater than -f 0-062 (relative to the hydrogen electrode). The retina proljably constitutes an almost ideal radiation detector, since the stimulation of a small number out of a very large array of identical molecules leads to a recognizable biological response. At the other extreme, the chain of events leading to loss of prolifera- tive capacity in bacteria, in higher plants and in mammalian cells is a long one, and this is the subject of this paper. CONSIDERATION OF DOSE-RESPONSE RELATIONS AS A GUIDE TO THE ANALYSIS OF THE REACTION CHAINS WHICH MAY BE IDENTI- FIED BY PHYSICAL AND CHEMICAL MEANS When allowance is made for experimental error, the analysis of dose- response relations cannot rigorously establish the number of initiating events. Zimmer (1960) has, for example, shown that it is possible to devise a model with a particular distribution of target size among a population of organisms, (mathematically related to the multiplicity of the events needed to inactivate each target), which would lead to a dose-survival curve which is indistinguishable, within the limits of experimental error, from an exponential curve. It is important to recognize this but, as Bertrand Russell has remarked, "It is a peculiar DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 23 fact about the genesis and growth of new disciplines that too much rigour too early imposed stities the imagination and stultifies inven- tion". In my view, the analysis of dose-response relations is still one of the most powerful means at our disposal for limiting our search — ■ among the tens of thousands of reactions which are proceeding simul- taneously in the irradiated cell — for those which lie in the pathway to the loss of reproductive integi'ity. We may remind ourselves that in genetics, the mathematical analysis of segregation frequencies pointed to the physical existence of permanent structures carrying the genes in a linear array before the microscopic study of Drosophila salivary gland chromosomes revealed differences in band pattern between mutant forms. On the basis of an analysis of loss of reproductive integrity by vaccinia virus Lea and Salaman (1942) reached the conclusion that the genetic material of the virus must occupy a small fraction of the total volume before a nuclear body was demonstrated by electron micro- scopy. Similarly, Preer's (1948) analysis of the radiation inactivation curve of kappa in Paramecium yielded an estimate of its size which exceeded the limit for visibility in the light microscope, and led to the identification of the kappa particles. By the criterion of the observed dose-response curves, loss of repro- ductive integrity would be judged, in almost all cases so far studied, to proceed from energy dissipated in the cell, either by a single particle or by, at most, a few ionizing particles. Dr. E. L. Powers and his col- laborators (1957, 1961) have published some extremely well defined linear relations between the log surviving fraction and the dose for spores of Bacillus megaterium irradiated in the dry state. A very similar relation for Serratia marcescens irradiated in aerobic suspension has been observed recently by my colleague Dr. Dewey (unpublished). After a small curvature near the origin, the experimental points define a straight line. Dewey's observations are remarkable in showing that the strictly linear relation is maintained over almost ten powers of ten. The logical interpretation of the linear part of this curve is that when a population of organisms is exposed to a certain increment of dose, the probability of loss of reproductive integrity is the same for each organ- ism and independent of the dose to which it has previously been ex- posed. For small increments of dose it is also strictly proportional to the dose increment. In physical terms, loss of reproductive integrity in any given bacillus depends only on the energy delivered to the cell by a very small number of ionizing particles. If the experimental observa- tions had defined a line which passed accurately through the origin, we should be able to infer that the loss of reproductive integrity was strictly independent of the reaction chains initiated by all other particles 24 L. H. GRAY than the one which is effective. It would not inijDly that one ionization (or one excitation) is the initiating event at the physical level, but only that one particle is involved. From the slope of the log survival curves, we know that the target within which the chemical reaction chains leading to loss of reproductive integrity are initiated must contain a great many molecules. Either the molecules themselves must be uniquely important to cellular prolifera- tion, or the reaction chains to which they give rise must involve the inactivation of uniquely important molecules. iVttention is thus focused on molecules of the DNxA.-RNA-protein class. A second deduction which can be made from the slope of the log survival curves is less agreeable. It is that, for all organisms as large as or larger than bacteria, the target is a small fraction of the volume of the cell. The total number of ions generated in the cell for each electron transit through the target is therefore large (Table 1). It follows that Table I. Fast electron transits per cell at a dose ivhich gives 37 per cent survivors Radius Aerobic Number of elec- Total number of cell -D37 tron transits of ion clusters (microns) (rads) (LET = l-7ekV/V) formed througli the cell Drv spores 1 2-5.104 3000 T.IO* Vegetative £. co/j 1 5.103 600 10* Plant or mammalian cells 10 100 1500 10^ when physical methods, such as electron spin resonance spectroscopy, are used to detect free radical intermediates, the chances that the signal is characteristic of the actual radicals which initiate loss of reproductive integrity is correspondingly small. BIOLOGICAL SYSTEMS IRRADIATED IN THE DRY STATE Loss of reproductive integrif/t/ by spores irradiated in the dry state I should like to take the exceedingly beautiful work with dry spores of B. megaterium by Dr. E. L. Powers, and his collaborators (this Sym- ]30sium) as a starting point for my remarks concerning loss of repro- ductive integrity in aerobic and anaerobic bacteria, and the proliferat- ing cells of higher plants and animals, with which I am personally more familiar. Davis and Hutchinson (1952), working with the closely joaMage in aerobic and anaerobic systems 25 related B. subtilis, inferred from their studies witli very slow electrons that the radiobiological ly iin])ortant material comprises only part of the spore, being surrounded by a shell of very insensitive material 230 A thick. The log survival curves observetl by Powers are, under all conditions of irradiation, strictly linear over almost their entire length, but show a small curvature towards the origin. The extrapolation number (as well as the slope) varies with the conditions of the irradiation, but is always less than 2. Loss of reproductive integrity in the spore has, therefore, the general features that I mentioned earlier. Certain features of Powers' work which are relevant to the present discussion may be summarized as follows : There are four recognizable classes of events which may lead to loss of reproductive integrity. These are characterized by the chemical reactivity, thermal stability and lifetime of intermediates in the reac- tion chains. Two involve molecular oxygen and two do not. Classes of event leading to reaction chains in ivhich the participation of molecular oxygen is not essential Class A events lead to reaction chains which are equally toxic under all conditions so far tested. Class B events lead to reaction chains which are equally toxic under all conditions tested, except treatment with H2S. They react with H2S, the simplest of the sulphydryl compounds, if present during irradiation, to give a product which is non-toxic. The lifetime of these species is such that reaction with H2S is no longer possible after the end of an irradiation of a few minutes' duration. Classes of event leading to reaction chains in ivhich the participation of molecular oxygen is essential Class C events generate intermediates that react readily with oxygen to give products leading to loss of reproductive integrity. In the absence of oxygen the intermediates in question have a comparatively long life, at room temperature, and may be stabilized against subsequent reaction with oxygen by thermal treatment, or exposure to NO or H2S after the end of irradiation. The stabilization is less complete if nitric oxide is present during irradiation (see p. 34). Class D events generate intermediates which only become toxic by reaction with oxygen. They have, however, a much shorter lifetime than the intermediates derived from Class C events, and are only in- fluenced by oxygen and H2S if these gases are present during irradiation. The physical reality of intermediates having lifetimes and chemical 26 L. H. GRAY properties closely resembling those inferred from the l)iological obser- vations, have been most beantifnlly demonstrated by Dr. Powers and his collaborators by means of electron spin resonance (ESR) spectroscopy. Further research may well lead to a further subdivision of these classes. As they stand, we may assign relative probabilities to the recognized classes of initiating event as: A-0"24, B-0'14, C — 0*38, and D — (V2-t. Relative probabilities of reaction chains in which the participation of oxygen is essential to those to which it is not, are thus C + D 0-62 = = 1-63 A + B 0-38 The sensitivity of the spores irradiated in oxygen to that when partici- pation of oxygen is excluded, l)oth during and after irradiation, viz: 1 = 2-63, a figure close to the values usually observed for the A+B ^ relative sensitivities of plant and animal cells irradiated aerobically and anaerobically. BIOLOGICAL SYSTEMS OF INTERMEDIATE WATER CONTENT The radiosentivity of seeds, with special reference to wafer content The influence of varying amounts of water on the sensitivity of cells has been studied in seeds (see reviews by Ehrenberg 1955, Caldecott, 1960, Nilan et al. 1960, Konzak et al, 1960, Sheldon Wolff, 1960, and Davidson, 1960). In evaluating this work it must be borne in mind that the l)iological damage induced in the seeds (as also that in spores) has always been examined after hydration and germination. When seeds are irradiated dry, intermediates of short and long life- time are produced which have much in common with those that lead to loss of reproductive integrity in irradiated spores. H2S and nitric oxide present during irradiation decrease the extent of the injury. This is largely due to the influence of these treatments on the course of those reactions which involve participation of oxygen. By ESR spectroscojjy it has been established that long-lived radicals induced by the irradia- tion of dry Agrostis seed disappeared fairly rapidly in the j^resence of nitiic oxide (Sparrman et al., 1959). If irradiated seeds are hydrated in water which is free from dissolved oxygen, the long-lived intermediates which are present at the end of ii-ra- diation are stabilized against subsequent reaction with oxygen, or with any other molecule so far tested. The level of damage is approximately DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 27 the same as if water (~ K) per cent in the embryo) had been present dnring irradiation. If. however, the water used for hydration contains dissolved oxygen (at or beU)w 300/liM/I), greater damage is observed. Evidently, if the oxygen and the water enter the embryo simiUtaneously, the oxygen competes successfully for long lived radicals, as it does in all cells of normal (high) water content. It was observed by Caldecott that after thermal annealing for 15 min at 85°C the long-lived intermediates induced by the irradiation of dry seed are no longer responsive to the presence or absence of oxygen in the water in which the seed is soaked. In this respect also the seed closely resembles dry spores, since it was found by Powers that ther- mal annealing eliminated the post -irradiation effect of oxygen. In this connection the interesting observation has been made by .:^ 1 1 1 1 cr 60 - — g 4-" U x'-x. X /' \ L- -5 60 ^ ~x 5 / \ M O f \ • L_ / \ cn 4-> S 40 _ a) / / O y / to ai / / i 20 _ / / > / p ^ ^>^ 1/) o o Q 1 1 1 0 4 8 12 16 Percentage H2O Fig. 1. — Influence of oxygen, nitric oxide and humidity on radiation damage to seeds. (Redrawn, by paraiission, from Sparrman el al., 1959). Caldecott (1960) that, whereas the yield of interchanges observed at meiosis in barley plants grown from irradiated dry seed increased linearly with dose when the seeds were irradiated and soaked anoxically : Exchanges = 1-94Z) Seeds which were irradiated and soaked in aerated water gave a yield of exchanges which contained an additional term in (Dose)^: Exchanges = l-76i)-f0-31i)2 28 L. H. GRAY The term in D^ is more important than that in D at closes above 6 krad. The rather close parallel between the influence of small amounts of water on the sensitivity of seed, and on the changes induced by radia- tion in non-living systems, as illustrated, for example, by the experi- ments of Clegg (1957) with cellulose fibres, supports the view that we are here concerned with a profound modification in the reaction chains brought about by the presence of water. When seeds of still higher water content ( ~ 15 per cent water in the embryo) are irradiated in the presence of nitric oxide, the radiation damage is enhanced to about the same extent as when the same seeds are irradiated in the presence of oxygen, i.e. the seeds now respond in a manner typical of vegetative bacteria and the cells of higher plants and animals. The influence of progressively increasing water content on the radio- sensitivity of Agrostis seeds, irradiated in the presence of nitrogen, oxygen and nitric oxide, is nicely illustrated by Fig. 1 taken from the paper by Sparrman et al. (1959). BIOLOGICAL SYSTEMS OF NORMAL (HIGH) WATER CONTENT Lifetimes of some of the radicals produced by irradiation of seeds having low and intermediate water content are long enough for study by means of conventional ESR spectroscopy. When we attempt to obtain information about the lifetimes of radicals produced in dilute aqueous solutions, or in cells of high water content, a number of difficulties arise, both in experimentation and in interpretation. Difficulties of experimentation Major difficulties arise on account of the short lifetime of the inter- mediates. We know from the observations of Howard-Flanders and Moore (1958) that essentially all the intermediates involved in the aerobic chains leading to loss of reproductive integrity in Shigella flexneri have lifetimes less than '20 msec, and estimates based on the way in which the sensitivity of these cells varies with the concentration of oxygen in the surrounding medium suggest that the lifetimes of the intermediates which react with oxygen may be in the 100 /^sec range (Howard-Flanders, 1958). In our laboratory we are attempting to investigate these lifetimes directly, both in chemical systems and in living cells, by means of a pulsed radiation source and pulsed analytical techniques. The source (Boag and Miller, 1959) delivers 1*5 MeV electrons in jmlses of 2/xsec duration. The dose, which can be delivered in a single pulse, depends on DAMACxE IN AEROBIC AKD ANAEROBIC SYSTEMS 29 the volume of material to be irradiated. Suspensions of bacteria have been exposed to 30 krad (Dewey and Boag, 1059, 1960) and volumes of solution suitable for spectrosco])ic analysis have been exposed to over 100 krad in a single pulse (J. W. Boag and 11. E. Steele, un|)uh- lished). For spectrosco[)ic analysis a light-source has been developed (Boag, 1957), the output from which during a flash of a few micro- seconds' duration, enables absorption spectra to be recorded at medium dispersion. Such records show the reagents present at a given time after a 2 fjLsec exposure to ionizing i^adiation. By means of a photomultiplier, set to record at the absorption peak of a known reagent, the rate of appearance or disappearance of that reagent may be recorded as illus- trated in Figs. 2 and 3. These flgures show certain aspects of the oxidation of a 10~3M FeS04 solution in the presence of OSN H2SO4 and in the presence or absence of NaCl. It will be seeii that the absorption band at 305 m/x character- istic of the ferric ion makes its appearance 20 times more slowly than the absorption (as yet unidentified) at 240 m/x. The values of the times required for half the final optical density to appear are 1200 /xsec and 60/xsec respectively for the two wavelengths. It is evidently quite feasible to carry out kinetic studies for species which occur in the visible and u.v. for lifetimes of this oi'der, or even considerably smaller, with this type of equipment. The apphcation of similar methods to cell suspensions is, of course, very much more difficult on account of non-specific light scattering, but the methods developed by Britton Chance (1952) and others are, in principle, applicable. ESR spectroscopy is likely to prove more widely applicable than light spectroscopy to the study of radical kinetics in cells which have been exposed to ionizing radiation. In order to take advantage of the high dose-rate during the pulse, Dr. Boag is constructing in our Laboratory a spectrometer in which the magnet has an axial hole through which the electrons may enter the resonant cavity. It is hoped that this will make radicals of lifetimes greater than 100 jLisec open to investigation. In general, the relatively slow process of diffusion across the cell envelope limits the speed with which the chemical composition of the intracellular fluid may be changed. In certain respects, oxygen is exceptional, since it is consumed in radiochemical reactions (cf. next section). Loss of reproductive integrity by the bacillus Serratia marcescens Several aspects of the loss of reproductive integrity by Serratia marces- cens have been studied by my colleague, Dr. D. L. Dewey. In all the 30 L. H. GRAY o E o o c i— M— ? o< o ro c CD o O d) ro X) ■•— o D o en 0) (U n. aj ■o c a> o o i_ Q. o O en ;_! r* .^ ^ i« o Q c ■* c o ^^ O c/: *c« 'o -t^ ^ OJ Cj £h o o &< o 3 o ^ c ^ P5 o ^S C3 '-3 o3 > ^ ^ o O Sh 5 ® O, 05 o i CI •=; -n 32 .5 Qju ;5 '^ bD .a DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 31 o o o a. O CO Hi K "A 00 o o CI © a, •- o O . CO t. CO S- o ^ << o '■+3 S-i o cc IJ o o CO lO cc 'O Xi O Oh b. ® o o u O 32 L. H. GRAY experimental conditions which have been investigated so far, the log survival curve is linearly related to dose, except near the origin, where a slight curvature is generally observed when the cells to be irradiated are suspended in a phosphate buffer. In the case of aerobic irradiation at room temperature, the log surviving fraction is linearly related to dose over almost ten powers of ten. Do = 1'7 krad, and the extrapola- tion number is 14:. These vegetative bacteria are thus 25/1*7 (= 15) times as sensitive as spoi*es of B. megaterium irradiated in the dry state under conditions that permit the maximum participation of oxygen. When oxygen is rigidly excluded at the time of irradiation, the sensi- tivity oi Serratia marcescens is reduced by a factor of 4. The sensitivity rises, however, very rapidly with increasing O2 concentration. Assuming the equation projDOsed by Howard-Flanders and Ali)er (1957) to be applicable, viz: S Oo = l = (m-l)- (1) Sn O2 + A where S is the sensitivity, measured by the slope of the log survival curve, at a concentration of oxygen in the medium equal to O2 /iuioles/l, and Sn the sensitivity when oxygen is rigidly excluded, then m = 4 and K = 4/xmoles/l. Both constants are, to a first approximation, inde- pendent of temperature over the range 0 to 30°C. This very marked influence of oxygen on radiosensitivity is almost universally observed with proliferating cells, and the question naturally arises as to whether this influence is mediated through the cytochrome system. A number of circumstances make this improbable. For example, Moustacchi (1958) showed that the sensitivity of several cytochrome-deficient mutants of yeast showed the same oxygen dependence as wild type organisms. In the case of Serratia marcescens, an involvement of the cytochrome system can be definitely excluded by the observation of Dewey and Longmuir (unpublished) that the oxygen concentrations at which respiration begins to decline at room temperature is very much lower than 4/xmoles/l. At temperatures near 0°C it is less than O.l/x mole/1. Sensitivity varies by nearly a factor of 4 over a range of oxygen con- centration within which the. respiration of the cells is virtually inde- pendent of oxygen concentration. The fact that Q02 is maintained approximately constant to much lower oxygen concentrations than 4 /anoles/1 also shows that the intra- cellular concentration of oxygen cannot differ from that of the surround- ing medium by more than a small fraction of the value of K. We may, therefore, safely conclude that, unless the nuclear membrane offers substantial impedance to the movement of oxygen, equation (1) is an DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 33 expression of the effectiveness with which oxygen can compete at different intracelhilar concentrations for reaction with radicals or other intermediates to give rise to loss of re])roductive integrity. Measnrements between 0 and 30 C show no systematic variation of K with temperature and enables us to assign an upper limit of 6 kcal/ mole to the differences between the activation energies of the two re- actions represented by equation (1). If the lifetime of the intermediate in question is longer than the rate constant for diffusion into the cell (estimated at ~ 1 msec) it may be possible to determine the lifetime directly by the use of our ])ulsed source. Figure 4 shows the result of an experiment conducted by Dewey Fig. 0 5 10 15 20 25 30 35 Dose (kilorad) 4. Comparison between pulsed and low dose-rate irradiation of Serrntia tnnr- cescens. (Redrawai, by permission, from Dewey and Boag, 19(iO). O, 2 /xsec. pulse; • , 1,000 rad/min for 0-5 per cent oxygen in the gas phase and Boag (1959, 1960) in which the effects of doses of radiation de- livered at normal dose-rate, and in a single 2 sec pulse, were compared with respect to loss of reproductive integrity. The bacteria were in sus- pension in a medium maintained at an oxygen concentration of about 10 /xmoles/1 by bubbling. Under these conditions the bacteria displayed almost their maximum sensitivity when irradiated at normal dose-rates, but appeared progressively more and more radio-resistant with in- creasing doses above 5 krad when this is delivered in a single pulse. This was interpreted as due to the radiochemical utilization of all the oxygen initially present in the cells by the reactions arising from the 34 L. H. GRAY first 2 to 3 krad of the total dose. Thereafter the cell reacted as an anoxic system. One of the most striking differences between damage induced in cells of high water content and low water content (dry spores and dry seeds) M'hen each is irradiated in the absence of oxj'gen, is that nitric oxide enhances the radiation damage in the former, and depresses it ia the latter. In dry spores of B megaterimn Powers and his co-workers observed less depression when the nitric oxide was present during irradiation than when it was added after irradiation and from this con- cluded that the nitric oxide slightly enhanced the toxicity of some inter- mediates while greatly depressing that of the others. The enhancement was first observed by How^ard-Flanders in Shigella flexneri (1957). As regards the magnitude of its influence on sensitivity at any given con- centration, Howard-Flanders and Jockey (1960) find the molecule of nitric oxide to be essentially equivalent to a molecule of oxygen. After the discovery of this phenomenon by Howard-Flanders, a similar effect of nitric oxide on the sensitivity of Vicia faba roots was observed by Kihlman (1958) : on the sensitivity of tumour cells by Gray et al. (1958) and in human liver cells by Dewey (1960a); (see p. 38). In Serratia marcescens and Proteus vulgaris, Dewey (unpublished) has observed that nitric oxide substantially enhances the sensitivity of anoxic cells, but falls a little short of oxygen in its effectiveness. In each case cells which are exposed to nitric oxide after the end of an anoxic irradiation, show only the normal anoxic sensitivity. Nitric oxide, having one un- paired electron in an outer orbital, readily coml)ines with free radicals to form stable compounds. In this respect it differs from oxygen, which has certain of the characteristics of a bi-radical, so that the product of a reaction between oxygen and a free radical is itself a free radical. H H C-+0.i C-0-0. i 1 H H By virtue of its paramagnetism, nitric oxide, like oxygen, readily catalyses transitions in either direction between singlet and triplet states (Porter and Windsor, 1958). Nitric oxide is also a very effective substitute for oxygen in reactions leading to the production of chromo- some structural damage by visible light absorbed in Vicia faba meris- tem cells which had ju'eviously been exposed to acridine orange (Kihl- man, 1959). Since niti-ic oxide is known to combine rather I'eadily with certain DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 35 haem pigments, including cytochrome oxidase (Keilin, 1055), the possibility of a hiocliemical role cannot be ignored, especially since post -irradiation respiration sometimes has an important influence on radiosensitivity ()). 41). In all the experiments with vegetative bactei'ia, and with normal and malignant mammalian cells referred to in this section, this ])ossibility was taken into account by control experiments in \\ Inch the nitric oxide was added after irradiation. One further investigation with Serratia marcescens, carried out by Dewey (1960b), may be mentioned as possibly indicating the extent to which the radiolysis of water contributes to loss of reproductive integrity in this organism. A number of j^ears ago Burnett et al. (1951) studied the influence of high concentrations of alcohol on the radiosensitivity of E. coli B/r, and these mvestigations were later extended by Marcovich (1958). In re- peating the experiments under controlled oxygen tensions Dewey (1960b) finds: 1. A progressive increase in the 37 per cent inactivation dose D (i.e. a progressive decrease in sensitivity) with increasing glycerine concentrations up to 2M, which conforms to the relation D G — = l + (in-l) Do G + K where Do is the inactivation dose in the absence of glycerine. 2. That when the bacteria are aerobic at the time of irradiation the influence of a given concentration of glycerine is completely inde- pendent of (a) oxygen concentration in the range 14 to 1400 /zmoles/1, and (b) temperature over the range 20 to 37°C (Fig. 5). Under all these conditions jx = 5-25 and K = 0*9 mole/1. 3. Increasing concentrations of glycerine also progressively lower the sensitivity of bacteria irradiated under strictly anoxic conditions. A two-fold depression of sensitivity below the normally anoxic level has been observed, and extrapolation to infinite glycerine concentration indicates a maximum depression of 2-66. The con- stant K was estimated to be 0*8 mole/1, and does not differ significantly from that for aerobic irradiations. Both constants are independent of temperature over the range 20 to 37°C. 4. The influence of glycerine is essentially the same for cells which are irradiated in the presence of nitric oxide as for cells which are irradiated in the presence of oxygen. 5. Equal molarities of ethyl alcohol, glycol and glycerine, which have respectively 1 , 2 and 3 OH groups per molecule, are of com- parable, though not exactly equal, efficiency. 36 L. H. URAY The fact that a given concentration of glycerine is equally effective in the presence of 1400^moles/l oxygen and in the presence of 14 /xmoles/1 oxygen, which is only a little higher than that necessary to D o o 0-8 m/l. f k I 2 3 Glycerine concentration -4 ^ 053 0-47 CO Molar Fig. 5. — Effect of glycerine on aerobic and anaerobic sensitivity of Serratin nuircescens. (Dewey, 1960 a, b) bring the bacteria to their maximum level of sensitivit}^ as far as oxygen is concerned, shows that the mechanism involved is not one in which glycerine and oxygen are competing for a radiation-induced radical. It is fairly clear that the action of the glycerine is antecedent to that at which oxygen enters into the reaction chain. One possibility which is at present under investigation is that, at the molarities in question, the alcohol molecules compete with biologically important molecules for radicals which result from the radiolysis of water. If this hypothesis should prove to be correct, then it would follow from the data presented in Fig. 5 that 0"47/r25 ( = 38) per cent of the anoxic sensitivity ofSerratia marcescens is due to energy deposited in the biological molecules themselves and 62 per cent to the radiolysis of water. For cells in-adiated anaerobically the corresponding figures would be 0'53/4: (= 14 per cent) for energy deposited in biological molecules and 86 per cent for t]ie contribution from the radiolysis of \vater, DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 37 Loss of reproductive integrity by mammalian cells It is now clear that the radiation-induced loss of reproductive in- tegrity by mammalian cells has much in common with the same phen- omenon n\ micro-organisms. o > "> {_ Zi en 001 500 1,000 Dose (rad ) Fig. 6. — Colony-forming ability of human liyer cells after exjiosure to X-rays. (Dewey, 1960 a) Without excej)tion so far, the reported log survival curves are linear for all except the lowest doses and have small extrapolation numbers — mostly ~ 2 and occasionally 4 ~ 6 have been observed. Figure 6 presents the data of Dewey (1960a) for human liver cells cultured and assayed by the technique developed by Puck, and exposed to X-rays under various experimental conditions. Figure 7 presents the data of Hewitt and Wilson (1959) for mouse leukaemia cells grown and exposed to ^OQo y-rays in vivo and tested for the ability of the irradiated cells to regenerate a tumour. The shapes of all the curves in Figs. 6 and 7 are remarkably similar, and for cells irradiated at the same oxygen tension the difference between the slopes of the curves for human liver cells and mouse leukaemia cells is close to that expected on account of the different LET of the radiations to which the cells had been exposed. The ratio of aerobic to anaerobic sensitivity for both types of gell is ~ 2-5. In the case of the human liver cells a sensitivity 38 L. H. GRAY u ^ ' \ -1 - \ ^ • — c \ "* O \ > ^ 4J u o -2 - \ ^^v^Mouse dead - > -3 \ \ N, > \ l_ \ D \ o -4 Mouse alive \ en •X O — 1 -5 -6 i [ 1 1.000 2,000 Dose (r) 3.000 4,000 Fig. 7. — Tumour forming ability' of mouse leukaemia cells after exposure to '■"Co y-rays. (Hewitt and Wilson, 1959) approximately midway between the aerobic and anaerobic levels is observed when cells are in eqnilibrium with a gas phase con- taining 0-25 per cent oxygen (dissolved oxygen = 3-5 jLtmoles/1). Nitric oxide enhances the sensitivity of the human liver cells (Dewey, 1960a) as also that of Ehrlich mouse ascites tumour cells (Gray et al, 1958) to about the same extent as oxygen. The relation between sensitivity and the concentration of oxygen in the immediate vicinity of the Ehrlich tumour cells at the time of irradiation is shown in Fig. 8. Froese (personal communication), while working in our laboratory, investigated the dependence of the respiratory activity of these cells on oxygen tension. He ob- served that respiration was maintained at nearly maximum rate down to concentrations 1 /nmole/l of oxygen in the liquid phase at which radiosensitivity is not much greater than that observed in the complete absence of oxygen. After making full allowance for experimental error in both sets of data it is evident that, as in the case of bacteria, the variation in the sensitivity of the cells with oxygen concentration cannot be ascribed to an influence of the enzymes of the cytochrome system. The tumour cells change their sensitivity in less than 1 sec of transfer from one medium to another of different oxygen tension (Deschner and Gray, unpublished). In mammalian cells, therefore, as in bacteria, we may confi- dently infer that one, two or, at most, a few ionizing particles initiate loss of reproductive integrity, and that the influence of oxygen concentration during an irradiation of short duration DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 39 reflects the chemical properties and lifetimes of intermediates in the reaction chains. On account of the technical difficulties referred to earlier, no lifetimes liave yet been measured directly, but we may infer on o O ex. K 20 40 60 80 100 150 Oxygen concentration fim/l 1,350 10 30 90 -^ i- 50 70 mm Hq Fig. 8. — Influence of oxygen on radiosensitivity 760 kinetic grounds (Howard-Flanders and Moore, 1958) that since the influence of oxygen on sensitivity is represented by equation (l)witliK ~ 5|Limoles/l (Deschner and Gray, 1959; Dewey, 1960a), the species with which oxygen reacts probably have lifetimes of the order of 100ju,sec when no oxygen is present. Although the sensitivity of a cell is apparently unrelated to its respiratory activity at the time of irradiation, the sensitivity of plant cells which have undergone a long period of anaerobiosis (Beatty et al., 1956) or which are anaerobic for periods as short as 1 5 min after the end of a brief iiTadiation sometimes have a different sensitivity from that of cells cultured aerobically through- out. It is now clear that in this case respiration is important as a source of energy for speciflc kinds of metabolism, which will be briefly considered in the next paragrajih. 40 L. H. GRAY The influence of mefabolism on the aerobic and anaerobic sensitivities of cells, ivith special reference to loss of reproductive integrity Witkin (1956) showed that the yield of mutations in certain auxo- trophic bacteria, resulting from exposure to u.v. light, is fixed within the first hour after irradiation at a high or low level by the nutrition of the organisms. Low levels appear to be associated with the inhibition of protein synthesis, and in an extreme case no mutation was observed at all, although the loss of reproductive integrity of the cells was little, if at all, affected. Although loss of reproductive integrity in cells exposed either to u.v. light or to X-radiation has not been entirely prevented by any nutritional variants yet tested very marked changes in the slope, and sometimes also in the extrapolation number, of log survival curves have been obtained both by the use of chloramphenicol (Gillies and Alper, 1959) and by cultivation at reduced temperature (Stapleton et al., 1953) after irradiation. The shape of the log survival curve may also be greatly modified by control of the pH of the medium in which the cells were cultured before irradiation (Hollaender et al., 1951 ; Stapleton and Adler, personal communication) and in other ways. As a general rule (Alper, 1961), maximum loss of reproductive integrity occurs if cells are caused to grow fast immediately after irradiation by the use of optimal media and optimal temperatures : conversely, more cells survive when gro^^■th is slowed or protein synthesis depressed after irradiation. The yield of interchanges when plant cells are exposed to spaced doses of radiation is increased by the inhibition of respiration or chloram- phenicol treatment between the two irradiations (Wolff, 1959). Nu- tritional control for a period of about an hour before an anoxic irradia- tion may change the yield of interchanges produced in plant cells by a factor of seven (Beatty and Beatty, 1960): This is equivalent to changing the dose by a factor of about 3 since interchanges vary roughly as the square of the dose. Anaerobic metabolism was found to increase the yield, and aerobic metabolism, or the addition of ATP to cells which had been growing anaerobically, to depress the yield. The interpretation of these observations is in the province of radia- tion biochemistry, and beyond the scope of this paper. If it be granted that the nutritional control of radiation damage in some way concerns the replacement or repair by normal metabolic processes, of key mole- cules in the DNA-RNA-protein class, which have been damaged through ionization, excitation and radical formation, then the question may be asked whether or not the extent to which damage may be re- paired is the same for all chemical j)athways by which the damage was produced. Alternatively, we may ask if damage which is produced by DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 41 diflferent chemical pathways is equally reparable by a given form of metabolic control. There is now abundant evidence to show that it is not. An extreme example comes from recent observations of Laser (1960) that starvation oi Pseudomonas for 48 lir after irradiation com- pletely eliminates damage which is oxygen dependent, but leaves un- changed the damage which does not involve the participation of oxygen in the chemical chain. Alper and Gillies (1958; 1960a, b) have recently made a special study of this problem using loss of reproductive integrity in E. coli B, irradi- ated aerobically and anaerobically, as test material for the influence of post-irradiation nutritional factors, Alper (1961) finds that the ratio of the sensitivity of organisms irradiated in oxygen and in nitrogen (i.e. the constant m of equation (1)) is approximately linearly related to the value of Do observed for organisms that have been irradiated anoxically , irrespective of the culture condition used to control Dq. Alper estimates that whereas m = 3-6 under conditions that give maximum injury (no "restoration") m = 1-5 under conditions that give minimum injury (full "restoration"). In other organisms m may be unaffected by the conditions of culture. The more usual case, however, appears to be of the kind investigated by Alper and Gillies, and we have to consider that chemical control and metabolic control are interrelated variables as regards radiation induced loss of reproductive integrity. REFERENCES Alper, T. (1961). Int. J. Bad. Radiol. 3, 369. Alper, T., and Gillies, N. E. (1958). J. gen. Microbiol. 18, 461. Alper, T., and Gillies, N. E. (1960a). In "Immediate and Low Level Effects of Ioniz- ing Radiations," p. 305. Taylor and Francis, London. Alper, T., and Gillies, N. E. (1960b). J. gen. Microbiol. 22, 113. Baylor, E. R., and Smith, F. F. (1958). Radn Res. 8, 466. Beatty, a. v., and Beatty, J. W. (1960). Proc. nat. Acad. Sci., Wash. 46, 1488. Beatty, a. v., Beatty, J. W., and Collins, C. (1956). Amer. J. Botany, 43, 328. BoAG, J. W. (1957). Proc. 2nd Congr. Int. Fotobiologia, Turin, p. 109. BoAG, J. W., and Miller, C. W. (1959). "Proc. 2nd Int. Conf. Peaceful Uses of Atomic Energy, 1958," p. 437. Pergamon Press. London. Burnett, W. T., Stapleton, G. E., Morse, M. C, and Hollaender, A. (1951). Proc. Soc. exp. Biol. N .Y . 77, 636. Caldecott, R. S. (1960). Pro. Symp. "Effects of Ionizing Radiation on Seeds and their Significance for Crop Improvement." Karlsruhe. IAEA. In press. Chance, B. (1952) Nature, Lond. 169, 215. Clegg, R. E. (1957). Radn Res. 6, 469. Davidson, D. (1960). In "Radiation Protection and Recoveiy", p. 175. (A. Holl- aender, ed.) Pergamon Pi'ess, London. Davis, M., and Hutchinson, F. (1952). Arch. Biochem. Biophys. 39, 459. Deschner, E. E.. and Gray. L. H. (1959). Radn Res. 11, 115. Dem'ey, D. L. (1960a). Nature, Lond. 186, 780. Dewey, D. L. (1960b). Nature, Lond. 187, 1008. Dewey, D. L., and Boag, J. W. (1959). Nature, Lond. 183, 1450. 42 L. H. GRAY Dewey, D. L., and Boag, Z. W. (1960). Z. NaturJ. 6, 372. Ehrenberg, L., (195.5). Bot. Notiser 108, 184. Gillies, N. E., and Alper, T. (1959). Nature, Lond. 183, 237. Gray, L. H.. Green, F. O., and Hawes, C. A. (1958). Nature, Lond. 182, 952. Hewitt, H. B. and Wilson, C. W. (1959). Brit. J. Cancer 13, 675. Hollaender, a., Stapleton, G. E., and Martin F. L. (1951) Nature, Lond. 167, 103. Howard -Flanders, P. (1957). Nature, Land. 180, 1191. Howard -Flanders, P. (1958). Advanc. biol. med. Phys. 6, 553. Howard -Flanders, P., and Alper, T. (1957). Radn Res. 7, 518. Howard -Flanders, P., and Jockey. P. (1960). Radn Res. 13, 466. Howard-Flanders, P. and Moore, D. (1958). Radn Res. 9, 442. Keilin, J. (1955). Biochem. J. 59, 571. KiHLMAN, B. A. (1958). Expl. Cell. Res. 14, 639. KiHLMAN, B. A. (1959). Nature, Lond. 183, 976. KoNZAK, C. F., XiLAN, R. A., Legault. R. R., and Heiner, R. E. (1960). Proc. Sijnip. "Effects of Ionizing Radiation on Seeds and their Significance for Crop Improvement." In press. Laser, H. (1960). Brit. J. Radiol. 33, 341 (Abstr.). Lea, D. E., and Salaman, M. H. (1942). Proc. ray. Soc. B123, 1. LiPETZ, L. E. (1960). In "Immediate and Low Level Effects of Ionising Radiations," p. 227. Taylor and Francis, London. Marcovich, H. (1958). In "Organic Peroxides in Radiobiology", p. 117. (M. HaissinskIy, ed.) Pergamon Press, London. MousTACCHi, E. (1958). Ann. Inst. Pasteur, 94, 89. NiLAN, R. A.. KoNZAK, C. F., Legault. R. R.. and Harle, J. R. (1960). Proc. Symp. "Effects of Ionizing Radiations on Seeds and their Significance for Crop Improve- ment." IAEA. In press. Platzman, R., and Franck:, J. (1958). In "Information Theory in Biology" p. 262. Pergamon Press. London. Porter, G., and Windsor, M. W. (1958). Proc. roy. Soc. A 245, 238. Powers, E. L., and Kaleta, B. F. (1961). In press. Powers, E. L., Ehret, C. F., and Bannon, A. (1957). Appd. Microbiol. 5, 61. Preer, J. R. (1948). Amer. Nat. 82, 35. Sparrman, B., Ehrenberg, L.. and Ehrenberg, A. (1959). Acta chem. scand. 13, 199. Stapleton, G. E., Billen, D., and Hollaender, A. (1953). J. cell, conip. Physiol. 41, 345. WiTKix, E. M. (1956). Cold Spring Harbour Sipnp. quant. Biol. 21, 123. Wolff, S. (1959). Radn Res. Suppl. 1, 453. Wolff, S. (1960). Proc. Synip. "Effects of Ionizing Radiation on Seeds and their Signifi- cance for Crop Improvement." IAEA. In press. ZiMMER, K. G. (1960). "Studien zur ciuantitativen Strahlenbiologie." Akademie der Wissenschaften und der Literatur. DISCUSSION EiDus : At what doses was there a bend in the dose curves in the experiments under anaerobic conditions ? GRAY: The Hnes were straight throughout. EIDUS : I would like to point out that in well-known experiments by Dr. Hollaen- der on E. colt the dose curve under anaerobiosis after the initial bend runs, over several orders of magnitude, parallel to the first curve characteristic for the aero- bic conditions, whereas in your exiaeriments there is always a divergence of the curves. What is the explanation for this difference in the results? GRAY : For the cell suspensions we have used, the form of the curves may differ slightly, but in general they are very similar. DAMAGE IN AEROBIC AND ANAEROBIC SYSTEMS 43 tumerman: I would like to i^oint out the possibility that the important role of the water in radiobiological effects is due not only to the formation of the i-adio- lysis i^roducts, but also to the fact that water has a structure, damage to which may contribute to the formation of the long lived triplet states. passynsky: Probably with the aid of the micro-impulse technique Dr. Gray used it would be possible to determine the minimum time for the interaction of oxygen Math the irradiated substance, which ought to depend on oxygen pressure. ALEXANDER : I caniiot agree with Dr. Gray that dose-response curves can be used to give quantitative information concerning the nature of the initial lesion in cells. There is now abundant evidence that every type of radiation lesion is capable of restoration after irradiation and that the magnitude of this restora- tion can be altered experimentally. Even if no special steps are taken to effect restoration, some is always taking jolace following irradiation. Moreover, it seems highly probable that radiation is also affecting the repair mechanism and this cannot therefore be assumed to introduce a constant factor which merely affects the size of the target calculated. The probability that ionization will produce a l^articular lesion is thus not governed only by "target size" considerations, but by a whole host of post-irradiation metabolic factors quite apart from protection and energy transfer phenomena which smear out the size of the target. In this complex chain of events, there would seem to be no direct or constant relation- ship between the end-effect and the initial act of ionization such as is necessary for the interpretation of dose-response curves along the lines you indicated. The interpretation of the general shajae of the curve in terms of multiplicity of events also seems to be unjustified since the effect of irradiation on the effect- iveness of repair may well cause a curve to steepen progressively or to make an otherwise hyperbolic curve appear linear. I believe that it is quite likely that exponential dose-response curves of the type you showed for bacteria are the consequence of several factors acting in conjunction. This view derives support from the important work of Hollaender in which he showed that the shape of the dose-response curve of bacteria could be altered drastically by small changes in experimental conditions. gray: I cannot understand why the fact that different dose-response relations are observed under different metabolic conditions should be a ground for reject- ing an interpretation of each of these dose-response relations in terms of the nmnber and probability of the initiating events which are radiobiologically significant vmder the respective metabolic conditions. In my view the dependence of dose-response relations on metabolism is, in part, a natural consequence of the facts mentioned in the earlier part of Dr. Alexander's remarks, namely that many of the injuries sustained by cell com- ponents are repairable under certain metabolic conditions, but not under others. In part, it is probably a reflection of the fact that the significance, for the chosen criterion of biological damage, of an injury sustained by a particular organelle will depend on what metabolic activities the cell has to engage in after iiTadiation e.g. whether or not it has to adapt to a set of culture conditions different from those under which it was grown previously to irradiation. Propei'ly used, the dose-response relation can be a valuable guide to the investigation of the mech- anisms which underly the modification of biological response by post-ii'radiation metabolism. By a dose-response relation I mean a curve, or its mathematical expression, 44 L. M. ClRAY which gives a satisfactory fit to the experimental observations. The reUance which may be placed on this expression is detei'mined by the accuracy of the fit and the range of dose over which the fit is maintained — as discussed fully by Zimmer ("Studien zur quantitativen strahlenbiologie"", Steiner Verlag, Wies- baden, 1960) and also by Dittrich ("Trefermischkurven"' Z. NaturJ. 15b, 261, 1960). As explained by Lea, a given dose-response relation may, in jDrincijile, be interjDreted either as an expression of the number of ionizing particles concerned in the initiation of the injury, or as representing a sensiti\ity distribution of the cell population to equal doses. When the form of the dose-response curve is simple, embodying a linear relation between the logarithm of the survivors and dose and a small extrapolation number (intercept between this line and the zero dose axis), I have no hesitation in choosing the former interpretation and inferring a low multiplicity of initiating events. To intei'pret a relation such as that which Dr. Dewey has observed for the proportion of survivors among an irradiated pojDu- lation o{ Serratia marcescens (shown as a lecture slide but unpublished), which is linear with dose over 9 powers of 10, in any other way seems to l^e luu-easonable. From the slope of the linear portion of the dose-response curve we obtain directly an estimate of the chance that at least one of the i3articles set in motion when a cell is exposed to unit dose of radiation will initiate the chain of events which lead to the biological effect under consideration. By the methods described in detail by Lea, and with the limitations pointed out by Lea, a knowledge of the slopes of the dose-response curves for different types of ionizing radiation can yield approximate information as to the size and shape of the critical organelle. The purpose of a theory is to jDrovide a basis for fiu'ther experiments. If, having exercised due caution in deriving the best dose-response relation from the experi- mental data, we refuse to make the most obvious inference, we throw away a valuable means of deciding which, aniong the multitude of conceivable mechan- isms, are the ones most worthy of fiu'ther investigation. ACTION OF RADIATION ON PROTEINS AND NUCLEIC ACIDS IN SOLUTION AND AT INTERPHASES A. G. PASSYNSKY A. N. Bach Institute of Biochemistry, U.S.S.B. Academy of Sciences, Moscoiv, U.S.S.R. SUMMARY Measurement of the oxidation of the -SH groups of proteins, the linkages of 33S-methionine, the radiation destruction of DNA and many other methods allow us to establish the presence of molecular changes in thousands of irradiated molecules of proteins even for a dose of 400 to 500 r and in nucleoproteins for 20 to 50 r. However, the application of the statistical theory of the action of radiation shows that the primarily irradiated volumes in the cell only have the dimensions of a few molecules. When establishing the mechanism of biological "amplifi- cation" of the action of radiation it is necessary to take into accovmt the par- ticular significance of damage to the molecules of the biological macromolecules which enter into the composition of the intracellular surfaces of the section (cytoplasmic and nuclear membranes) and also the properties of the living cell as an open system for which the alteration of the transfer constant is of great significance. It has been shown that the appearance of chemical "cross-links" in monolayers of DNA substantially destroys the structure and increases the area of the monolayer, and radiation damage to the thin siu-face layers of RNA which separate the enzyme from the substrate (peroxidase-ascorbic acid, H2O2) leads to considerable acceleration of their interaction. The variation caused by the damage of only several molecules in the surfaces of the section may be a source of all the subsequent biochemical disruptions and radiation damage to the living cells. Living cells and organisms are constantly interchanging matter and energy with the surronnding medium, i.e. they form so-called "open systems". Therefore the theory of the action of radiation on living organisms must take into consideration the general properties of reactions in open systems. According to the theory of open systems (Burton, 1939; Denbigh et al., 1948; Passynsky, 1957a) stationary concentrations of the components are determined not only by the rate- constants of chemical reactions, as in closed systems, but also, to the same extent, by the constants of the transfer of matter in the process of free diffusion or of penetration through membranes. Consideration of the constants of transfer, along with the coefficients of reaction rates is an essential feature of the theory of open systems which reveals the 45 46 A. G. PASSYNSKY indivisible relation between the spatial and temporal organization of metabolic processes. The action of radiation on open systems, like that of other extex'nal factors, consists, in general form, in a disturbance of the stationary state of the system which, within a definite range of changes, can be compensated by the open system, while for more extensive changes it becomes impossible to estalilish a ncAv stationary state and the system is degraded. The specific peculiarity of the biological action of I'adia- tions, however, when compared with other jihysical factors, is their ability to induce considerable physiological disturbances or even the death of the organism by small amounts of energy. The lethal dose for mammals (500 to 600 r) is known to correspond to the absorption of energy which, by heat equivalent, would cause a temperature rise of only some 0-002°C. But most of even this small amount of the absorbed energy does not have a lethal effect. For example, with a dose that pro- duces an average of about 1,000 ionizations in each yeast cell, only some 10 per cent of the cells perish. It is beyond any doubt that so far as the effect of radiation is concerned the total amount of either chemically or structurally changed polymer molecules — such as proteins, nucleic acids — per cell is many times greater than that of changed molecules which effectively determine radiation damage to the cell. By means of trivial physico-chemical methods (viscosity, electro- phoresis, solubility, absorption spectra, etc.) the action of radiation on protein solutions can be detected only with very heavy irradiation doses, (75,000 to 100,000 r and higher). A new, isotopic method for the investigation of protein changes on radiation, has been developed in our laboratory. This is based on the ability of irradiated protein to show an increased binding of some organic substances, including 35S-labelled methionine (Passynsky et al. 1955). The high sensitivity of the isotopic procedure has enabled us to lower considerably the threshold of the observed action of radiations. In most experiments 30 mg of protein was dissolved in 1 ml of borate buffer (M/15, pH 8-5), and 0-5 ml. of an aqueous solution of 35;^- methionine (about 30,000 to 50,000 counts per min per ml of the mixture) was added to the solution. After irradiation the protein was precipitated with an equal t^ohime of 10 per cent TCA, and the pre- cipitate washed on a filter 50 times with a mixture of equal volumes of 10 per cent TCA and borate buffer; the constancy of the specific activity of the precipitate served to control the procedure. Thereafter the precipitate was dried and its activity measiu'ed on an end-window counter (evaluated per 10 mg of dry protein precipitate). It was shown in this way that appreciable molecular changes in protein (in 0-1 per PROTEINS AND NUCLEIC ACIDS IN SOLUTION 47 cent solution) could be revealed after as small an irradiation dose as 400 to 500 r. The isoto])ic procedure was also a|)])lied to the detection of changes in serum albumin and serum globulin after y-irradiation or after mixed neutron irradiation in a nuclear reactor, as well as to the X-irradiation of lipoproteins isolated from horse serum (Volkova and Passynsky, 1955). The data obtained are given hi Table I. Table I. Thresholds of detect able radiation action Substances Irradiation Thresholds in thousands of r by trivial phys.-chem. by isotopio method methods (viscosity, elec- trophoresis, etc.) Human serum albumin Lipoproteins (from horse serum) Nucleoproteins (from the thymus) j^-rays y-rays Neutrons X-rays 75 to 100 100 250 1,000 005 0-4 to 0-5 5 5 50 Thus, the threshold of the action observed could be lowered 20-fold for lipoproteins and up to 200-fold for proteins. When irradiating a high molecular weight nucleoprotein isolated from the thymus (N/P 3-3; 0-2 per cent solution in 1 M NaCl), no iso- topic measurements were taken, since an appreciable radiation action was observed with as small a dose as 25 to 50 r by the decrease of the relative viscosity (Table I). At this dose the decrease of the relative viscosity was 4-5 per cent, while after 500 r it was 20 per cent (Volkova and Passynsky, 1955). Thus, by means of sufficiently sensitive methods, the presence of definite molecular changes in the main classes of biological polymers can be revealed even with sublethal doses, with 400 to 500 r for proteins and 25 to 50 r for nucleoproteins. This result thus deprives the nucleic acids and nucleoproteins of the peculiar position ascribed to them by various theories of the biological action of radiations where these sub- stances appeared to be the sole well-founded target for the action of sublethal doses of radiation, while other substances appeared either as inert media or as objects of secondary changes in the organism. On the conti-ary, protein molecules undergo change after these small doses, and it is evident that primary changes in cellular materials must be of a sufficiently general and wide-spread character. Hundreds and thousands 48 A. G. PASSYNSKY of ionizations arising in the cell at even comparatively Ioav irradiation doses certainly do not pass without trace but remain in the cell in the form of hundreds of changed or damaged molecules of various sub- stances, including such important ones as nucleic acids and nucleopro- teins, proteins, enzymes, etc. One of the main types of chemical change in protein molecules on irradiation seems to be the oxidation of SH-groups. In the work with Pavlovskaya (1956) we showed that the action of X-rays on 2 per cent aqueous solutions of crystalline egg albumin, gives an oxidation of SH-groups with an ionic yield of 1:1. The same result was obtained when dry preparations of crystalline egg albumin were irradiated ; the total amount of oxidized SH-groups was 6 x lO^^ per g protein, while the ionization number for the irradiation dose of 5 x lO^r was about 8-5x1018 (Passynsky and Pavlovskaya, 1960). With a 20 per cent protein solution (assumed conditionally in the form of egg albumin) in the protoplasm the lethal dose of 600 r may lead to the oxidation of only 1 SH-group per 3,000 molecules. When taking into consideration the distribution of ionizations within the cell it may be assumed that the j3roportion of chemically changed protein molecules in the cell can hardly be greater than 0-01 per cent of the total number of j^rotein molecules. Similar results are obtained, according to available data, for other chemical changes in protein molecules, such as the breakage of peptide bonds, deamination, etc. when doses of the order of 600 to 1,000 r are used. It seems that the transition of a great number of protein molecules to the excited or activated state at the expense of the absorbed radiation energy, which is accompanied by a re-grouping of some of the links in polypeptide chains and by corresponding structural changes, is of a considerable importance. These changes result in the increase of the aggregation of particles, decrease of solubility, activity augmentation of functional groups of protein, revealed, in particular, in the experiments described above by the increase of ^sg -methionine binding. It should be noted that the effects of the oxidation of SH-groups in protein molecules (in which these groups are the most radiosensitive areas) can hardly be explained by the formation of organic radicals. On irradiation of dry trypsin with fast electrons or y-rays in oxygen, Alexander (1957) observed an increase of inactivation which he inter- preted by the formation of -RO2 radicals from the primary -R ones, without, however, quantitatively analysing this suggestion. In our work (Passynsky and Pavlovskaya, 1960) the number of oxidized SH- groups and that of radicals measured by the ESR method was com- pared for one and the same preparation of dry egg albumin under the PROTEINS AND NUCLEIC ACIDS IN SOLUTION 49 same conditions of y-irradiation in vacuo. It appeared that up to 45 SH-groups were oxidized per radical on irradiation in vacuo. On irradiation in air this discrepancy increases, since the number of peroxidized radicals (after the addition of O2), according to Alexander, is equal to that of primary radicals, while the number of oxidized SH- groups under these conditions increases twofold. This quantitative dis- crepancy is so considerable that the direct oxidation of SH-groups by radiation can hardly be explained by the formation of free organic radicals through the breakage of valency bonds, since in this case a closer correspondence between them and the number of oxidized SH-groups should be expected. The suggestion of Alexander (1957) on the role of molecular O-i" ions in the oxidative effects observed seems to be more likely. However, it may be expected that lethal or sublethal radiation doses (500 to 1,000 r) will lead to the appearance in the cell of hundreds or thousands of protein or nucleic acid molecules changed to a greater or lesser degree in chemical or structural respects. Moreover, the primary inactivated volumes in the cell calculated according to statistical theory of the action of radiation have in many cases the size of only one or several molecules. These calculations are usually made on the grounds of the so-called "target-theory" which, in its physical basis, gives just the method of calculating the value of some inactivated volume with respect to various conditions of radiation (kinds of radiation, intensity of the dose, etc.) without defining the nature of this volume. Within this range the application of this theory does not have serious objections. In a paper by Pollard and his co- workers (1955) it was shown that statistical theory enabled one to determine directly by means of radiation the molecular weight of enzymes, hormones, viruses, etc., in a good agreement with the results of other experimental procedures. In our laboratory similar results have been obtained for preparations of lipoxidase (Budnitzkaya et al., 1956) and insulin (Volkova et al., 1957). Thus the fundamental question of radiobiology is why does the damage to only a fcAv molecules or of some cell portion of only mole- cular size lead to the death of the cell, while damage to hundreds of similar molecules in the cell produces no lethal effect, or, from the point of view of the theory of open systems, how can damage to a few molecules bring about essential disturbances in the stationary state of the cell? The investigation of these questions was mainly directed in the litera- ture to the establishment of specifical structural changes arising after irradiation of the molecules of such biochemically important substances 50 A. G. PASSYNSKY as proteins, nucleic acids, lipids, corresponding proteins, etc. Despite the significance of this work, it should be noted that in essence they did not take into consideration the specificity of cell structures and the complicated heterogeneity of the internal structure of the protoplasm. Primary mechanisms of action of radiation were often analysed as if the cells were just a homogenous solution of various substances but in fact it is the complexity of the intracellular structure that underlies the qualitative peculiarities in the action of radiation on the living cell in comparison with its action on isolated cell substances and separates true radiobiology from radiation chemistry of complex molecules. Current theories of radiobiological action associate the effect of the action of radiations with the damage of either nuclear structures (chromosomes), or microscopic cytoj^lasmic structures (mitochondria microsomes, etc). Chromosomes being unique cell structures, the first group of theories makes it possible to explain the death of cells with a "target" volume of molecular size, and the genetic effect of radiation, but it does not allow for physiological changes in cells after radiation and for many other radiobiological phenomena. Considerable dependence of the number of chromosomal aberrations on the dose intensity, temperature and kind of irradiation, as well as many instances of their deviations from the exponential, show that they themselves may be of a secondary character (Bacq and Alexander, 1956). The second group of theories (Bacq and Alexander, 1956) attributes a major role to the damage of structurally-conjugated enzymic systems in mitochondria, microsomes, etc. These theories are also of certain interest ; they encounter difficulty, however, in the fact of the multiplicity of mitochondria and microsomes in the cell. A sharp increase of radioresistance in polyploid cells, the chromosome number of which is only two to four times that of the normal, shows that in the presence of scores or hundreds of parallel functioning structures it is in fact very difficult to explain the high sensitivity of cells to the action of radiations by the damage of several molecules in one of these struc- tures. One of the attempts to take into consideration the influence of intracellular structure in the light of the theory of open systems is the elucidation of the possibility of destroying the stationary state in the cells through essential change in diffusion parameters of the cell brought about, however, by damaging a small number of molecules. It is evident that the cause of such an effect cannot consist in the destruction of observed structures formed by thick polymolecular membranes or films, since the destruction of several molecules in them would not essentially change their coefficient of permeability. It would seem that breaks in the structure of thin monomolecular or bimolecular PROTEINS AND NUCLEIC ACIDS IN SOLUTION 51 intracellular interphases in which a destruction of but one or two mole- cules can directly change the permeability of the layer, are more likely. It is w^ell known that the proto])lasm is characterized by a consider- able heterogeneity and multiphase nature of its iimer structure. At the interphases of media of different composition thin molecular layers necessarily arise which are oriented at the interphase and contribute to the spatial organization of metabolic reactions. The presence in the protoplasm of some inner interphases (ergasto- plasmic membranes) forming in the protoplasm a number of internal zones or areas which sometimes sharply differ in pH or redox-potential values may be of great imj)ortance for the sequence of metabolic reactions. In this case damage to only some molecules in the bimolecular interphase switch on a chemical potential difference of much larger volumes or of relatively considerable numbers of components of border- ing media, similar, for example to the situation in which a small hole in a dam can lead to the ultimate levelling of the very large water masses on both sides of it. The role of the change in the permeability of mem- branes has already been stressed by Bacq and Alexander (1956), but these authors meant mainly the membranes of mitochondria, while, in our opinion (Passynsky, 1957b), an essential change in the constants of the transfer in the cell as a whole which is required by the theory of open systems should rather be expected when one takes into consideration the molecular heterogeneity of the structure of the protoplasm. A dis- turbance of the normal sequence of substrate transfer in a number of enzymic conversions with a possible switching off of a number of re- actions at once can be of a more specific or unique character than the damage of one or two enzyme molecules in one of the many hundreds of mitochondrial particles each of which contains hundreds of enzyme molecules. The mathematical formulation of the target theory thereby pre- serves its significance both for the case when the primary inactivated volume actually corresponds to the size of molecules, and those cases when it corresponds to the size of a membrane (for example, in the work of Zirkle and Tobias (1956), /• = 800 A, a = 0-26-2-1 A). At the same time a direct relation between the inactivated volume and meta- bolic processes and the physiological state of the cell is evident. From the same viewpoint some other fundamental problems of radiobiology can be interpreted (direct and indirect action, analogy with radiomimetic substances, activation of some enzyme systems on irradiation, influence of different kinds of radiation, etc. (Passynsky, 1957b) . X-ray structural investigation of thin films (300-400 A) of crystalline 52 A. G. PASSYNSKY polymers, e.g. polyethylene, irradiated with a beam of fast electrons (Karpov and Zverev, 1955) was carried out. The formation of but one chemical bond or "cross-link" between molecular chains as a result of contraction of the distance between them from 4-5 A to 1-54 A was shown to bring about a disturbance of the regularity of structure at a distance of scores of atomic groupings as a result of the transmission of tensions arising along molecular chains. Formation of double-bonds in an iiTadiated polymer acts in the same fashion. It was pointed out by these authors that a relatively small number of strong distortions of the lattice in polyethylene crystals as a result of radiochemical reactions in some 1 per cent of polymer links results in a conversion of the bulk of the substance from the crystalline to the amorphous state. In our work with Tongur (196U) the action of transverse "cross-links" in a monomolecular layer of DNP (deoxyribonucleoprotein) on the structure of the monolayer was studied. The DNP monolayer was obtained on a Langmuir balance by applying microdrops of an 0-04 per cent DNP solution (mol. weight 4-5x106 and 8 x 10^; N/P-3-8) in 1 M NaCl to a 38 per cent aqueous solution of (NH4)2S04. This lower phase was chosen in order to decrease the solubility of the nucleoprotein api^lied to facilitate the conditions of its spreading. The thickness of the DNP monolayer was about 23 A. Formation of transverse "cross- links" was achieved by the addition of 2 per cent formaldehyde to the lower phase. It was shown that without formaldehyde in the lower phase pressure-area curves which characterize the mechanical properties of the monolayer were rather constant for native and degraded (down to M = 20,000) DNP preparations, while the value of the monolayer area was, respectively, 0-36 and 0-30 m^/mg (some decrease of the area is due to the dissolution of a portion of the most altered molecules in the lower phase). On the contrary, in the jjresence of "cross-links", i.e. in the lower phase with formaldehyde, stable distortions of the monolayer structure arise, accompanied by an appreciable increase in the area/mg. For example, for native DNP the area of the monolayer increases from 0-34 to 0-43 m2/mg, for the treated DNP from 0-26 to 0-36 m2/mg; the monolayer being compressed, it remains in a somewhat expanded state. This result, which is very similar to the data already mentioned for thin films of polymers, shows that radiation-induced formation of "cross- links" between polynucleotide chains in DNA and DNP monolayers leads to stable distortions of monolayer structure which, by their very nature, must result in a change in the permeal)ility of these thin layers. The influence of this factor on the course of enzymic reactions was studied by Passynsky and Volkova in the following model system, A preparation of crystalline peroxidase, in the form of a fine dry powder, PROTEINS AND NUCLEIC ACIDS IN SOLUTION 53 was suspended at 4 to 5°C in 0-1 per cent RNA solution (Merck prepar- ation, M = 26,000) in acetate buffer (0-1 M; pH3-7). Under these condi- tions each grain of enzyme powder was covered with a thin surface layer of ribonucleoprotein (RNP) which enveloped the grain and inhib- ited its further dissolution. The suspension thus obtained was centrifuged and the supernatant poured off and replaced by the same buffer, the suspension was then stirred again and the process repeated two or three times to wash away all the excess RNA. The pure enzyme suspension in acetate buffer thus obtained was stabilized by a thin surface layer of RNA with particles of a diameter of 0-4 to l-O ir (Fig. 1). The nitrogen and phosphorus contents were determined. The N/P ratio in the pre- cipitate of the suspension was 6-5; since all the phosphorus belonged to RNA, while the nitrogen contriljuted 13-2 per cent in protein and 14-5 per cent in RNA, then from the values of N and P the RNA y Fig. 1 54 A. f!. PASSYNSKY content could be calculated and the thickness of its layer on the par- ticles. For example, with a mean diameter of particles of 0-4/t the thickness of the surface RNA layer was 160 A (Fig. 2) or about 8 RNA monolayers. A known amount of ascorbic acid and H2O2 in equimolar ratio was RNP — I6OA Substrate — Fig. 2 t(min) Fig. 3 added to half of the enzyme suspension stabilized by the surface RNA layer and the kinetics of enzymic oxidation of ascorbic acid were deter- mined on the contact between the enzyme and the substrate through PROTEINS AND NUCLEIC ACIDS IN SOLUTION 55 the thin separating RNA layer (Fig. 3, curve 1). The second half of the enzyme suspension in acetate buffer was exposed to X-irradiaton with various doses; thereafter the substrate was added to this half of the suspension and the rate of enzymic oxidation of ascorbic acid through the RNA layer damaged by radiation was determined in the same way (Fig. 3, curve 2). In the second case the oxidation of ascorbic acid always proceeded faster. The activity of pure aqueous solutions of peroxidase changed but little with the doses of irradiation used, (28,000 to 70,000 r); besides, inactivation of the enzyme could only lead to the slowing down of the rate of ascorbic acid oxidation but not to an increase in this rate. In control protein RNA suspensions, where the enzyme preparation was rej^laced by human serum albumin or on mixing RNA and ascorbic acid solutions, radiation did not alter the course of spontaneous ascorbic acid oxidation (Fig. 4), very much unlike 50 t (mm) Fig. 4 100 the course of the curves in Fig. 3. Therefore, it seems that the accelera- tion of the enzymic oxidation of ascoi'bic acid in irradiated peroxidase- RNA suspension could be regarded as a result of an increased perme- ability of the damaged thin RNA layer separating the enzyme and the substrate, and of an acceleration of the diffusion stage of enzymic reaction. Applying as the measure of acceleration of enzymic reaction the ratio of the areas of curves 2 and I in Fig. 3 (and in similar experi- ments with other doses) one can plot a further curve (Fig. 5) characteriz- ing the dependence of the effect observed on the dose of radiation. The dotted part of the curve in Fig. 5. corresponds to scattering of points (about 7 per cent) in the control, in various unirradiated enzyme suspensions. Thus, the dose of about 18,000 r can be seen to be the thres- hold of the experimentally observable effect. It may seem that this threshold lies above the doses of biological importance; it should be taken into consideration, however, that in the suspensions tested the 56 A. G. PASSYNSKY surface layer consisted of 8 molecular layers of RNA, i.e. of those where scores of damages can arise in various places attenuating the total effect of the permeability increase which is undoubtedly of a statistical character. In a mono- or bimolecular layer each damage must consider- ably change the permeability of the layer ; therefore it can be expected (J a. 30 20 10 20,000 40,000 60,000 D Fig. 5 that the effect discussed can take place in mono- and bimolecular layers directly wdthin the limits of biologically important doses. When obtaining enzyme-substrate systems with finer molecular separating layers of different nature, a considerable lowering of the threshold of the action observed can be expected. At any rate, from the viewpoint presented, which was developed in detail earlier (Passynsky, 1957b), it is evident that the key, specific importance of certain damage to molecules in the cell can be explained not by some unique peculiarities of their structure but by their j)artici- pation in molecular interphases. In the light of the theory of open systems the damage of a few molecules in cell interi^hases can lead to a considerable change in the constants of transfer and in the destruction of the organization of biochemical processes in the cell even in those cases when the bulk of enzyme and substrate molecules remain intact (for example, in the action of sublethal doses of radiation). It should be noted that similar conclusions were drawn in our laboratory from the study of enzymic oxidative processes in living objects, in leaves of various plants (Budnitskaya et al., 1956; Passynsky and Vyrovetz, 1959). The source of biological strengthening of the action of radiation can thus reside in the sjjecificity of the fine intracellular structure. It seems that this factor may play an important part in the mechan- ism of biological strengthening of the action of radiation and. tlierefore, in the theory of the biological action of radiation. PKOTEINS AND NUCLEIC ACIDS IN SOLUTION 57 REFERENCES Alexander, P. (1957). Kadn Res. 6, 653. Bacq, Z. M., and Alexander, P. (lO.K)). "Fundamentals of Radio])iology", ]^i). (i9-71, 1S5-187. Pergamon Press, London. Burton, A. (1939). J. cell. comp. Physiol. 14, 327. BuDNiTZKAYA, E., BoRisovA, I., and Passynsky, a. (1956). Biochemistrij, Leningr. 21, 702. Denbigh, K., Hicks, M., and Page, F. (1948). Trans. Faraday Soc. 44, 479. Karpov, v., and Zverev, B. (1955). "Collected Papers on Radiation Chemistry", p. 215. Academy of Science, U.S.S.R. Passynsky, a. (1957a). Adv. mod. Biol. Moscow, 43, 263. Passynsky, a. (1957b). Biophysics {Russ.) 2, 566. Passynsky, A., and Pavlovskaya, T. (1960). C.R. Acad. Sci. U.R.S.S. in press. Passynsky, A., and Vyrovetz, O. (1959). Biochemistry, Leningr. 24, 922. Passynsky, A., Volkova, M., and Bloxhina, V. (1955). C.R. Acad. Sci. U.R.S.S. 101, 317. Pavlovskaya, T., and Passynsky, A. (1956). Colloid J., Voronezh, 18, 583. Pollard, E., Guild, W., Hutchinson, F. and Setlow, R. (1955). Progr. Biophys. 5, 72. ToNGini, A., and Passynsky, A. (1960) Biophysics {Russ.) 5, 517. Volkova, M. and Passynsk:y, A. (1955) Biochemistry, Leningr. 20, 665. Volkova, M., Tongur, A., Tchunaeva, N., and Passynsky, A. (1957) Biophysics {Russ.) 2, 465. ZiRKLE, R., and Tobias, K. (1956). Arch. Biochem. Biophys. 47, 282. DISCUSSION BACQ : Wliat is the relative efficiency of the a-particles and X-rays witli regard to the enzyme system you have studied? passynsky: Up to now X-rays alone have been studied. Comparison with the effects of a-particles is vmdoubtedly of the greatest interest, since until now different models always differed from the living cell by the relative efficiency of different irradiations. gray: How do the radiation effects depend on the thickness of the RNP layer around the enzyme particle? passynsky : We tried to obtain layers as thin as possible, but the influence of the irradiation up to now has only been studied for the minimum thickness obtained — about 8 molecular diameters. ARDASHNiKOV: What is the explanation for the irradiation dose effect? PASSYNSKY : At a given dose in every monolayer an equal number of the structural lesions would be produced, irrespective of the number of the successive layers. But the effect of these lesions on the permeability of a separating layer of course would not be the same, if the layer's thickness varied. In a layer consisting of one row of molecules every lesion would change the permeability ; whereas in the case of a multi-molecular layer it is necessary that lesions of different layers should form a kind of channel, and that is an unlikely occurrence. Just because of this it may be expected that the sensitivity of the system to irradiation would in- crease rapidly. ELECTRON SPIN RESONANCE (ESR) INVESTIGA- TIONS ON RADIATION-INDUCED CHEMICAL EFFECTS IN BIOLOGICAL SPECIES L. A. BLUMENFELD AND A. E. KALMANSON Institute of Chemical Physics, U.8.S.R. Academy of Science, Moscow, U.S.S.R. SUMMARY The ESR method may be used to investigate radiation-produced unpaired electrons in lyophilized biological structures. The authors have studied nearly all the amino acids, a number of di- and tripeptides and various proteins, nucleo- proteins and tissvies. The number of free radicals produced in y-irradiated lyophilized proteins is some two or three orders of magnitude less than in amino acids and peptides. It is suggested that "conductive channels" may exist and that electrons can migrate along these, thus "healing" the injuries. The appearance of a new method of investigation, especially if based on exact theory, often leads to considerable progress. This is exactly so in the case of the ESR method, which was invented by the Soviet physicist, Zavoysky, in 1944. There are a number of articles on the theory of ESR and its experi- mental techniques (Frenkel, 1954; Gordy, et al., 1955; Ingram, 1955, 1958; Van Vleck, 1948; Semenov and Bubnov, 1959). These problems will not, therefore, be discussed. It is known that the ESR method permits us to obtain the following information about the substances investigated ; 1. The presence of unpaired electrons at concentrations of about 10-11 to 5 X 10-12JVI per gram, or, in other words, IO12 to IQi^ unpaired electrons in the sample ; 2. A sufficiently correct quantitative estimation of the concentration of unpaired electrons (by comparison with standards) ; 3. The analysis of the form and the width of ESR spectral lines permits important conclusions to be drawn about the interaction be- tween unpaired electrons and the surrounding atoms ; the influence of structural particularities of the substance being investigated on the 59 60 L. A. BLUMENFELD AND A. E. KALMANSON behaviour of unpaired electrons; the magnitude of exchange inter- actions and the degree of delocalization of such electrons, and other important parameters ; 4. The analysis of hyperfine ESR structure (resulting from the inter- action of unpaired electrons and nuclear magnetic moments of surround- ing atoms) allows us to make the structure of paramagnetic particles clear. Free radicals formed by ionizing radiations have unpaired electrons. For this reason the ESR method has now become one of the most wide- spread and effective methods of investigating initial radiation-induced chemical effects. The investigation of the kinetics of free radical for- mation, while the dose rate, the energy of radiation and the intensity of emitting sources are varied ; the study of the temperature effect and the effect of oxygen ; the study of the effects of humidity ; the investi- gation of anti-radiation compounds ; the determination of the quantum yields of radiation -induced chemical reactions; the evaluation of the recombination energy of radicals by use of kinetic curves; all these l^roblems and others can be solved by means of ESR spectroscopy. Hutchison (1949) was the first to observe the ESR spectra of irradia- ted organic compounds. He irradiated alkyl halides with neutrons and found them to form r-centres, which give ESR signals. ESR investigations of irradiated compounds, important biologically, were begun in three countries independently: by Combrisson and tJbersfeld (1954) in France, by Gordy and his collaborators (1955, 1958) in the U.S.A. and by us in the U.S.S.R. (Blumenfeld, 1958; Blumenfeld and Kalmanson, 1957a, b,c; 1958a, b; Kalmanson and Blumenfeld, 1958). Combrisson and tJbersfeld irradiated amino acids in an atomic pile directly. The wide energy spectrum of the source, the high total dose rate and the high intensity of the irradiation lead to extensive destru- tion of the amino acids. In sucli conditions the ESR spectra of the free radicals were rather similar with only a slight hyperfine structure. Since 1955 moi*e complete and interesting investigations of the ESR sjjectra of iiTadiated biological materials have been carried out in the U.S.A. by the group headed by Gordy, who investigated such important irradiated biological compounds as amino acids, proteins, hormones, vitamins, fats and nucleic acids. The most interesting and unexpected result of this work proved to be that although the amino acids, of which proteins are constituted gave ESR spectra after irradiation that were characteristic for each amino acid, irradiated ])roteins always gave rise to only two types of signal: either to a doublet with a splitting of 15 to 16 oersted, or to a ELECTRON SPIN RESONANCE INVESTIGATIONS 61 signal characteristic of comiiounds containing sulphur. The doublet was observed in proteins that did not contain sulphur, such as, for example, collagen. Signals characteristic of irradiated sulphur-containing amino acids, were observed with those irradiated proteins which contain a consider- able amount of sulphur, such as wool, horn, hoof, and nails. No other hyperfine structure in sulphur-containing j)roteins was found by Gordy and his collaborators although all these proteins con- tain enough protons and nitrogen nuclei in various positions. It is known that the magnetic moments of such protons and nitrogen nuclei are responsible for the appearance of the hyperfine structure of irradiated amino acids. Gordy was quite right in suggesting a possible migration of "injuries" along the protein molecules towards certain more vulnerable points whose properties in this respect, however, were not made clear. Gordy gives no explanation of the "migration of injuries" in his early work. In his later work Gordy develops various assumptions of the possible radio-protective role of sulphur-containing compounds. Such studies are justified and to some extent are connected with the numerous radiobiological investigations on the anti-radiation effect of sulphur- containing substances. In spite of numerous experimental data and a quite profound theoretical evaluation of these data from the point of view of quantum chemistry and the ESR theory, we think that Gordy's work has some defects. First, no quantitative estimation is given of radical yields for irradiated biological structures of different complexity and in different native states. The second defect, apparently due to the physical rather than biological treatment of the problems being studied, is that Gordy's group often use proteins without clearly defined biological properties. So far as radiobiology is concerned some research paj)ers published by the Swedish scientists Lars and Andreas Ehrenberg (1958) together with the noted German radiobiologist Karl Zimmer (Zimmer, 1960; Ehrenberg and Zimmer, 1956) are far more interesting. In these papers ESR spectra of irradiated seeds and sprouts of different water content were investigated. The authors gave a quantitative estimation of the radical yield as a function of the radiation dose, and also traced the rate of decay of free radicals after the irradiation as a function of the percentage of water of the samples. The main results of the Ehrenbergs' and Zimmer's work are: 1. A linear de]3endence of the free radical yield on the radiation dose was established. 62 L. A. BLUMENFELD AND A. E. KALMANSON 2. A protective action of water was discovered; the yield of radicals was halved as the water content of the irradiated seeds increased from 3 per cent to 10 per cent. This protective action, accoixling to the anthors, is connected with the greater possibility of recombination between free radicals as the water content in the samples is increased. 3. Some protective action of water was also observed by the anthors in expei'iments on the germination of irradiated seeds of diffei^ent hnmidity. In these experiments seeds of lower water content perished if iiTadiated by smaller doses. 4. ESR studies of the rate of decay of free radicals in samples of different water content showed an exponential decrease, the coefficient before the exponential factor being larger for samples of higher water content. The work of the Ehrenbergs and Zimmer successfully combines the possibilities of new physical methods and ordinary radiobiological experiments, and thus help to approach one of the most debatable problems of radiobiology — the problem of the direct and indirect (with water px'esent) action of irradiation. Any theory that takes into account the participation of the low mole- cular products of water radiolysis must consider the extremely high reactivity and short life-time of these products at the temperature of living cells. It would be right to mention, that according to the ESR data atomic hydrogen can be observed in irradiated ice only at liquid helium tempei'atures, and the free OH radical only at liquid nitrogen temperatures. We shall now proceed to give an account of our own exj^eriments and of some general conclusions which we believe can be drawn from them. First of all it must be pointed out that we used ionizing radiation just to obtain and accumulate unpaired electrons in biological structures of increasing complexity and different native states and not at all for the investigation of the initial radiochemical aspects of radiobiological problems. We hoped that the investigation of ESR spectra in such a system would help us to find out what structural peculiarities give rise to the surprising activity of biological structures. We proceeded from the general hypothesis that the important role of free electrons in a number of most important biological processes is connected with energy migration within the biological structures. Initially we supposed that enzyme action and muscle contraction be- longed to such phenomena. It is quite clear, however, that all our results are also closely related to the nature of initial radiation-induced effects. Having these general considerations in mind in 1955-1957 we systematically investigated a wide range of iri'adiated biological objects : ELECTRON SPIN RESONANCE INVESTIGATIONS 63 nearly all amino acids, a number of di- and tripeptides and various proteins and tissues, some of them biologically active, and some of them destroyed beforehand by boiling. The source of irradiation was cobalt-(JO. The samples were irradiated with a dose of 10^ to IC^ roentgens. The amino acids were investigated in the crystal state, while proteins and tissues were in the form of lyophi- lized specimens retaining a maximum of their native properties. Quanti- tative measurements were carried out by comparing the areas of a standard signal with that of the signal from the sample being tested. The structure of the free radicals fox^med by irradiating amino acids especially were not investigated in detail. However, in a number of cases we were able to reveal their structure sufficiently correctly and completely. A more detailed study of the structure of free radicals, formed by irradiating complex amino acids would require a special in- vestigation with additional modification of the amino acids, for instance the introduction of isotopic atoms such as deuterium, which has a different nuclear spin from that of a proton and which can be introduced into certain groups in amino acids. We feel, however, that such additional complications would probably not help us much to solve the main problem of ascertaining the peculiarities of the macrostructure of the most important biopolymers, proteins and nucleic acids. The main experimental results may be considered: (i) Dry specimens of amino acids as a rule produce intensive ESR spectra with a width of tens or hundi'eds of oersteds and with a clearly defined hypei'fine structure, due to the interaction of unpaired electrons with protons and nitrogen nuclei, which are part of the free radicals produced. With a dose of about 10'^ r the yield of free radicals is about 1019 paramagnetic particles per gram. It is possible to assert that every ionization event in the amino acid leads to the production of about one free radical. As a rule, most amino acids evolve ammonia and CO2 following irradiation, and sulphur-containing amino acids evolve hydro- gen sulphide. The spectra of some simple irradiated amino acids can be interpreted as spectra of amino acid fragments which remain after such decomposition products have been removed. However, the un- paired electron of a sulphur-containing compound can be considered to be localized near the sulphur atom; this results from the value of the gr-factor for such compounds. It is worthwhile to note that in irradiated dry crystalline preparations of amino acids ESR spectra, and that means free radicals also, have been ke^Jt unchanged at room temperature (and in the presence of air) for more than four years already ; however, if the ciystals are dissolved in water the radicals vanish immediately. 64 L. A. BLUMENFELD AND A. E. KALMANSON (n) In the ESR spectra of irradiated native proteins and lyopliilized tissues (60 to 80 per cent of proteins) two special features may be dis- cerned. First, the number of free radicals produced in proteins and tissues is from one to three orders of magnitude less than in amino acids and peptides (with equal doses of y-irradiation). Second, the signal is not a superposition of ESR specti'al patterns (this might be expected since the energy of y-quanta is quite sufficient to break any peptide bonds in proteins), but is usually a single naiTow peak with a half- width of 4 to 10 oersted and without any hyperfine structure. The position and the width of the signal is usually the same as that for ESR spectra of enzyme specimens frozen and lyopliilized at the moment the enzyme action takes jjlace. The narrowness of the signals cannot be explained by exchange interactions, because of the small concentration of unpaired electrons. We must admit a translational mechanism for the narrowing (Anderson 1954). The small intensity of signals in irradiated proteins and the absence of hyperfine structure, in comparison with signals from iiTadiated amino acids, are of si3ecial interest to radiobiologists. In the case of irradiated amino acids and peptides the ESR signal is due to "holes" with unpaired electrons, or to neutral free radicals arising when bonds are broken homogeneously. However, from the point of view of our hypothesis about the exis- tence of energy levels of a molecule as a whole or, as we called them, "conductive channels", it may be suggested that in native proteins eliminated electrons can re-enter the previously mentioned "bands", formed by an orderly net of hj^drogen bonds, and return along them to the "holes", recombining with them and "healing" the injuries. This hyx^othesis about the role of regular hydrogen bonds in creating "conductive bands" and in increasing the radio -resistance of proteins, was investigated in special expei'iments. It is known that heat denaturation sharply disturbs an orderly system of hydrogen bonds, and makes it chaotic. When denaturation is sufficiently complete proteins lose their biological activity completely. A number of protein prej^arations were exposed to heat denaturation before lyophilization and irradiation. In all cases intense signals were obtained with clearly expressed hyperfine structure, characteristic of free radicals with localized electrons, instead of a weak narrow singlet. By increasing the amount of denaturation, an increase of radical yield by one to three oixlers was produced. We suppose that the disturbance of the secondary structure of protein molecules during the denaturation leads to the disappearance of "conductive channels" along which the electrons ejected by the ELECTRON SPIN RESONANCE INVESTIGATIONS 65 radiation might recombine with "holes" and this is shown by the in- creasing concentration of unpaired electrons in the sample, snch un- paired electrons being already localized. In 1958-1959 the ESll spectra of irradiated nucleic acid compounds was investigated. This work was carried out in collaboration with Passynsky and Shen-Pei-Gen (Shen-Pei-Gen et al., 1959). Nucleic acids and their components were irradiated under the same conditions as before. We also irradiated nucleoproteins and some artificial complexes of nucleic acids with proteins and other compounds. As in the investigations of amino acids and proteins we found a dis- tinct dependence of free radical yield on the complexity of the struc- tures. For instance irradiation of nucleic acid bases and ribosides with a dose of 10"^ r gave a free radical yield of lO^^ to 10^9 particles per gi'am. The free radical yield is one to three orders less for high molecular weight preparation of nucleic acids. The most important result, from the point of view of radiobiology, IS that although the radical yield per gram for low molecular weight compounds is much higher than for those of high molecular weight, the number of damaged molecules per ionization event is, however, much higher (50 to 70 times) because of the great dimensions of the nucleic acids. This conclusion is in excellent agreement with other purely biological experiments in radiation cytology and genetics, which showed that a disturbance in even one section of a high polymeric nucleic acid, which is a part of the chromosomes, can lead to non-reversible and even lethal damage to the cells. We can explain the small radical yield in irradiated high-polymer nucleic acids on the basis of our hypothesis about the existence of molecular "conduction channels". It is interesting that in one of their last papers on the irradiation of nucleic acid compounds, Shields and Gordy (1959) also conclude that nucleic acids have semi-conductive properties. In conclusion it is necessary to point out that all the experimental data given above were obtained on lyophilized, solid specimens. Be- cause of this, direct comparison of these data with those for biological species containing much water is not quite correct. In aqueous media additional difficulties, due to the secondary action of short-lived active free radicals created by water radiolysis, can occur. However, we are sure that these special featui'es of the ionizing action of radiation on biological polymers are due not to aggregation, but to properties of their molecular structure. From this ]3oint of view molecules of i:)rotein and nucleic acid may be considered in aqueous medium as particles of a solid body. So we think 66 L. A. BLUMENFELD AND A. E. KALMANSON that, without takmg into consideration the influence of short-hved radicals, the mechanism of the initial action of ionizing radiations on biological structures does not depend on their state of aggregation. Some papers have recently been published, testifying to the specific role of molecular and supra-molecular order in the peculiar properties of biological structures, for example the work of Arnold and Sherwood (1957) on the semi-conductive properties of biological structures, as shown in particular by photosynthetic properties ; our work on abnormal magnetic properties of nucleic acids and nucleoproteins (Blumenfeld et al., 1959; Blumenfeld, 1959); and the work of Polonsky et al. (1960) and Duchesne and Monfils (1955) on the magneto-electrical properties of nucleic acids. We believe that all these phenomena are due to the high degree of order of these materials. Evidently this high degree of order in biological structures conditions the peculiarity of their radiolysis, and is responsible for the characteristics of their ESR spectra. REFERENCES Arnold, W., and Sherwood, H. K. (1957). Proc. nat. Acad. Sci., Wash. 43, 105. Blumenfeld, L. A. (1958). Bull. Acad. Sci. U.R.S.S. 9, 22. Blumenfeld, L. A. (1959). Biophysics (Russ.) 4, 5. Blumenfeld, L. A., and Kalmanson, A. E. (1957a). C.R. Acad. Sci., U.R.S.S. 117, 1. Blumenfeld, L. A., and Kalmanson, A. E. (1957b). Biophysics (Rtiss.) 2, 5. Blumenfeld, L. A., and Kalmanson, A. E. (1957c). Btdl. Acad. Sci. U.R.S.S. (Biol.) No. 3. Blumenfeld, L. A., and Kalmanson, A. E. (1958a). Biophysics (Rtiss.) 3, 1. Blumenfeld, L. A., and Kalmanson, A. E. (1958b). In "Proceedings, II International U.N. Conference on Peaceful Uses of Atomic Energy". A. (15) (R 2079) 837. Geneva. Blumenfeld, L. A., Kalmanson, A. E., and Gen, Shen-Pei- (1959) C.R. Acad. Sci, U.R.S.S. 124, 1144. CoMBRissON, J., and I^bersfeld, J. (1954). C.R. Acad. Sci., Paris, 258, 1397. Duchesne, J., and Monfils, A. (1955). C.R. Acad. Sci., Paris, 241, 749. Ehrenberg, L., and Ehrenberg, A. (1958). Arcfiiv. fiir Fisik, 14, 133. Ehrenberg, L., and Zimmer, K. G. (1956). Hereditas, Lund., 42, 515. Frenkel, I. (1945). J. exp. theor. Phys. 15, 409. Gen, Shen-Pei-, Blumenfeld, L. A., Kalmanson, A. E., and Passynsky, A. K. (1959). Biophysics (Russ.) 4, 263. GoRDY, W. (1958). In "Symposium on Information Theory in Biology". Pergamon Press, London. GoRDY, W., Smith. W., and Trambarulo, H. (1955). "Radiospectroscopy", Foreign Literature Pubhshing House, Moscow. Hutchison, C. A. (1949). P/i^/s. Rev. 75, 1769. Ingram, D. J. E. (1955). "Microwave Spectroscopy", Foreign Literature Pubhshing House, Moscow. Ingram, D. J. E. (1958). "Free Radicals as studied by Electron Spin Resonance", But- terworths, London. Kalmanson, A. E., and Blumenfeld, L. A. (1958). Biophysics (Russ.) 3, 4. Polonsky, J., Douzen, P., and Scadrom, Ch. (1960). C.R. Acad. Sci., Paris, 20, 250, 3414. Semenov, a. G., and Bubnov, H. H. (1959). Apparatus ami Technique of E.vperiment. Shields, H., and Gordy, A. (1959). Proc. nat. Acad. Sci. Wash. 45, 269. Van Vleck:, G. H. (1948). Phys. Rev. 74, 1168. Zavoysky, E. K. (1944). Dissertation Physical Inst. Acad. Sci. U.S.S.R. Zimmer, K. (1960). "Studien zur quantitativen Strahlenbiologie", Heidelberg University Press, Wiesbaden. ELECTRON SPIN RESONANCE INVESTIGATIONS 67 DISCUSSION BACQ: You have worked witli absolutely dry proteins. Can you give us the exact temperature and oxygen pressure during the irradiation. BLUMENFELD : Irradiation was carried out at room temperature. Oxygen pressure during irradiation was about 10"^ mm Hg. Our samples were freeze-dried at a temperature of — 50°C, ESR spectra were studied both at room temperature and at lower temperatures, down to liquid nitrogen temperature. I did not dwell here on ESK siiectrum changes in irradiated amino acids associated with temperature decrease: it could be the subject of a special report. As for an oxygen effect, it was j)ractically non-existent as far as amino acids and low molecular weight compounds were concerned, but oxygen affected considerably the high molecvilar weight comi^ounds, particularly proteins : the intensity of the ESR signals de- creased considerably on admission of air. As to the form of the ESR signals, since we did not perform experiments with irradiation at low temperatures but applied irradiation at a sufficiently high room temperature, we did not observe any changes in the form of the ESR signals. ALEXANDER : In our experiments we have observed that when DNA from sperm heads is irradiated at a low temperature, a high yield of radicals occurs. After irradiation at room temperature there is a drop in the radical yield. Our explana- tion was that at high temperatures a recombination of radicals is possible dvie to molecular movements. Could your theory account for this phenomenon? blumenfeld: Your observation that during irradiation the radical yield in- creases with the lowering of temperature, is a very interesting one. I believe that it fits in well with our conceptions. For uncoupled electrons to migrate through formed structures, it is necessary that some quite definite structural conditions be fulfilled, which, generally speaking, could be unfulfilled (not realized) in the structure of the proteins and nucleic acids. In order for uncoupled electrons (which are 2p electrons), to migrate through the hydrogen bond system, it is necessary that on all the centres through which they migrate their wave fimctions be parallel. This condition may be unfulfilled in molecules of the nucleic acids and proteins at equilibrium configuration. ALEXANDER: May the signal be more intense at low temperatiu'es? BLUMENFELD : In Order that semiconductivity properties become manifest, an orderly structure of the hydrogen bonds in protein and nucleic acid molecules is necessary. It is necessary that in proteins all the peptide groups by which migra- tion occurs, should lie in the same plane. If they are oriented at right angles migration is impossible. If they are oriented at different angles, the probability of the migration is proportional to the cosine of the angle. At liquid nitrogen temperatures these conditions may be unfulfilled under equilibrium conditions for all the structm-e. At room temperature owing to vibrational and rotatory movements there always would be moments when different peptide grou]3s are in the same plane and migration may occur. passynsky: What is the significance of the g^-factor in all the systems studied? Is it possible to relate the observed intensity of the signals only to the number of the uncoupled electrons, without taking into account their interaction? blumenfeld: In all the cases for all the ESR signals in proteins and nucleic acids elicited by the ionizing radiation — I emphasize it — the g^-factor of the 68 L. A. BLUMENFELD AND A. E. KALMANSON signal practically coincides with g^-factors of the free electron except for several proteins with high sulphur content, which give signals with ^--factor of 2-024, testifying to the localization of the uncoui^led electrons on the sulphm- atom. I believe that in all the cases mentioned here the magnitude of the signal is determined only by the nvmiber of the imcoupled electrons, as in all the com- pounds with paramagnetic properties. passynsky: To what extent are all -molecular electron levels utilized in proteins and nucleic acids unexposed to irradiations under their basic natural con- ditions? BLUMENFELD : I believe that in all the proteins and nucleic acids there are potentially these all-molecular levels, but under the basic conditions of these compoimds they are unpopulated. In the case of proteins they become populated when enzymatic processes occvu-, owing to the formation of the comj)lex sub- strate-enzyme. Or they become populated as an effect of irradiation. But protein molecviles as such do not possess populated all-molecular levels and from this standpoint are not semiconductors. THE ACTION OF X-RAYS ON INTRACELLULAR BACTERIOPHAGE FORMATION F. HERCIK Biojihysical Institute of the Czechoslovak Academy of Sciences, Brno, Czechoslovakia SUMMARY 1. Inactivation of the capacity of E. coll B for phage T 3 by the action of soft X-rays was measured. 2. The dose-effect curve is biphasic. After small doses, rapid inactivation occurred, but the inactivation was markedly slower after large doses. 3. The capacity of irradiated E. coU B was affected by addition of chloram- phenicol. On adding chloramphenicol to irradiated bacteria together with the phage during the logarithmic phase, the decrease in capacity was relatively smaller than might have been expected if the deleterious effects of the two factors were cumulative. In stationary cultures the protective effect of chloram- phenicol was less marked. The ability of bacterial cells to form phage was defined by Benzer and Jacob (1953) as the capacity. Earlier observations by Anderson (19J:4, 1948) showed that cells of Escherichia coli sterilized by large doses of ultraviolet radiation could still form phage T4. Later it was found that the capacity of E. coli for other phages was much more sensitive to the action of ultraviolet radiation (Benzer, 1952; Garen and Zinder, 1955; Labaw et al., 1953; Tessman, 1956). Similar results were obtained with ionizing radiation (Rouyer and Latarjet, 1946; Tobin, 1953; C4aren and Zinder, 1955; Pollard et al., 1958). Recent measurements (Stent, 1958) have shown that, for phage T 2, a dose of 650,000 r is required to inhibit two-thirds of the bacterial phage-forming capacity. A deeper insight into this remarkable phenomenon will not only elucidate, to a certain degree, the mechanism of the basic action of radiation on living matter but will also reveal the processes of phage formation. In our laboratory the inactivation by X-rays of the capacity of E. coli for phage T3 has been studied. The immediate effect of ionizing radiation was compared with the delayed effect. In another series of experiments the protein synthesis of the irradiated bacterial cells was 69 70 F. HERCIK stopped by chloramjihenicol either before or after infection of the cells with phage (Hercik, 1959, 1960). The bacteria were irradiated on broth agar, from which they were washed off either immediately after irradiation or 24 hr later. They were then tested for the ability to form phage by adding phage T 3 and determining the titre of the phages developed. The survival of the capacity after irradiation was determined from the ratio of phage yield of irradiated and of non-irradiated controls. To avoid formation of colonies out of surviving cells, the bacteria destined for the observa- tion of the delayed effect were kept for 24 hr at a temperature of 6°C. This procedure was in accordance with that of Stapleton et al. (1953), who found that if cells of E. coli were cultured at temperatures between 4°C and 37 °C after irradiation, the inhibition of division was the same at 4°C as at 37°C (with a maximum of recovery at 18°C.) By using this method with doses up to 20,000 r it was possible to avoid errors that would otherwise have occurred as a result of capacity in bacteria that had developed by proliferation of surviving cells. The bacteria were irradiated with a tube of 60kV, 4 mA and a dose rate of 4,000 r/min. The range of doses used was from 4,000 r to 960,000 r. The survival of E. coli B corresponded to the customary value of 3,500 r for the half-value dose (one-hit curve). After a dose of 120,000 r only one cell in 10'^ is capable of forming colonies. The results of this series of experiments are shown in Fig. 1. The inactivation of the capacity by ionizing radiation differs according to whether it is determined immediately after irradiation or 24 hr later. Immediately after irradiation the decrease in capacity in relation to the dose, on a semilogai'ithmic scale, is only approximately linear. This is in agreement with the results obtained by Pollard et al. (1958) for phage T 1. This is even more manifest after 24 hr. After small doses (4,000 r and 8,000 r) a sharp linear decrease in capacity occurs. The response to higher doses is quite dissimilar. There is also a linear de- crease, but the drop in the capacity is much slower. The explanation of this phenomenon is difficult. It should be taken into account that the inactivation of the bacterial capacity to produce phage is the result of the irradiation reaction which has its own course. It appears that recovery processes develop in the case of the immediate effect of high radiation doses (as a result of the long exposure period) which reduces the degree of inactivation of the capacity. On the other hand with the delayed effect the change in the capacity follows the course of the completed irradiation reaction and, therefore, relatively small doses of irradiation produce a large drop in the capacity. With doses higher than 80,000 r the bacterial capacity becomes more radio- INTRACELLULAR BACTERIOPHAGE FORMATION 7i resistant. This probably indicates a second mechanism of phage for- mation. At the present time there is not enough evidence to support the view that there are two distinct mechanisms of phage formation Capacity N,/No(22) 4 8 12 24 48x10 Dose(r) 96x10' Fig. 1. — Effect of X-rays on the capacity of E. coli B to form phage T 3. O Survival of the bacteria O Change in capacity immediately after irradiation. # Change in capacity 24 hr after irradiation. Numbers in brackets : age of the culture in hours. with differing radio -resistance. However, several examples do exist where dose-effect curves are non-uniform, indicating processes with different radio-resistances. Another possible explanation based on the non-homogeneity of the particular population of E. coli B resulting in two different types of capacity appears to be even less probable. The reason is that in such a case the inactivation curve of the colony-forming ability should be also biphasic, and this is not the case. In lookuig for an explanation of the biphasic nature of the bacterial capacity, we tried to influence the phage production of the irradiated bacterial cells by chloramphenicol. It is known that chloramphenicol inhibits protein synthesis in £". coli B (Wisseman et al., 1954). In E. coli B cells irradiated with X-rays or u.v.. Gillies and Alper (1959) found a higher survival rate in cells incubated on agar containing chloram- phenicol. Phage formation by bacterial cells is also influenced by chloramphenicol (Bozeman e^ aL, 1954; Crawford, 1957, 1959). It was. 72 F. HERCIK therefore, to be expected that chloramj^henicol would influence the production of phage in irradiated bacterial cells. The action of chloramphenicol on the capacity of irradiated E. coli was studied in two series of experiments. In the first series, the chlor- amphenicol was added to the irradiated bacteria at the time of in- fection with the phage, or 15 min later at the end of the latent period. Bacteria were infected for the determination of the capacity immedi- ately after irradiation or 22 hr later. In the second series of experiments the irradiated bacteria were exjDosed to the action of chloramphenicol for different times before determining the capacity. 10' 10' > Capacity Nzch/Nch(3,0) Nzch/N,H(22,0) N Nzch/Nch(22,l5) _L 24 48 Dose (r) 96x10' Fig. 2. — Relationship between capacity of E. coli B for phage T 3 in the presence of cliloraniphenicol after irradiation with X-rays. Nch — capacity of non-irradiated bacteria treated with chloramphenicol. Nzch — capacity of irradiated bacteria in the joresence of chloramphenicol. Numbers in brackets : the age of culture in hours and the time in minutes at which chloramphenicol was added. The toxic effect of chloramphenicol on the capacity of E. coli B was investigated without previous irradiation. It was stated that the addition of chloramphenicol during the logarithmic phase caused a marked reduction of capacity (222-fold), while during the stationary phase the reduction in capacity was about tenfold. Under these circumstances the combined effect of chloramphenicol and radiation is bound to be manifested in a further decrease of the capacity. In general it may be stated that in the presence of chloram- phenicol the capacity of the bacteria decreased with the radiation dose, INTRACELLULAR BACTERIOPHAGE FORMATION 73 and that the biphasic character of the dose-effect curve was maintained. Very interesting results are found when the data are plotted in such a way that the capacity of irradiated and chlorani])henicol-treated cells is compared with non-irradiated cells treated with chloramphenicol. The effect of irradiation on the capacity was lowest in the 3-hr culture to which chloramphenicol was added, together with the phage, immedi- ately after irradiation. It has already been stated that the chloram- phenicol itself has a high toxic effect; it would, therefore, be expected that heavy irradiation doses would lead to complete inactivation of the cai^acity. This is evidently not the case (see Fig. 2), and chloramphenicol must, therefore, have a restorative effect. These experiments have clearly shown that chloramphenicol and radiation do not supplement one another in inhibiting the capacity of E. coli B to sustain phage growth but that under certain conditions they can be antagonistic to each other. This problem was investigated in a second series of experiments with the aim of diminishing the toxic effect of cliloramphenicol. The method of Gillies and Alper (1959), using cellophane carriers on which the bacterial culture was spread, was used. This method enabled us to remove the carriers, after certain time intervals, from the surface of the agar medium containing chloramphenicol. In these experiments, which have not yet been completed, it can be shown that a short stay of heavily irradiated cells (80,000 r) on chloramphenicol agar increases the capacity ofE. coli for phage T 3. It is quite possible that this favourable effect is due to the fact that chloramphenicol inhibits protein synthesis, and as the polymerization of nucleic acids is also inhibited, a certain amount of DNA precursors are accumulated, which can be used for restoration. REFERENCES Andekson, T. F. (1948). J. Bad. 56, 403. Anderson, T. F. (1944). J. Bad. 47, 113. Benzek, S. (1952). J. Bact. 63, 59. Benzer, S., and Jacob, F. (1953). Ann. Inst. Pasteur, 84, ISO. BozEMAN, F. M., Wisseman, Jr., C. L. Hopps, H. E., and Danaskaus, J. X. (1954). J. Bact. 67, 530. Crawford, L. V. (1957). Biochem. J. 65, 17 P. Crawford, L. V. (1959). Virology, 7, 359. Gaeen, a., and Zinder, N. D. (1955). Virology, 1, 347. Gillies, N. E., and Alper, T., (1959) Nature, Lond. 183, 237. Heecik:, F. (1959). i^oZia. Biol., Prague, 5, 328. HEReiK, F. (1960). Folia. Biol., Prague, 6, 269. Labaw, L. W., Mosley, V. M., and Wyckioff, R. W. G. (1953). J. Bact. 65, 330. Pollard, E., .Setlow, J., and Watts, E. (1958). Radn Pes. 8, 77. RouYER, M., and Latarjet, R. (1946). Ann. Inst. Pasteur, 72, 89. Stapleton, G. E., Billen, D., and Hollaendbr, A. (1953). J. cell. comp. Physiol. 41, . 345. 74 F. HERCIK Stent, G. S. (1958). Advance Virus Res. 5, 95. Tessman, E. S. (1956). Virology, 2, 679. TOBIN, J. O'H. (1953). Brit. J. e.vp. Path. 34, 635. WissEMAN, C. L., Smadel, J. E., Hahn, F. E., and Hopps, H. E. (1954). J. Bad. 67, 662. DISCUSSION maecovich: There was an experiment in whicli chloramphenicol was added before the release of phage. How could it exert any influence, if the phage par- ticles were already completely formed? HERCIK: Chloramphenicol was added at the end of the latent period. marcovich: Wliat is the explanation for the results obtained? HERCIK : It is very difficult to exjilain, since it should be borne in mind, that the cjuantity of the phage is very small — only about two or three phages are released in the Escherichia coli suspension. Becau.se of this, two possibilities pi'esent them- selves: the first one is that phages ai'e released by one or two siu'viving cells, since there are always surviving cells; the second possibility is that phages are released at a very slow speed by several hundred cells. I cannot say with certainty which of these two possibilities is more probable. pollard : When you changed over to agar with chloramphenicol, what was the mediimi — the same as in the case of the suspension? HERCIK : Yes, the same. MARCOVICH: Did you allow the bacteria to gi'ow before seeding with the phage? Under these conditions the quantity of the phages formed would have had no influence. HERCIK : It would depend upon the number of bacteria which had been damaged by the irradiation. THE ACTION OF IONIZING RADIATION ON THE CELLULAR SYNTHESIS OF PROTEINj ERNEST POLLARDJ Biophysics Department, Yale University, Neiv Haven, Connecticut, U.S.A. SUMMARY The experiments described are all in accord with the idea that the immediate synthesis of protein is caused by organelles which are sensitive to ionizing radi- ation in the manner expected for ribosomes in an extended form. The volume of the sensitive regions agrees with the volume of an SOS ribosome and this, inde- pendently, substantiates the work of McQviillen, Roberts and Britten (1959) who have shown that the short term incorporation of ^^S04 is into 70S ribosomes. The long term effects of radiation are much more difficult to interpret. In separate experiments we have found that there is a reduction in the rate of in- crease of DNA in irradiated cells. Possibly this means that there is some kind of disruption of the bacterial nucleus which produces an unbalance in the cell and reduces cellular synthetic action later on. Partially successful attempts to express this theoretically have been made (Pollard, 1960), but it cannot be claimed that the process is fully understood. Synthesis of protein is an essential characteristic of every Hving system. It is now known that it takes place in several stages and that different mechanisms are involved with each. The present studies were made with the aim of using the disruptive action of ionizing radiation to give some information on the nature of the process of protein syn- thesis. The work reported is not complete: it is still in i^rogress. Never- theless some definite facts have been established and some conclusions can be drawn regarding protein synthesis, and also on the sensitivity of some parts of the cell to ionizing radiation. The cell employed throughout the work is the bacterium EschericJiia coli. PHYSICAL ACTION OF IONIZING RADIATION The pioneer work of Lea and others in the pre-war years has been greatly extended in the past ten years in the Yale Biophysics Depart- ment. The work has been reiiorted in various review articles (Pollard, t The work reported here was largely supported by the U.S. Atomic Energj' Com- mission. J For 1960-Gl visiting Professor at the Pemisylvania State University. 75 76 ERNEST POLLARD Guild, Hutchinson and Setlow, 1955; Pollard, 1959) and the important conclusions are as follows. 1. An ionization within the volume of a protein molecule or a nucleic acid molecule has a very high probability, in excess of 0-5, of re- moving its biological action. The mechanism of this action in protein is beginning to be understood and is probably related to the migra- tion of excitation energy to an -S-S- linkage which thereby becomes sensitized. In nucleic acid it is probably l)reakage of the chain, or possibly cross-linkage. 2. A nucleoprotein molecule, such as a small virus, can also be inacti- vated by ionization. The probability of inactivation is less, but still high. 3. Estimates of the volume of biologically important molecules based on the theory that for activity to remain after irradiation they must have wholly escaped any ionization whatever, are very in- formative and have proved to be correct within a factor of two, with few exceptions. 4. Within cells, the radicals formed l)y ionizing radiation do not diffuse more than 30 A before they encounter some structure or molecule which removes them. The evidence in support of the above four conclusions cannot be given in this paper because time does not permit. Table I shows the results of irradiation of several enzymes, with the conclusions drawn from the statistics of complete escape. Table II shows some results of irradiating viruses. Table III shows the results of experiments largely by Hutchin- son (1960) on which the distance of 30 A has been estimated. It will be useful later to see what conclusions can be drawn about protein synthesis using the same analysis. Table I Material Molecular weight deduced Reported molecular by radiation weight Penicillin 050 356 C'atalase 110,000 250,000 Invertase 120,000 120,000 Pepsin 39,000 36,000 ChymotryiJsin 50,000 23,000 Insulin 23,000 6,000 Tiypsin 34,000 24,000 DNase 62,000 63,000 Alpha-amylase 145,000 100-200.000 /3-galactosidase 290,000 360,000 RADIATION AND THE CELLULAR SYNTHESIS OF PROTEIN 77 Table II A'irus Diameter deduced from lieported diameter radiation (m/x) (m^i) 42 80-90 56 110 40 44 58 140 18 30 Influenza A (infectivity) Newcastle Disease (infectivity) Shops papilloma (infectivity) Measles Southern bean mosaic virus Table III Enzyme Cell Diameter(m/x) Dry Wet Diffusion distance in angstroms Invertase Yeast 6 12 29 Alcohol dehydro- Yeast 1-3 28 31 genase Coenzyme A Yeast 3 200 35 E.coli 15 200 17 /3-galactosidase E.coli 9-6 11-4 9 STATISTICAL ANALYSIS OF RADIATION DATA If the idea of complete escape from ionization is to be used it is tirst necessary to determine the nature of the distribution of ionization. If the agent causing ionization is a fast electron the primary ionizations are widely separated along the track, which is itself very much subject to scattering. Thus there is every reason to treat the ionization pro- duced by fast electrons, which includes the effects of gamma rays which generate fast electrons, as being spread statistically throughout any volume. The original tracks do not contribute regions of locally high ionization in any way that matters much. The ionizations them- selves are not wholly vuiderstood for solid or liquid material. If we apply the results found for gasses to more dense material then the average energy release at a primary ionization is 100 eV. There is a wide distri- bution of values around this average, but it can be used as a basis for statistical reasoning. We suppose that all the secondary ionizations consequent on the primary process occur within a distance of a few angstrom units from the primary ionization. This will not be true for the more energetic secondary electrons, but it is true for those near to the average. It is not a bad first approximation. Using this value, we take the energy lost per cubic centimeter in the material bombarded as a result of the radiation, express it in electron volts and divide by 100. This then gives /, the number of clusters of ionization per unit volume, 78 ERNEST POLLARD If the sensitive region which we are interested in has a volume V cubic centimeters, then the average number of chisters of ionization occurring within V is / V. We are interested in the probabihty of com- plete escape. This can l^e estimated from the Poisson formula, from which we deduce that if IV is the average number of "hits" then r(0) the probability of no hit at all is P(0) = e-iv. If we state that the ratio of activity remaining, ii, to that initially present, no, is a measure of P(0) we deduce that n no - P{0) - e-^^ or (1) In many cases it is found that the activity remaining is related to the dose by the relation /n\ In I — I = — (constant) (Dose) This can be converted into the same form as equation ( 1 ) by calculating / from the dose. The constant is then immediately expressible as V. The volume T^ is informative in regard to the nature of the biological unit responsible for the activity which is lost because of radiation. STATISTICS OF HEAVY PARTICLE RADIATION If irradiation by heavy particles is used then the ionization cannot be considered to be randomly distributed in volume, as the above reasoning cannot be used. The heavy particles are much more nearly line probes which are randomly distributed in area. Unfortunately this is only approximately true, because the secondary electrons along the path of the particle spread ionization away from the track. The amount of this spread can be estimated and it is possible to use heavy particle radiation to make estimates of the area of a molecule or molecular system, and of its thickness. Neither are to be considered as precise, yet it is clearly possible to tell whether an organelle is long and thin, or has one dimen- sion small, or whether it is more nearly spherical, with no thin dimension. When such irradiations are performed, then, the dose is expressed as particles per square centimetre, D, and if the effective cross-section is S an area which is now the equivalent of the volume, V, used formerly, RADIATION AND THE CELLULAR SYNTHESIS OF PROTEIN 79 we have, as before (2) We first correct S for the secondary radiation off the path, using the method given by Pollard and BaiTett (1959) and then, iftis the effective thickness and i the number of primary ionizations per cm path of the particles, S=So{l-e-it) (3) With sufficient variety in the linear energy transfer, which deter- mines i, we can estimate both So and t. These figures are then useful to examine the character of the cell organelle responsible for the effect observed. RADIATION ACTION ON AMINO ACID UPTAKE Bacterial cells were grown on minimal medium, using phosphate for buffer, and 5 grams of glucose per litre. At a concentration of 4 x 10^ cells per ml samples were taken and irradiated either in a cobalt source, or in a cyclotron. For cobalt radiation they were irradiated in screw-top culture tubes. Some radiation was done in the frozen state at — 80°C. For cyclotron radiation the cells were placed on fine grain filters backed by a porous layer soaked in minimal medium. The temi)erature during radiation was 2°C. After radiation the samples, including unirradiated controls, were incubated in minimal medium with addition of the particular labelled substance under study. At two minute intervals 2 ml samples were withdrawn and either filtered at once on a bacterial filter (collodion membrane, average pore size 0-85 jli), constituting the "whole cell" fraction, or allowed to stand in cold trichloroacetic acid (TCA) for an hour at 2°C before filtering. The whole cell fraction was washed with minimal medium, the TCA insoluble fraction with 5 per cent TCA. The filters were then dried and counted under a thin window counter. The results for one case, arginine, are shown in Fig. 1. The five graphs show the uptake in the whole cell (upper gi-aph) and TCA in- soluble fractions. It can be seen that the normal behaviour is a rapid uptake in which all the radioactivity is rather quickly incorporated. The difference between the whole cell and TCA insoluble fraction, designated as "pool", is small. This is in agreement with the findings of Roberts et al. (1957). For small doses of radiation there is no readily detectable effect at all. At 192,000 r the whole cell uptake rises nearly normally and then falls, probably because the cell membrane develops 80 ERNEST POLLARD L-Arginme- C incorporation hy E coli after Cobalt-60 irradiation 30 0 2 15 0 3 6 9 12 15 Fig. 1. — Incorporation of L-arginine into tlie whole cell (upjjer) and TCA insoluble fraction as affected by various doses of '""fo y-radiation. There is always a steady in- crease in the TCA insoluble fraction, but the whole cell fraction rises and falls. leakage dne to faulty growth. The fall is not observed in the same way for all amino acids. The uptake into the TCA insoluble fraction is re- duced and also delayed in its rapidity of uptake. Steadily increasing the dose steadily increases all the effects. The doses used, it must be noted, are very large. In Fig. 2 the amount of uptake at 6 min is plotted against dose using a logarithmic scale for the uptake. It can be seen that the relationship obeyed is in accord with the requirements of equation (1) and so a value for the inactivation volume, V, can be deduced. In Fig. 3 is shown the effect of deuteron bomliardment. The control behaviour is slightly modified by the fact that the cells are on a mem- brane and have to be removed before incubation, but the effect of radiation is clearly the same. Again, the doses used are veiy large. This same procedure was followed for histidine, leucine, isoleucine, proline and methionine. In addition, the uptake of i^C-uracil and ^^C- glucose was studied. RADIATION AND THE CELLULAR SYNTHESIS OF PROTEIN 81 YiG. 2. — The percentage uptake of L-arginine at 6 min as a function of dose. The plot of the percentage is on a logarithmic scale and it can be seen that if nlno is the ratio of uptake to original uptake, then the relation ln(w/«o) = constant X dose is obeyed. The results are shown in Fig. 4. In Fig. 4 we plot the nncorrected cross-section found from the relation \n{nlno) = —SD against the number of primary ionizations per cm generated by the bombarding particle. We can include the data for cobalt bombardment by realizing that if i is the number of primary ionizations per cm per heavy jDarticle then for particles of very low linear energy transfer the equivalent volume ionization density is the number of j^articles per cm'^ (D) times the ionization per cm for each one (^) so that / = Di. Since equations (1) and (2) are equivalent we can set VI = SD or VDi = SD or V =Sli. Thus V appears as an initial slope, on the S versus i graph and has been so represented. It can be seen from Fig. 4 that uracil and glucose be- have quite differently toward heavy particle bombardment than the other metabolites, with the possible exception of methionine. The sensitivity to cobalt irradiation is quite high and yet the expected high cross-section does not develop for heavy particle bombardment. The sensitive region is roughly sj^herical of radius 160 A in the case of uracil and 90 A for glucose. Since some radical migration must be occurring it w^ould be expected that the actual organelle size is rather less. Both 82 ERNEST POLLARD L-Arglnine- C incorporation by E. coli after deuteron bombardment 150 100 50 / * S-'"'^^^ / /^ 8-2x|0'° d/cm^ 3 6 9 12 IS l6-5x|0'°d/cm2 0 3 6 9 12 15 0 3 6 9 12 15 Fig. 3. — IncoqDoration of L-arglnine as affected by various deuteron bombardments. u 40 •x|0" \2 cm' • ^ <* 30 ^--'''^^ • Argmme V Methionine X j/^ ° Histidine D Isoleucine B Uracil 20 / ^ Leucine • Proline * Glucose 10 "/? fr a V h / a ^ 400 800 Radiation energy loss 1.200 1.600 electron ^ volts per 100 A Fig. 4. — A plot of the sensitive cross-section for seven metabolites against the rate of energy loss. The initial slojae is found from •'"Co inactivation. Three groupings appear: arginine, histidine, leucine, ^soleucine and proline are all characterized by higli sensi- tivity at high rates of energy loss; methionine and uracil have rather low sensitivities for such radiation, and glucose is consistentlj^ low. Probably naetabolites of the first grouping involve long thin objects, but the others are more nearly spherical. KADIATIOX AND THE CELLULAR SYNTHESLS OF TKoTKIX 83 these could be rejDreseiited by one or other of the various classes of ribosoiue. It is possible that methionine also fits into this class. In any event, the sensitivity of methionine uptake markedly differs from the other amino acids, suggesting that some other mechanism is involved for the incorporation of methionine. The data for the other 5 amino acids agree with a sensitive region having a molecular weight of between 3-5 and 5 x 10^ and having one thin dimension, roughly estimated as 30 A thickness. The molecular weight agrees rather well with that of the ribosomes, being equivalent to a particle of 70 Svedberg units. The shape does not agree and it is suggested that for these 5 amino acids the operating condition is one which is unfolded, while in preparations in broken cells, the particle is rolled up or folded in some way. Perhaps it is the function of the energy source to provide the unfolding. This w^ork was done in collabora- tion with Dr. E. S. Kempner. RADIATION ACTION ON THE FORMATION OF AN ENZYME The previous study is concerned with the almost immediate incor- poration of a labelled amino acid in the presence of a minimal medium. The radioactive tracer method is very sensitive and the studies are ^ T Unirr. 10 - /^ OJ E M C / /I5kr O / -t-J D 1 / 3 / y ^2lkr / --a 45 kr ^-Z::-::"--"'75kr 1 ._i 1 1 1 30 60 90 120 ISO Time (mm) 180 210 Fig. 5. — The effect of various doses on the time course of production of /3-galactosidase in cells already induced. For low doses there is a small increase initially. In general the enzyme is depressed and delayed. relatively simple. If we turn to the formation of an actual enzyme, ^-galactosidase, the sensitivity of assay is harder and it is not so easy to detect differences at short times. Using cells already induced by growth 84 ERNEST POLLARD on lactose,, no effect of radiation up to 75,000 r could be seen up to 45 mill after irradiation. To make the enzyme assay it is necessary to open the cells. The method used was rapid expansion under pressure and Avhile the recovery of enzyme was always good the data scatter more than for the radioactive uptake experiment. Nevertheless there is no reason to suppose that the immediate effect of ionizing radiation on enzyme formation differs greatly in sensitivity from that observed for amino acid incorporation. At later times after irradiation the story is quite different. A dose as low as 15,000 r quite clearly gives a reduced yield of enzyme. Data for different doses and different times are shown in Fig. 5. It is quite clear that a process which involves the develop- ment of the cell is at work. A clear decision as to the nature of this process cannot, at the moment, be made. RADIATION ACTION ON THE PROCESS OF INDUCTION For this particular enzyme it is necessary that cells which have grown on glucose become adapted, or induced, before they are able to make the enzyme if they are grown on lactose. The above described procedure can be applied to cells which have not been induced and radiation action on the process of induction can be studied. A typical time sequence is shown in Fig. 6. The first clear reading of the enzyme _o o cr 21,000 r Control turbidity Control enzyme Irr. turbidity enzyme 160 Time (min) Fig. fi. — The effect of 21.000 r on the larocess of induction of ^-galactosidase. The effect on turbidity is shown for comparison. in the control appeared at about 60 min and thereafter rose sharply and steadily. The irradiated cells also showed a small reading at 80 min which corresponds to the same as the control, but thereafter rose much RADIATION AND THE CELLULAR SYNTHESIS OF PROTEIN 85 less definitely and a])])eared to flatten somewhat between 140 and 240 niin. even though the culture was showing an increased turbidity. After 4 hr there is an increase in enzyme, but we also noticed some enz3^me leaking from the irradiated cells into the medium, a feature not observed in the controls. Increasing the dose had the effect of depressing the development of enzyme into the region hard to measure. After 320 min, a culture which had received (30,000 r barely gave a readable amount, although small readings had been recorded when the control began to show the presence of enzyme. The data are summarized to some extent in Table IV. The ratio of enzyme for irradiated cells to control cells is shown for various times. It was also observed that the amomit of enzyme, for both control and irradiated cells approximately follows the development of turbidity. It is once again clear that a process involving cellular development is involved. Table IV Dose Ratio of enzyme to control at intervals after induction (r) Omin 30 min 90 min 180 min 21.000 1 0-5 0-26 013 30.(l 1 0-7 0-5 0-04 60,000 1 0-3 0-12 0-06 UPTAKE OF 35S AS SULPHATE Radioactive sulphate is primarily incorporated as protein. A general idea of protein synthesis can be therefore obtained from an observation of sulphate uptake after irradiation. It w^as found that very little effect was observed on the ability of the cell to incorporate sulphate for times up to 12 min unless doses over 200,000 r were used. A rough estimate of the 37 per cent survival dose is 300,000 r. The immediate effect of radiation is thus very much like the effect on the uptake of amino acids and so is probably due to the disruption of ribosomes in some way. If longer times of uptake are considered there is a marked effect as can be seen from Fig. 7. The uptake follows the normal and then, rather sharply, deviates from the exponential line and becomes linear, in- creasing at lower rates w ith higher dose. The aj)pearance is rather like the production of enzyme and confirms the general behaviour seen there. Some secondary development process is again taking place. The irradi- ations described were under conditions which were nearly anaerobic. If oxygen was bubbled, a given dose had definitely more effect, both in 86 ERNEST POLLARD 10 20 30 40 50 Time (min) 60 Fig. 7.— The uptake of 3^804 after various doses. The linear uptake can be seen, also the fact that for very high doses there is an initial effect. .E u 0) J) 1^ o o ^ g o o o CC -O 20 40 60 80 j'-ray dose 100 kr Fig. 8. — The radioactivity observed in the ribosome fraction as a function of dose. The relative amount of ^-P rises slightly and then falls. the stage of departure from exponential incorporation, and also in the reduced slo])e. INCORPORATION INTO RIBOSOMES In order to attempt to see what mechanism in the cell is damaged in such a way as to cause the delayed effects the incori3oration of 32p and 35S into ribosomes was measured. If cells are broken open in the presence of sufficient concentration of Mg++ the ribosomes remain in- tact and can be separated by differential centrifugation. After clearing RADIATION AND THE CELLULAR SYNTHESIS OF PROTEIN 87 cell debris, the pellet formed after three hours' centrlfngation at 1 20,000 g was taken to be ril)osomes, following the work of Roberts et al. (1958). The amount of 32p and ^sg in the ribosomes of irradiated cells was com- pared to that in normal cells, in both cases after incubation for 60 min with labelled PO4 and SO4. The results obtained, in work in collabor- ation with R. Wax, are shown in Fig. 8. In the case of 32p there seems to be a very slight increase in the incorporation of 32p into the ribo- somes for small doses. For higher doses the proportion falls, with a sensitivity which corresponds to that found for the uptake of amino acids. In the case of ^^S there is a fall to a constant level. REFERENCES Hutchinson, F. (1960). A7ner. Nat. 94, 59. McCrea, J. F. (1960). Ann. N.Y. Acad. Set. 83, 692. McQuiLLEN, K., Roberts, R. B., and Britten, R. J. (1959). Proc. nat. Acad. Sci., Wash. 45, 1437. Pollard, E. C. (1959). Rev. 7nod. Phys. 31, 273. Pollard, E. C. (1960). Amer. Nat. 94, 71. Pollard, E. C, and Barrett, N. (1959). Radn Res. 11, 781. Pollard, E. C, Guild, W. R., Hutchinson, F., and Setlow, R. B. (1955). Progr. Bio])hys. biophys. Chem. 5, 72. Roberts, R. B., Britten, R. J., and Bolton, E. T. (1958). In "Microsomal Particles and Protein S^Tithesis", pp. 84-94. (R. B. Roberts, ed.) Washington Academy of Science. Roberts, R. B., Cowie, T>. B., Abelson, P. H., Bolton, E. T., and Britten, R. J. (1957). Carnegie Institution of Washington, Publication 607, Washington D.C. DISCUSSION HERCiK : Is there really a bend in the first curve? POLLARD : It is possible that it may depend on the precision of measurements and at jjresent we should be cautious about it. We have as yet but few data pointing to the presence of a bend. MARCOViCH : Is there a formation of the inductive enzyme within the cell or does an inhibition of the enzymatic activity take place? POLLARD : This is not yet clear. It would be interesting to find such an inducing agent which would not itself be a metabolite. ALEXANDER: While calculating the sizes of the protein particles in complex systems did you take into consideration the possibility of energy transfer from one molecule to another? POLLARD : Experiments carried out on jDure substances and on non-purified pre- parations (for example on pure DNA and on DNA in yeast) have shown that the accompanying substances do not alter the effect considerably, only about two- fold. ALEXANDER : Protection by transfer of energy to other substances may alter the effect not twofold, but up to tenfold. 88 ERNEST POLLARD POLLARD : It seems to me that within the cells there is no considerable protective effect. EREERA: Did the cross-section of the protein synthesizing particles for the ^sg incorporation coincide with that for the methionine? Were the cross -sections comj^ared for the cells with deficient and adequate amino acid and nucleotide supply? POLLARD : The first question we may answer in the affirmative, although it would be interesting to determine the specificity of the methionine's behaviour com- pared with that of its homologue. The second problem we have not yet studied although we intended to in order to determine whether ribosomes can exist in the extended as well as in the contracted form. PASSYNSKY: In the basic ec(uations \)i (h/do) = — ]I and In, {n/i^a) = SD the values V and S are not the volume and real surface of the target in the strict meaning of the word. They are but factors of probability, possessing the dimen- sions of volume and surface, but they differ from these by dissimulated non-de- nominated coefficients of proportionality K. If /v = 1, then these concepts coincide. For pure proteins results may be compared with those obtained by other methods and it could often be assumed that K = 1; this approach then gives some in- teresting results. But with regard to such complex intracellular systems as systems of protein biosynthesis or amino acid incorporation we cannot be sure that the condition K = 1 always holds true, and in such cases the dimensions calculated may differ considerably from the real values. POLLARD : That is cjuite correct. In model expei'iments on simple substances the agreement was gootl enough. Unfortimately, for the study of protein synthesis in a living cell there are no other ai:)proaches available to evaluate the dimensions of the structures involved. We believe that our investigations contribute sonie information and deserve some consideration along with other joroofs. It is possible that they have some importance. BACQ : It is 2^ossible that the peculiar behaviour of methionine is accounted for by its utilization by the cell not only for protein synthesis but for other functions, for example, connected with methyl group transfer. POLLARD : This possibility exists for other amino acids too. TOBIAS: Irradiation with doses as large as 2 x 10^ r could produce membrane lesions and loss of a quantity of certain substances (ions, nucleotides) from the cell which could alter the calculated results. POLLARD: Within 1.5 minutes we have not seen any loss of substances with the exception of arginine. Since there is an increase in cell mass we Ijelieve that active protein biosynthesis was taking j:)lace. TUMERMAN : Are there data available on the use of the electron microscope for the direct measiu-ement of the areas studied, since their flimensions are within the resolution range of this method. POLLARD : While studying irradiated viruses and ribosomes of the irradiated cells witli the electron microscope, no difference was found comjoared with non- irradiated preparations. It is unlikely that this approach would allow us to discern differences in the process of amino acid incorporation. RADIATION AND TJiK CELLULAR SYNTHESIS OF PROTEIN 89 EiDus: Is it. possible to relate the values for the diffusion tracks in dry objects given in the report to dclinite radicals (for example, those profluced flaring radio- lysis of the residual water) or to migration phenomena recorded by the ESH method? POLLARD: We have not studied the detailed picture of the process but ha\e just tried to observe effects on biological action. CHEIMICAL SPECIES INDUCED BY X-RAYS IN CELLS AND THEIR ROLE IN RADIATION INJURYf E. L. POWERS Division of Biohxjiad and Medical Research, Argonne National L(d) oratory, Argonne, Illinois, U.S.A. SUMMARY The experimental evidence for the existence of several chemical species in the dry bacterial spore after X-irradiation is reviewed and examined critically. One very short-lived species is recognized only if oxygen is present during the time of irradiation; another becomes toxic anoxically and is recognized only if hydrogen sulphide is present during irradiation. Certain characteristics of the long-lived free radicals that become toxic to the cell if they combine with oxygen are examined with special attention to reconciliation of biological and chemical evidence with jahysical evidence gained from ESR techniques. Post-irradiation thermal annealment and post-irradiation treatment with nitric oxide reduce the biological effectiveness of X-rays to the same degree; but the fonner approx- imately halves ESR signals, whereas the latter removes all signals almost com- pletely. One interpretation of these results is that two general kinds of radicals are formed, one of which is thermally annealable and biologically important the other of which is not. Both react with oxygen and with NO. While the removal of the two kinds of radicals with NO results in obliteration of the ESR signal, the biological result is the same as that seen after thermal annealment because only those radicals are important biologically. Another interpretation is that reaction of one kind of radical with NO results in a harmless complex, whereas reaction with a second kind results in a harmful complex. These studies and others similar to them, especially those involving the role of water in these effects, should lead eventually to some vmderstanding of the early effects of high energy radiations in cells. INTRODUCTION The "initial" effects of high energy radiations in living systems depend npon the same parameters as those in non-living ones, and any one interested in miderstanding the biological effects of irradiation should find assistance from studies on the interactions between radia- tions and purely physical systems. For instance the relationship be- tween stopping power and atomic number is undoubtedly the same in protoplasm as it is in a plastic, the very early chemical changes (i.e. those strictly non-enzymatic in nature) should not be different, and the t This work was performed under the auspices of the United States Atomic Energy Commission, 91 92 E. L. POWERS fates of the new chemical species must be governed by the same physical laws. In order to apply the knowledge of the physical systems to the bio- logical, one must design experiments to test parameters of the types ntilized in ordinary jjhysical and chemical radiation experiments. It is clear, however, that most functioning biological systems operate successfully only within narrow environmental limits, and it is difficult to apjily many physical experimental techniques to them. In conse- quence there has been little progress in the understanding of early events induced by high energy radiation in living systems. Very recent studies of two general kinds provide hope that descrip- tions will be forthcoming of some of the early I'adiation-induced events in cells that are important in bringing about biological effects. In aqueous systems the use of very short pulses of radiation coupled with fast, sensitive, detecting devices can circumvent the severe difficulties one meets in analyzing a series of events that goes to completion in a fraction of a second (see L. H. Gray in this Symposium). The other general approach that promises early success is the use of dry biolog- ical materials that can l)e exposed to a wide range of environmental circumstances like those useful in physical experiments, and in which, in addition, reactions are sufficiently slow to allow analysis with ordinary techniques. Direct extension of the results obtained with this kind of material to the wet biological system is difficult, because water undoubtedly modulates the series of reactions. But the evi- dence from the dry system aaIH provide a basis for asking many questions, one of which is the difference made by the introduction of water. The second approach is being pursued in several laboratories in which a variety of biological materials such as plant seeds, pollen, dry bac- terial cells and spores, and viruses are being investigated. In this lal)oratory we have been systematically investigating the response of dry l)acterial spores to X-rays under a variety of environ- mental circumstances. We have evidence for several of the early events, and have been able to describe the characteristics of some of the pro- ducts of irradiation and their relationship to radiation-induced damage. In this paper we shall summarize briefly the evidence for several kinds of identifiable chemical species, for the relationship of oxygen to them, and the extent to which they participate in the biological damage caused by X-rays. The results demonstrate the existence of the follow- ing: an oxygen -independent portion (Class I) that can be sub-divided into a radical s})ecies (lb) and one that may not be radical-like (la); an oxygen-dependent portion that consists of very short-lived species CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 93 (Class 11); and an oxygen -dependent poilion that consists of long-lived free radicals (Class 111). THE BACTERIAL SPORE SYSTEM We use spores of a strain (ATTC #8245) of Bacillus megaterium that has demonstrated good spornlating capacity. The spores are mounted in known numbers on precipitated cellulose discs (called the Millipore Filter) O-l nun thick (Powers et al. 1957 ; Kaleta and Powers, 1958). The discs carrying the spores are dried in a chamber at less than 1 mm Hg. pressure over a drying agent for several hours, and are kejjt in this chamber until ready for use. Colony formation is induced by putting the paper disc on an absorbent pad saturated with liquid nutrient medium. The spores germinate and give rise to colonies on the surface of the disc. Control, unirradiated discs demonstrate 100 per cent re- covery of mounted spores. Experimental methods When exposing the spores to X-rays, we utilize the chaml)er shown in Fig. 1. (Webb et al, 1958). The discs are put on the bottom of a stain- less steel cylinder. The gas surrounding them can be controlled by X-ray tube Vacuum seal — Liquid heliufTi Rubber sleeve Potentiometer Membrane filters 2in exposure cylinder Thermocouples Liquid helium Liquid nitrogen Fig. 1. .—Diagram of the basic exposure chamber, for controlhng atmosphere and tem- perature before, dm'ing, and after irradiation. (From "Webb et al., 1958). means of pumps and valves connected to the manifold that is between the cylinder and the X-ray tube. The temperature within the cylinder 94 E. L. POWERS can be maintained by means of constant-boiling liquids in the surround- ing vessels. The temperatures during irradiation are monitored by means of thermocouples placed at the bottom of the cylinder. The measure of radiation sensitivity in all the studies described here is the slope of the survival curve expressed in reciprocal kiloroentgens. The response curves usually have small shoulders in the low-dose regions. A convenient expression for describing them is Fraction surviving = 1 — (1 — e -^'Dyn in which the two constants have the following meanings : k is the slope of the curve in kr~i and is the measure we use of radiation sensitivity ; w is a measure of the size of the shoulder in the particular experiment, and in some other papers is referred to as the "hit number". We refer to the slope as the "inactivation constant", and to 7i as the intercept number, because of the fact that extrapolation of the straight line por- tion of the response curve on a semilog plot gives the value of w as the interce]5t on the y axis. All of the experimental variables are tested with complete survival curves, and all I'esponse curves are reduced to their respective slopes. The values of n in these experiments vary about a mean of 1-30. They are regarded as constant and without significance in the studies we review in this paper. The biological response being measured in these experiments is the ability of the irradiated spores to germinate and to produce visible colonies; no other endpoint is being considered. THE LONG-LIVED RADICALS (CLASS III) The radiation response can be divided into a number of categories, that is, the individual cell may be damaged in a number of ways. The first of these that we shall consider is that damage which is brought about as the consequence of the production of chemical sjiecies with unpaired electrons, termed "free radicals'". In our system these have appreciably long lifetimes, and are related to oxygen in a particular way in the production of the radiation damage. Thennal evidence We repeated with the spores the study of the basic tem])erature re- sponse of radiation sensitivity in an oxygen-free environment studied earlier in T 1 virus (Bachofer et al., 1953), and noted that, as the tem- perature is varied from very low temperature (5°K) to liigher tempera- tures, radiation sensitivity does not change until the temperature reaches 12r)'K (Webb et a).. 1958; Webb and Powers. 19(U). At that CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 95 point, radiation sensitivity increases slowly to a maximum at 30°C (303°K) (Fig. 2). It tluMi decreases markedly, and reaches a minimum at 80°C at a level that is appreciably below that ol)served even at the very lowest temperatures. While it is not ])ossible to interpret these results in terms of free radicals from this evidence alone, we shall see that this is consistent with and contributes to, the free-radical hypothe- sis. The marked inversion of radiation sensitivity at the higher tempera- tures is the consequence of annealment of free radicals. The effect of post-irradiation exposure of the spores to heat is described in the next paragraph. n O o u c: g +-> o > u o 0 50 100 150 200 250 Temperature Fig. 2. — The relationships among radiation sensitivity of spores (the ordinate), tem- perature during irradiation (the abscissa), and post-irradiation thermal and NO treat- ments. (From Powers et al., 1960a). HaO-free Spores Curve Exposure gas Post-irradiation treatment • O A X N2, He O2 N2, He N2, He 80°C NO Nitric oxide In another series of experiments, w^e used the gas nitric oxide as a modifying agent (Powers et al, 1960b). This gas is most effective when 96 E. L. POWERS presented to the spore after irradiation. In Fig. 2 we show that apjjroxi- mately 50 per cent of the total effect of irradiation can be removed by post-radiation treatment of spores with nitric oxide. This effect is inde- pendent of the temperature during irradiation, and the effect of expos- ing the spores to X-rays over the temperature range and treating them with nitric oxide after irradiation is to shift the entire curve down without changing its general form. The same figure demonstrates that coincident values are obtained by post-irradiation heating of the irradiated sjDores for 15 minutes at 80°C. Heat and nitric oxide exposure after irradiation accomplish the same tiling; namely, they induce changes in the irradiated spore that pre- vent the development of part of the damage expected from the given radiation dose. The coincidence of these results, the well-known action of nitric oxide in reducing free radical concentrations in physical systems, and the well-known effect of heat in reducing free radical con- centrations, lead us to conclude that the portion of radiation-induced damage that is not observed following these treatments is caused by radiation-induced free radicals that have appreciably long lives. It should be noted that this radical component is independent of the temperature (below 30°C) at which the spores are irradiated — the over- all I'elationship between temperature and radiation sensitivity is the same in the restored and in the unrestored spoi'es. When nitric oxide is present during irradiation, the degi'ee of pro- tection observed is less than that seen when nitric oxide is given after (Powers et al., 1960b). In Fig. 3 we see that the level (inactivation con- stant) to which nitric oxide protects the cells when present during irradiation is approximately 25 per cent higher than that seen when nitric oxide is given after irradiation. This result is understandable if we postulate two actions of nitric oxide : one, a protective action that is due to the scavenging of radicals by nitric oxide that prevents their becoming toxic to the cell ; and the other an enhancing effect of nitric oxide on tlie action of X-rays. The enhancing effect is much smaller than the protective effect, and the net result of the two actions is a lower protective action of nitric oxide when present during irradiation compared to its action afterwards. Hydrogen suliMde Because of the interest in protective chemicals containing sulphydryl groups, and because of the possibility that these may act by scavenging radicals, we have tested a number of sulphydryls for their action against the long-lived radical component. Using the gas hydrogen suljihide (Powers and Kaleta, 1960), we have been able to show that CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 97 when given after irradiation it is equivalent to nitric oxide given after irradiation, and to post-irradiation heat. The level to which the cells are protected by the gas is a]>proximately the same as that to which radiation sensitivity is reduced by heat and NO. Tliis result is consistent with the interpretation that this part of the effect is due to long-lived free radicals : in this instance H2S is donating a hydrogen atom to the free radicals, repairing these before they can become damaging. When hydrogen sulphide is present at the time of irradiation, a degree of protection is seen exceeding any that we have observed in any other 42 1 1 1 1 1 38 ' k ■0 0 ./^'^o. ^ X 1 34 / I- - / . 4-> c 30 N2(0-0290 kr" ') 0 4-> in 26 - 0 u " 0 0 22 — NO during 0 > '4^ 18 ~ ^ /-> r 0 ^ u McrS^— 0 0 c 14 _ ^^^ 0 9 ^•^^ >^ _ ^NO after 10 1 1 1 1 1 0 5 10 15 20 Percentage gas in He or N2 O - o •4- 100 jrig_ 3. — Changes in I'adiation sensitivity with concentration of O2 and NO present at the time of irradiation. (From Powers et al., 1960b). circumstance. This indicates the presence of another kind of radical that can accept hydrogen atoms from hydrogen sulphide. The lifetime of this species must be very short compared with the lifetime of the radicals that can be removed by post -radiation treatment. This is dis- cussed below. The role of oxygen The relationship of oxygen to these effects is revealed by the follow- ing series of experiments. When we irradiate spores in the presence of oxygen, we see a gradual increase in radiation sensitivity with an in- crease in oxygen concentration to a constant level that is reached at about 10 per cent oxygen (Powers et al, 1960a, b and Fig. 3). The ratio 98 E. L. POWERS of the values at saturation is 1-25; that is, in the presence of oxygen, spores exhibit 35 per cent more radiation sensitivity than they do when irradiated in the presence of nitrogen and exposed to oxygen imniedi- atel}^ after. This is a small oxygen effect compared with the oxygen effects of about 300 per cent usually observed in wet systems (Gray, 1957-8). This, however, is not the total oxygen effect that can be demonstrated in the bacterial spore. As shown in Fig. 2, the radiation sensitivity in the presence of oxygen increases more rapidly with in- creasing temperature than it does in its absence. At room temperature the ratio observed is about 1 -25 as noted on the jDrevious figure. Above 30°, the dramatic decrease in radiation sensitivity observed in the absence of oxygen cannot be demonstrated when oxygen is present (Powers et al., 1959). This indicates that oxygen prevents the thermal reversion of the free radicals we observed in the other experiments. Verification of this can be obtained by irradiating the spores in nitrogen at any one of the temperatures below 30°, exposing them to oxygen briefly, removing the oxygen, and then treating with nitric oxide or heat. In these instances, the radiation sensitivity is not affected by heating or by the gas. Removal of the free radicals is possible, then, only if they are manipulated prior to their exposure to oxygen. These facts mean that the free radicals and oxygen form complexes that are irreversible, and that are damaging to the cell. This oxyradical or per- oxyradical should be very strongly oxidizing, and should undergo a secondary reaction to produce damage to the cell. If the free radical is disposed of before oxygen is admitted, the damaging complex cannot be produced. The i)hysical evidence We have physical evidence in the form of electron spin paramagnetic resonance (ERR) analysis that supports our interpretation that part of the radiation damage is brought al)out bj^ long-lived free radicals to- gether with oxygen (Ehret et al.. 1960; Powers et al., 1961). When the spores are irradiated at low tem])erature and an ESR spectrum is re- corded, we observe a second derivative tracing with three peaks with 30 (t spacings (Fig. 4). If these spores are l)rought to room temperature for brief periods, and then returned to low temperatures for reading, twin ])eaks 10 G on each side of the centre line grow in at the expense of the central peak. After about 20 minutes at room temperature, the fully developed spectrum can be seen: it consists of the originally observed triplet with a 1:2:1 configuration, and a newly developed doublet with a 1:1 configuration. CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 99 Therefore, the s])ecies produced iininediately in the spore are not the only ones that are finally observed — migration of energy within the spore after irradiation must take place. Also, theESR apparatus reports to us only two general kinds of rad- icals, one an electron associated with two protons and the other associated with a single proton. The simplicity of this result is quite unusual, and probably indicates that in the well-ordered biological system, the energy from the X-rays, although deposited at random, migrates and becomes fixed in a limited number of ])laces. We have treated the irradiated spores by the methods that influence 4xl0*r in He at-l95°C Warmed in He at 25 C (00 gauss ^ Fig. 4. — Changes in the ESR spectrum (2nd derivative) with time after irradiation of dry spores at 77°K. The spores were returned to 77 "K for reading after each warming period. The arrow marks the position of g = 2-003. (From Ehret et al., 1960). radiation sensitivity as judged by biological criteria, and the results are shown in Fig. 5 (Ehret et al, 196U; Powers et al, 1961). When the irradiated spores are heated for 15 min at 80°, there is a loss in ampli- tude with no qualitative change in the signal. The decrease appears to be approximately 50 per cent, in agreement with the biological experi- ments. When the spores are exposed to oxygen, we see the gradual growth of a single oxyradical (peroxyradical) signal from the finely split signals of the two other radicals. The radicals become oxygen com- plexes; they seem to be irreversible, for removal of oxygen from the system does not affect the signal. This is expected according to the bio- logical result. Furthermore, the oxygen signal that develops from the heated spores is small, in correspondence with the fewer number of radicals available to form the complex. Now we discuss the question as to whether the radicals that cannot be annealed by heat are responsible for damage when they are finally 100 E. L. POWERS exposed to oxygen. When the irradiated spores are exposed to nitric oxide, abnost complete obHteration of the radical signal is observed (Fig. 5). At first glance this seems to be anomalous, for the biological consequence of heat and the nitric oxide treatment is the same, whereas the physical result appears to be different. However, instead of indi- cating limited usefulness of the experimental approach, the result might be amenable to an interesting explanation. Either of two possibilities exist. First, the compound formed by the reaction of oxygen and the radicals remaining after heat annealment H (e) 100 gauss Fig, 5. — The effect of various treatments on the E8R signal seen in irradiated bacterial spores, (a) The fully developed doublet-triiDlet signal, (b) The signal observed after exposure of the irradiated spores to oxygen. The dotted line is the signal intei'mediate Ijetweea (a) and the heavy line of (b). (c) The effect on (a) of heating the spores at 80"C for 15 minutes, (d) The effect of exposing the heated spores of (c) to oxygen, (e) The signal observed after tlie spores of (a) are exposed to NO. (From Ehret et al., 1960). may not be important in the damage that we see, for in the NO-treated cells these cannot be formed, yet the biological result is the same in the two instances. Or, second, reaction of nitric oxide with these radicals brings about cellular damage — that is. the reaction between nitric oxide and the residual, non-annealable radicals is equivalent to the reaction l)etween these residual radicals and oxygen. The reason there is no evidence of the nitric oxide-radical complexes is that after reaction there are no unpaired electrons, but the chemical or biochemical con- sequences are the same as those seen following reaction with oxygen, forming compounds with un])aired electrons that can be measured by the ESR spectrometer. If this is true, we can subdivide the long-lived CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 101 radicals we observe once again into those which can be liarnilessly removed by reacting with nitric oxide bnt are toxic after reaction with oxygen, and those which become toxic or damaged by virtue of their reaction with nitric oxide or with oxygen. There are then two ways in which the subdivision of Class III into two components can be viewed, each implying different characteristics of the radicals in question. One is as follows: Ilia 1. In presence of *02* Ra* > Ra02* (toxic) 2. Removable by heat (Anneal.) Ra' "^ > RaH (not toxic) 3. Reacts with NO* Ra* > RaON (not toxic) Illb Not annealable Rb' > Rb02* (toxic) Reacts with NO' Rb* > RbON (toxic) In this case the radicals Rt,* are not sufficiently mobile to be annealed by heat, or they are in some way shielded from H* atoms, or some other characteristic prevents their annealment, so that they persist and be- come active RO2* or RO' radicals when oxygen is admitted to the system. These very radicals are, however, available to NO* as shown by the ESR data, and if the foregoing statement is true, i.e. if they do become toxic radicals even after high temperature treatment, then the combination Rt,ON must be equally as toxic as Rb02*. Heat treat- ment and NO treatment produce the equivalent results biologically when used alone, and no additive effects of sequential treatments of the two agents has been demonstrated. If the above explanation is true, we see a distinction between the two radicals on two counts: one (Ra*) is heat annealable, and the other (Rb*) is not, both being toxic when in the form RO2*; and one (Rb*) is toxic as RON and the other (Ra*) is not. Another general possibility is seen by setting up the characteristics of the two radicals as follows : Ilia — All toxic when in Ra02* form 1. Annealable Ra' > RaH (not toxic) 2. Reacts with NO* Ra* > RaON (not toxic) Illb — Not toxic when in Rb02* form 1. Not annealable, but Rb* > Rb02* (not toxic) 2. Reacts with NO' Rb* > RbON (not toxic) In this scheme, the diff"erences between Ra* and Rb' is that one (Ra') is annealable, the other (Rb*) is not; and only the annealable 102 E. L. POWERS radical forms the toxic RO2* radical. The radical that is not effectively available for annealment apparently is not available for secondary reaction after formation of an RO2' radical. It is trapped with respect to annealment, and, though available to O2, as shown by the ESR result, it remains effectively trapped after reaction with O2. Both are available to NO, and are converted into non-radical RON species, but smce only one (Ra*) is biologically important, the removal of Rb' by NO, an effect that results in an ESR spectrum different from that caused by annealment, does not cause reduction of damage beyond that observed after annealment. A choice between these two general possibiUties is not required by the evidence at hand today. THE SHORT-LIVED, OXYGEN-DEPENDENT SPECIES (CLASS II) Another class of radiation -induced chemical change can be infei'red from the data previously examined. We have noted that O2 when present at the time of irradiation increases radiation sensitivity by 30 per cent. We have also seen that the presence of nitric oxide at the time of irradiation decreases the protective capacity of the gas by 20 per cent, as compared with nitric oxide given after irradiation; or. in other words, nitric oxide at the time of radiation increases radiation effect by 20 per cent, as compared with nitric oxide given after irradia- tion. It can be allowed that oxygen and nitric oxide accomplish the same thing in increasing radiation sensitivity. If oxygen and nitric oxide are in some way preserving energy within the cell for damage that is absorbed primarily by other molecules, then the lifetimes of these "excited" molecules must be very short. We are considering at the present time one possibility, namely, the action of nitric oxide and oxygen in catalyzing certain degradations of excited species (Powers et al., 1960a). One of these could be the quenching of fluorescence, i.e. the non-radiative transformation of an excited species to some other form (e.g. an excited singlet to a triplet). In this case energy that in the absence of the two gases escapes from the cell in the form of a photon is i^reserved within the cell and becomes damaging when nitric oxide or oxygen is present. THE SHORT-LIVED, OXYGEN-INDEPENDENT SPECIES (CLASS lb) One other class of radiation-induced chemical change can be postu- lated on the basis of the results of irradiating the spores in the presence of hydrogen sulpliide. As noted earlier, radiation sensitivity appears to be below that observed when hydrogen sulphide treatment is given after irradiation (Powers and Kaleta, 1960). We conclude from this CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 103 that hydrogen sulpliide can donate hydrogen atoms to a radical, or some species, that has a very short lifetime, and that can very rapidly become toxic to the cell in the absence of oxygen. This constitutes part of the oxygen-independent portion of the general response. THE RADIATION SENSITIVITY PROFILE The studies described above enable us to construct a diagram inter- relating the various kinds of damage we can infer from some of the experiments to date. We call this the "radiation sensitivity profile" (Fig. 6; Powers and Kaleta, 1960). The one we demonstrate in this O2 -independent-*' 3S7o la Oj- dependent 62% lb NO 'after in H2S during Heat after n N2 during O2 during 0 9-0 14 -24%— l47o- Very short- lived or 'immediate" species Short- lived radicals 29-0 38-0 38% Long-lived radicals 24% Very short lived or 'immediate" species — I-3IX^ 2-60 X- 4-22 X Fig. 6. — The radiation sensitivity profile of bacterial spores irradiated with soft X-rays (18 keV mean) at about 16 kr/min at room temperature. On the heavy horizontal line are indicated the inactivation constants observed under the described circumstances. The Roman numerals are the designations for the various components of radiation injury. (From Powers and Kaleta, 1960). discussion is for X-rays of mean energy about 1 8 kV delivered at a dose rate of approximately 16,000 r/min at room temperature. It is necessary to specify these three items, since the relative response of each of the classes diagrammed may change with changes in these variables. COMMENTS These and other experiments already performed or planned should result in a partial understanding of some of the early events in this cell caused by high-energy radiation. The experiments under way include the effect of dose-rate, linear energy transfer, and the inteiTelation of temperature with these. The very important problem of moisture is 104 E. L. POWERS being studied to attempt to bridge the gap between our dry system and the wet, metabolizing cell (Webb and Powers, 1961; Tallentire and Powers, 1961). ■]" In this way we hope to make some contribution to the understanding of the interactions between high-energy radiation and functioning, living cells. Even at this time, we can describe a mechanism of action of sulphydryl comjaounds in protecting the bacterial spore, and can wonder as to its applicability to other biological systems. In the case of the spore, the sulphydryl (H2S in this instance) j^rotects by reducing O2 tension and by donating H atoms to free radicals making them harmless. The immediate O2 effect (Class II) cannot take place, and the long-lived radicals (Class III) are scavenged before O2 tension is increased again to the point that O2 can react with the 02-dependent radicals. Also, the 02-independent radicals of short lifetimes can be removed harmlessly as they are formed. Thus, the effect of H2S is the equal of the total oxygen effect, and may exceed it somewhat. REFERENCES Bachofer, C. S., Ehret, C. F., Mayer, S., and Powers, E. L. (1953). Proc. nat. Acad- Set., Wash. 39, 744. Ehret, C. F., Smaller, B., Powers, E. L., and Webb, R. B. (1960). Science, 132, 1768. EiDUS, L. K., and Ganassi, E. E. (1959). Biojjhysics (Riiss.), 4, 215. Gray, L. H. (1957-8). "'Lectures on the Scientific Basis of Medicine," Volume VII, pp. 314-347, The Athlone Press, London. Kaleta, B. F. and Powers, E. L. (1958). Report of Division of Biological and Medical Research, Argonne National Laboratory. ANL # 6U93 pp. 78-81. Powers, E. L., and Kaleta, B. F. (1960). Science, 132, 959. Powers, E. L., Ehret, C. F. and Bannon, Anne (1957). Appl. Microbiol. 5, 61. Powers, E. L., Ehret, C. F. and Smaller, B. (1961). In "Free Radicals in Biological Systems", pp. 351-366, Academic Press, New York. Powers, E. L., Webb, R. B. and Ehret, C. F. (1959). E.vp. Cell Res. 17, 550. Powers, E. L., Webb, R. B. and Ehret, C. F. (1960a). Eadn Res. Suppl. 2. pp. 94-121. Powers, E. L., Webb, R. B. and Kaleta, B. F. (1960b). Proc. nat. Acad. Set. Wash. 46, 984. Tallentire, A., and Powers, E. L. (1961). Radn Res. 14, 510. Webb, R. B., and Powers, E. L. (1961). Radn Res. 14, 515. Webb, R. B., Ehret, C. F., and Powers, E. L. (1958). Experientia, 14, 324. DISCUSSION bacq: At what dose-rate were the spores irradiated? POWERS : In all the experiments reported here the dose-rate was 20,000 r/min. We considered it imjDortant that this condition be always fulfilled. It could be shown that as the dose-rate changes the general picture of the radiosensitivity is altered also. t Tlie work of Eidus and his colleagues (Eidus and Ganassi, 1959) is of interest in this connection. The loss of enzymatic activity of aqueous solutions (1 per cent) of mysoin after irradiation parallels our resxxlts on the bacterial spore. They observe an inunediate oxygen effect, and a post-irradiation oxygen effect, tliat correspond closely in magnitude to these effects in the bacterial spore. CHEMICAL SPECIES INDUCED BY X-RAYS IN CELLS 105 BACQ : At what time after the exposure to radiation were the spores treated with nitric oxide or hydrogen sulphide? ro\\EKS : A\'e found tliat for 1 -5 to 3 In- at room temperature the relative concen- tration of the radicals we dealt with did not change considerably. This j^oint was carefully studied and in our last paper the kinetics of these phenomena at different temperatures are given. BACQ : It is a great pity that mammals are not bacteria and could not be fitted into yoxir data. HOLLAENDER: Did you study any physiological or genetic phenomena? POWERS : We were interested in morj^hological characteristics of the colonies and tried to get a deeper insight into the j^hysical i^arameters which are of importance here. Very little attention has been given to other biological processes outside the scope of our investigation. TARUSSOv : Can nitric oxide be regarded as an inert substance with regard to the objects you studied? POWERS: \^Tien oxygen is excluded the nitric oxide by itself is not toxic for bacteria. In other words the presence of nitric oxide i^roduces no harmful effect on the sjDores ; it does not affect them in any way. The same may be said for hydro- gen suljihide. I should like also to answer Dr. Gray's question. When we use nitric oxide, it niust be carefully removed afterwards from the system. Otherwise some toxic phenomena may occui'. TOBIAS: From the sm'vival curve it may be seen that a lethal effect is present. Can you determine the quantity of the radicals in one spore, in ten thousand or in other great quantity of spores? "WTiat is the precision of the experimental tech- nicjue you have used? POWERS : We are very hajopy that we can seek the advice of the jDhysicists who use ESR methods. passynsky: Is it jDossible to determine the radicals' concentration using more simple substances or some fractions of the spores studied? Or do they appear only when all the complexity of the .spores' composition is retained? POWERS : Of course, there is the possibility of carrying out these measurements on cellular fractions, but we have not done it. I may call your attention to the fact that quite recently in Dr. Gordy's laboratory there was observed, while studying caesin, an increase in the size of the doublet signals. It is almost identical to what I have shown today for spores. PASSYNSKY : Was the quantity of the radicals determined directly vuider the beam? POWERS: No. All the measurements were performed after the exposure and, by the way, in c^uite another building. Experimental material was transferred to other conditions. BARENDSEN: What could you tell about the cross-section and the effects of the linear energy transfer? POWERS : Our experiments with irradiations of different ionization density were carried out together with Dr. Tobias' gi-oup in California using the HILAC. At present I can tell you that as linear energy ti'ansfer increases, radiosensitivity 106 E. L. POWERS changes, and the general outUne of the survival curve depends on the ioniza- tion density. I would like to show our preliminary data but they are as yet too few. ALEXANDER: ^'\^ly do you think that ESR spectra are in some way related to chemical transformations that cause damage? When ESR measurements are taken, it is impossible to distinguish, whether the reactions in progress are important to the cell or whether they are trivial biological processes. POWDERS : It is very difficult to distinguish which mechanisms are more important and have crucial significance and which play a trivial role. I tried to present my material here very cautiously. I said that the role of the free radicals may be estimated from the biological effects. All the experiments listed above throw some light on the biological effects also. ESR data are only used as supporting evidence. ALEXANDER: It is with satisfaction that I hear that ESR methods could help in the elucidation of biological problems. The data you have presented on the free radicals are in favour of this method and justify the hopes it arouses. POWERS: My attitude towards EvSR methods is very reserved since here we enter the sphere of physics. This method should be used very carefully and employed as a control. I do not consider this method a universal one. FLUORESCENCE STUDIES OF THE CHANGES UNDERGONE BY NUCLEOPROTEINS AND THEIR DERIVATIVES IN IRRADIATED CELLS M. N. MEI8SEL, E, M. BRUMBERU, T. M. KONDRATJEVA AND I. J. BARSKY Institute of Biophysics, Institute of Badiation and Physico-chemical Biology, Academy of Sciences of the U.S.S.K., Moscoiv, and Central Scieyxtific Research Institute of Medical Radiology, Leningrad, U.S.S.R. SUMMARY Radiation-induced changes in nucleoproteins, nucleic acids and nucleotides in the cytoplasm and nuclei of living cells are described in the paper. These changes are detected on preparations vitally fluorochromed with acridine dyes, by fluorescent microscopy in the visible spectral region, as well as by an investi- gation of the auto -fluorescence registered in the ultraviolet spectral region. The processes described reiDresent labilization and denaturation of DNA- proteins, accinnulation of RNA and nucleotides in the cytoplasm, and changes in theii" x^hysico -chemical properties which have an effect upon the character of the fluorescence. The intensity and the spectrvim of ultraviolet fluorescence of the cells in radiosensitive organs undergo considerable changes soon after X- irradiation. Ultraviolet fluorescence microscopy aj)plied to the study of radiation damage to cells yields, especially in combination with ultraviolet absorption microscopy and fluorescence microscopy in the visible spectral region, new facts about the state of cellular nucleoproteins, and their early and later changes due to radiation. The first reports of the use of fluorescence microscopy in radio- biological studies appeared more than twenty years ago (Wels, 1938; Hercik, 1939; Biebl, 1942). Considerable advances made in this subject in the following twenty years extended its scope and led to a more knowledgeable use of the technique in various branches of biology, in- cluding radiobiology. Progress was facilitated firstly, by the introduc- tion of fluorescent stains, fluorochromes, of low toxicity, suitable for vital and supravital investigations; secondly, by the development of fluorescence cytochemical methods, especially the cytochemistry of nucleic acids (Meissel and Korchagin, 1952; Armstrong, 1956; Schiim- melfeder et al., 1957; Bertalanffy and Bickis, 1956); and thirdly, by considerable improvements in the apparatus permitting fluorescence microscopy to be combined with phase contrast and ultraviolet absorp- tion techniques (Brumberg, 1955; Haselmann and Wittekind, 1957). In this respect special mention should be made of the procedure 107 108 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY whereby the microscopic object is irradiated with incident Hght de- scending through the objective, a special fluorescence opaque iHumin- ator being employed (Brumberg, 11)55). Besides a number of advant- ages of a fluorescence nature in this case, considerable possibilities are laid open by the ability to observe one and the same site of an object simultaneously by phase contrast, dark field and ultraviolet methods and the determination of the character of the absoi'ption. FLUORESCENCE MICROSCOPY IN THE VISIBLE SPECTRAL REGION Strugger's (1940) observations on the specific behaviour of acridine orange towards healthy, pathologic and dead cells led Krebs (1947), Krebs and Gierlach (1951) and others to use this fluorochrome in radiobiological investigations. It was found in a number of cases, par- ticularly with botanical material, that radiation-damaged cells accu- mulated larger amounts of acridine orange than normal cells and as a result emitted light of longer wave-length (yellow, orange or red). In 1953 Strugger, Krebs and CJierlach carried out studies on the early manifestations of injury caused by X-rays in plant cell cytoplasm. In 1947 we began our studies which were centred on unicellular plants, cultures of animal cells in vitro and the hemopoietic organs of various animals. In this report we shall attempt to give a generalized survey of the results. The first communications (Meissel and Zavarzina, 1947 ; Meissel et al., 1951) jDointed out that acridine orange binds diff"erently with the nucleic acids of the nucleus and of the cytoplasm, imparting to these compounds fluorescence of different colour. In 1952 Meissel and Kor- chagin showed, on nucleoproteins isolated from the microbial cell, that deoxyriliose comjDounds form green fluorescent complexes with acxidine orange, whereas under the same conditions this same fluorochrome forms red fluorescent complexes with compounds of the ribose type. These findings, confirmed later by a number of other investigators, made it possible to determine with certainty the distribution and be- haviour of nucleoj^roteins in irradiated cells under vital and supravital conditions. The very first reactions towards radiation of cellular nuclei showing a light green fluorescence in the normal condition are manifested in a marked increase in the intensity of fluorescence of the nuclear mem- branes. The delicate internal structures of the nuclei harden, the nucleolus and nucleoprotein granules swell, assuming a droplike or pooly apj^ear- ance with increased intensity of fluorescence. It is at that time that a FLUORESCENCE STUDIES OF NUCLEOPROTEINS 109 characteristic separation of the nucleoproteins into two fractions begins to make its appearance. One fraction weakly flnorescent, lias a green colour and the other fluoresces with a more 1)rilliant, whitish light. At first the latter fraction is in close contact with the former, but gradually, evidently turning more fluid, it becomes mobile, circumventing the larger nuclear structures, nucleoli, nucleoprotein granules and the internal surface of the nuclear membrane. The next stage is a sharper differentiation of the nuclear material. The amount of the brilliant white fraction increases, forming droiDlike accumulations that assume a greenish yellow colour and are often to be found touching the membrane. At this stage one may observe nodal enlargements of various shapes. At times this is accompanied by partial secretion of the brilliant white nucleoprotein fraction from the nucleus. The nuclear membrane becomes markedly thicker and begins to fluoresce with a yellowish green colour. The nuclear structures become undifferentiable. In separate areas of the nucleus may be found accumu- lations of the more fluid brilliant fraction in the form of two or three pools or droplets. In the next stage the nuclei disintegrate and in their place remain variously shaped accumulations of highly fluorescent brilliant white substance. We believe that the changes observed in the nuclear matter are associated with the various stages of denaturation of DNA-protein, with its labilization and with the separation of DNA from the protein. The initial stages of DNA-protein denaturation are evidently accom- panied on increase in complex formation with acridine orange with a resultant intensification of the fluorescence of the complex. The sej^ara- tion of DNA from the protein is characterized morphologically by the appearance in the nuclear chromatinic structures of a substance exhibit- ing a bright whitish-green fluorescence. Further intensiflcation of the fluorescence and a shift of the colour in the longer wavelength direction is accompanied by liquefaction of the substance (clearly seen in the microscope), and by a fall in viscosity, as a result of which the substance flows around the nuclear structures, forming drops and pools. In this way vital fluorochroming of the irradiated cells allows one to observe directly the various stages of denaturation and depolymerization of DNA-protein. Such in general are the impairments in the nucleoprotein structures of the nucleus proceeding at various rates depending upon the dosage and upon the time elapsing after the radiation, as well as upon the radio- sensitivity of the cells. A highly interesting effect, brought to light only with the aid of fluorescence microscopy is the change in the nature of the fluorescence of the damaged cell nucleus, a considerable increase in 110 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY fluorescence intensity and a characteristic shift in the fluorescence spectrum. The change in colour of the fluorescence of the acridine orange complex may be brought about either by the so-called concen- tration effect, described as long ago as 1940 by Strugger or through formation of dimeric and trimeric cations of the fluorochrome, a process investigated by Zanker (1952). Both these effects in the cases under consideration depend upon changes in the physico-chemical state of DNA-protein or of DNA. Their binding with diaminoacridine increases even in the initial stages of denaturation. It increases particularly on depolymerization of the highly polymeric DNA, complexes being formed of the type of those given by RNA with diaminoacridine. According to the concepts being developed by Bradley and Felsenfeld (1959) the degree of aggregation of acridine orange cations when the dye is being bound with poly anions dejiends upon the configuration of the polymer. It is highly probable that the passage of DNA from the rigid bihelical to the more mobile helical structure during denaturation is accompanied by intensification of the dimerization of acridine orange cations. This may serve as the explanation of the shift towards the right of the fluorescence spectrum exhibited by the complex formed. Whichever of the interpretations proves to be correct on further study one may even now assert that the changes in intensity and colour of the fluores- cence of nuclear structures in irradiated cells are manifestations of important changes in the physico-chemical state of the nuclear nucleo- proteins. These changes are evidently very widespread and may be observed both in the cells of yeasts and in a variety of animal cells. We have found them in the l)one-marrow of totally and locally irradiated animals (Meissel and Sondak, 1955, 1956; Kondratjeva, 1956). They appear immediately after irradiation, with doses from 100 r upwards, in the form of individual cells or of cellular aggregates with sharply changed and brilliantly fluorescent niiclei. Such aggregates formed as the result of coalescence of the cells we have termed "micronecrotic." The number of such micronecrotic foci increases with the dose and the post-irradiation time (Figs. 1 and 2). The maximum number is found 6 hr after irradiation, following which they gradually disintegrate and are resolved. The substance binding the cells into micronecrotic foci energetically absorbs u.v. Hght with a wave-length of 280 to 254 jnju and is apparently nucleic acid (Bukhman and Kondratjeva, 1959). In bone-marrow cells of irradiated animals (900 r, 4 hr after irradiation) there is a marked increase in absorption of u.v. rays of wave-length 365 m// (Kondratjeva and Bukhman, 1960). Similar cellular changes of focal character are found in the spleen (Sondak, 1957) and in the lymph nodes and thymus of irradiated FLUORESCENCE STUDIES OF NUCLEOPROTEINS 111 Fig. 1. — Microneci-otic foci in the bone-marrow of an irradiated rat (30 niin after irradi- ation, dose 500 r). Fig. 2. — Micronecrotic foci in the bone-marrow of an irradiated I'at (3 hr after irradiation, dose 500 r). 112 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY animals (Meissel, 1957; Meissel et al., 1958). They result largely from the direct action of the radiation, since screening of the hemopoietic organs obviates the reaction to a considerable degree. The aforemen- tioned nuclear impairments revealed by fluorescence microscopy were discovered soon after iiTadiation in leucocytes and lymphocytes of the peripheral blood vessels (Kondratjeva) and also after iri-adiation of blood in vitro (Kondratjeva and Pinto, 1961). In the cytoj)lasm and small vacuoles of the cell acridine orange com- bines with the nucleic acid and nucleotides to form complexes showing a bright red fluorescence. This fluorochrome binds not only with existing (pre-formed) granules but also forms new ones as a result of the separating out of the dye-nucleic acid complexes. Considerable amounts of nucleic acid compounds are observed to accumulate in the cytoplasm of irradiated cells. These compounds form with acridine orange numerous large and small granules showing a fiery red fluorescence. The large granules, or at least some of them, are dis- tinguished by greater density and stability than the granules of un- irradiated cells. They do not spread out under the action of various factors, including u.v. irradiation. The change in the nature of the cyto- plasmic granules in the irradiated cells is indicative either of physico- chemical changes in the nucleic acids forming complexes with acridine orange or else of the formation of a stronger bond between these sub- stances and the proteins of the cytoplasm. Irradiated cells continuing their metabolic activities accumulate in- creasing amounts of nucleotides and ribonucleic acids in the cytojDlasm, in a number of cases completely filling up the cells. This pertains equally to cells in vitro and in vivo, for instance the myeloid cells of the bone-marrow. It is interesting that in the cytoplasm and vacuoles of irradiated metabolizing yeast cells also basophilic substances and volu- tin accumulate, representing a complex of ribonucleic acid and poly- phosphates. Hence retardation of the metabolism of nucleotides and other high energy compounds is a quite widespread reaction of irradi- ated cells, belonging to the most varied cellular types. ULTRAVIOLET FLUORESCENCE Many biologically important sul)stances (aromatic amino acids, proteins, purine and pyridine bases, nucleotides, nucleic acids and some vitamins) possess absorption maxima in the u.v. region. In consequence one might exjiect that fluorescence of these substances could also be exhibited in the same spectral region. For a number of substances this has proved to be the case. FLUORESCENCE STUDIES OF NUCLEOPROTEINS 113 It was very interesting to ascertain whether the character of the ii.v. fluorescence of organs, tissues and cells changes under the influence of ionizing radiation. This was studied at organic and cellular levels. In the jDresent section we shall dwell only on the former. Khan-Mago- metova et al., (1960) recorded the integral u.v. fluorescence spectra obtained from considerable areas of organs without taking into account the complexities of their tissue and cellular patterns. Thin sections of the organs of an animal (rat) or an homogenate were prepared on a freezing microtome. Blood plasma was also investigated. The homo- genate of the organs, and the blood plasma, wei'e placed in a special quartz cell 0-8 mm high, owing to which the objects under observation were all of constant thickness. Measurements of fluorescence were made with the aid of a photoelectric microspectrofluorometer designed by Agroskin and Korolev. The light source was a mercury arc lamp (DRSh-lOO) from the spectrum of which separate sections were iso- lated by means of a mirror monochromator with a diffraction grating. Rays coming from the monochromator were further passed through chlorine and bromine gas filters (when working in the region of 260 to 280 m/(). The fluorescence spectra were analysed by a similar mono- chromator at the entrance slit of which a tenfold enlarged image of the object under investigation was produced with a quartz -fluorite micro - objective. The light intensity was determined by the magnitude of the signal from a photomultiplier mounted at the exit slit of the mono- chromator. Measurements were recorded as a deflection of a mirror galvanometer. The data were analysed in corresjiondence with the sj)ec- tral sensitivity of the system micro-objective-monochromator-receiver. The ultraviolet fluorescence -peak of the organs investigated lies in the region of 320 to 330 m^. Short wave u.v. irradiation causes a definite decrease in the fluorescence intensity and a gradual shift of the peak in the direction of the longer wave-lengths. With decrease in intensity of the initial peak there is an increase in intensity of a new peak of longer wave-length. This effect is undoubtedly due to active photo- chemical jDrocesses. The fluorescence spectra of the organs of animals given whole-body irradiation with X-rays at doses of 1,000 r did not differ significantly from those of controls. However the intensity increased markedly in the radiosensitive organs (bone-marrow, spleen, lymi^h nodes) and in the blood plasma (as tested 4 and 24 hr after irradiation (Fig. 3)). At present it is difficult to explain the increase in intensity of u.v. fluorescence of the radiosensitive organs. Perhaps this is associated with changes in the nucleoproteins or other proteins in which cyclic amino acids are liberated. 114 M. N. MEISSEL, E. M. BRUMBERG, T, M. KONDRATJEVA AND I. J. BARSKY 200 « ; ISO •7 / N^\ ■7 ' ' / y .-• - — •—" ■^. 100 1 1 , ; ; , ' — . 1 .y ... L. 1 i 309 349 389 429 A (m/Lt) Fig. 3. — Ultraviolet fluorescence spectra of the blood plasma of normal and irradiated rats. 1-normal (control), 2-4 hr after irradiation; 3-27 hr after irradiation; 4-shift of spectra after u.v. irradiation. With the aid of the photoelectric inicrospectrofiiiorometer descrihed above Agroskin d. al. (1960) investigated the low temperature fluor- escence sx^ectra of solid specimens of ribo- and deoxyribonucleic acids 1-0 U A C c * ••■/ '-^^ ■/' / *> / / v. 0-5 / • / / ,' / / / X \ \ ' / / / / / / / / / / t i \ \ N \ 0 / / 1 / 1 310 340 430 460 370 400 A (m//) Fig. 4. — Fluorescence spectra of ribonucleic acid (from yeast) and of its comiDonent bases (excitation with A = 2(J0 to 280 lufj.). A-adenine, G-guanine, U-uracil, C-cytosine. and their separate components. Of the data obtained the most interest- ing from the standpoint of the subject under discussion ai*e the spectra of the nucleic acids and their bases (Figs. 4 and 5). It was found that the component bases of the nucleic acids and also the nucleosides and FLIJORESCENCE STUDIES OF NUCLEOPROTEINS 115 nucleotides possess characteristic fluorescence spectra. The fluorescence of the nucleic acids covers the spectral region of emission of their l)ases and the difference in fluorescence of RNA and DNA is determined pre- dominantly l)y the spectra of thymhie and uracil. 440 A (rr.ii) Fig. 5. — Fluorescence spectra of deoxyribonucleic acid (from the erythrocytes of hens) and of its component bases (excitation with A — 260 to 280 m/u). A-adenine, G-guanine, C-cytosine, T-thymine. ULTRAVIOLET FLUORESCENCE MICROSCOPY In 1956 Brumberg described a new method of fluorescence micros- copy which allowed one to photograph the it.v. fluorescence of micros- scopic objects. He made the assumption that the monotonous blue or violet autofluorescence of the majority of biological objects are the "tails" in the visible spectrum of a fluorescence of which the peaks are located in the u.v. region. This assumption has been confirmed. It was found that many tissues of plant and animal organisms possess charac- tei'istic and definitely expressed u.v. fluorescence (Brumberg et al., 1958; Barsky et al., 1959). The version of fluorescence microscopy in which excitation and recording of fluorescence is carried out in the u.v. region is called u.v. fluorescence microscopy. Recently Brumberg and Barsky (1960) have described the application of the method to investigations of cytological objects. In this study, as well as in further investigations, the same site of the preparation was consecutively photographed in ultraviolet light, first in the light of its own u.v. fluorescence (on excitation by incident light coming through 116 M. N. MEISSEL, E, M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY the microscope objective) and then in transmitted liglit in order to ascertain the character of the n.v. absorption l)y the separate com- ponents of the object. The opaqne iUnminator over the objective had two interchangeable beam splitters of which one was characterized by high reflection of ulti'aviolet rays of wave-lengths 280 m//, or less and by good transmission of longer wave-lengths. This splitter was nsed for exciting and photographing the n.v. fluorescence of the specimen. The second beam splitter reflected n.v. rays in the range 360 to 300 m//, transmitting light of the visible spectrum. It was employed in studies of visible fluorescence. Excitation and recording of the fluorescence of the object was carried out with crossed filters. For u.v. fluorescence such filters were the following: a quartz cell with chlorine and bromine gas filters mounted between the light source and the microscope (transmits u.v. A = 250 to 260 m/f) and a Woods glass filter (transmits u.v. A = 340 to 380 ni/i) moimted over the ocular of the microscope. Visible fluorescence of fluorochromed objects was observed respectively with blue and yellow filters. The microscope employed in the work was fitted A\'ith a quartz- fluorite objective lens (58 x ) with an aperture of 0-8 (water immersion) and with a 3 x quartz-fluorite ocular. Theexi^osure during photomicro- graphy of the u.v. fluorescence was 10 seconds and of absorption 2 to 3 seconds. In a numl)er of cases the specimens were preliminarily fluorochromed with very dilute solutions of acridine orange (I : 10^). Such treatment did not interfere with the photography of ultraviolet fluorescence in- asmuch as acridine orange fluoresces only in the visible region. At the same time fluorochromed objects, particularly blood and bone-marrow cells and micro-organisms are more easily identified and discrimin- ated and also focused better during subsequent photography in u.v. light. A brief general account will now be given of the results of u.v. fluorescence studies of normal and irradiated cells arising from the joint work of Brumberg, Barsky, Kondratjeva, and their collaborators (I960) and of these authors in collaboration with Meissel and Gutkina. Ultraviolet autofluorescence has been revealed under normal con- ditions in cells of the most varied types and origins, l^eginning with micro-organisms (bacteria, yeasts) and including the cells of higher animals and man. The fluorescence is particularly noticeable in cells cultivated in vitro (cells of human amnion, of monkey kidney, of HeLa tumour), in the cells of Ehrlich's ascites tumour and of a number of other tumours. FLUORESCENCE STUDIES OF NUCLEOPROTEINS 117 t* • • 9 * ma-^M Fig. 0(a). Fig. 6(b). Fig. 6.— Ultraviolet absorption and autofluorescence of megakaryocytes and of cells of the myeloid series from the bone-marrow of a normal rat. (a) u.v. absorption A = 265 m^i. (b) u.v. fluorescence. lis M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY In normal bone-marrow and blood distinct u.v. fluorescence is exhibited by cells of the myelocytic series np to matm'e leucocytes, inclusively; monocytes, megakaryocytes and thrombocytes. Young cells of the erythrocytic series and mature lymphocytes have weak fluorescence. Young lymphocytes (lumphoblasts) in the lymph nodes show clear u.v. fluorescence. It is very significant that the fluorescent substance is found chiefly in the cytoplasm of the cells. It is either more or less uniformly distri- buted, or is concentrated for the gi'eater joart in definite regions of the cytoplasm, mostly those adjacent to the nucleus. In vital fluorochrom- ing with acridine orange the substance fluorescing in the ultraviolet partially enters into the composition of the newly-formed granules. Cell nuclei, evidently with some exceptions, do not exhibit marked u.v. fluorescence. At times nucleoli may fluoresce and a very faint diffuse fluorescence of the nucleus may be observed. But in these cases also the light emitted by the nucleus is incomparably weaker than that of the cytoplasm ; as a rule the nucleus stands out as a dark object on a bright cytoplasmic background. This is somewhat unexpected. We have already indicated that deoxyribonucleic acid isolated from the cells shows a definite u.v. fluorescence. Evidently when it forms a com- ponent part of the DNA-protein complex deoxyribonucleic acid loses its capacity for fluorescence. What is the nature of the substances responsible for the ultraviolet fluorescence of the cytoplasm? By means of special experiments on the selective extraction of various nucleic acid components from the myeloid cells, Brumberg et al., (1960b) showed that they are nucleo- tides, ribonucleic acid and, to a lesser extent, proteins. What then are the changes undergone by the u.v. fluorescence of cells subjected to u.v. and ionizing radiation? It was found that as a result of intensive irradiation by u.v. rays (A 250 to 280 m//) or X-rays (in doses exceeding 25kr) the u.v. autofluorescence considerably weakens giving place to clearly visible blue-violet fluorescence, excited by rays of 365 n\fi wave-length. This fluorescence may be studied in an ordinary fluorescence microscoj^e. It appears not only in the cytoplasm but also in the nuclei of the cells. The sharp fall in u.v. fluorescence in intensively irradiated cells is due to the resultant photo and X-radiochemical processes leading to labilization of the nucleoproteins and release from the cells of nucleo- tides and nucleic acids. The appearance of a bright visible fluorescence is evidently associated with some kind of structural changes in the nucleoiJroteins and proteins, which have not yet been investigated more closely. FLUORESCENCE STUDIES OF NUCLEOPROTEINS 119 r • • .'^ • • V- • ^« V • • •. -V^ r ; •••• Fig. 7(a). Fig. 7(b). Fig. 7. — Ultraviolet absoiption andautoflnoi'escence of cells of the lymph nodes of a rat irradiated with a dose of 900 r, 4 hr after irradiation. (a) u.v. absorption (A = 265 ni/x), (b) u.v. fluorescence. 120 M. N. MEISSEL, E. M. BEUMBEEG, T. M. KONDRATJEVA AND I. J. BARSKY We now pass to a discussion of the studies on the n.v. fluoi^escence of the cells of radiosensitive and radiostable animal organs. In our experiments, white rats were irradiated with X-rays (by the apparatus RUM-3, 190 kV. 10 niA with 0-5 mm copper and 1 mm aluminium filters) in doses of 900 to 1,000 r, the dose-rate l)eing 74 r/min. Cells of the various organs were investigated immediately, and 4 hr after, irradiation. Already, soon after irradiation, a marked increase was observed in the intensity of idtraviolet fluorescence of the lymphocytes in the lymph nodes (Fig. 7) and in the blood and the fluorescence of the leucocytes. After irradiation there is a considerable increase in the u.v. fluorescence of cells of the myeloid series in the bone-marrow. Barsky obtamed the fluorescence spectra of normal myeloc^^tes and of the same cells from the bone-marrow of irradiated animals (Fig. 8). Exam- ination of the spectra revealed considerable changes in those of the 300 340 380 420 Fig. 8. — Ultraviolet fluorescence spectra of individual myelocytes: 1, from the bone- marrow of normal and 2, irradiated animals (rats) 4 hr after irradiation witli a dose of 900 r. irradiated animals. The emission bands broaden and are somewhat shifted in the direction of the longer wave-lengths. This bears evidence of qualitative changes in the substances responsible for the fluor- escence. No changes in the character and intensity of the fluorescence could be detected in the liver cells either immediately after, a few hours after or 24 hr after irradiation. Even on the 9th or lOtli day after irradiation ^^"ith a dose of 500 r the liver cells fluoresced in the same way as the controls. The necrobiotic changes of the cells in the bone-marrow and lymph nodes of irradiated animals, wiiich we had described pi'eviously as micronecrotic and which are clearly observed on fluorochroming with acridine orange (on fluorescence microscojiy in the visible region), were FLUORESCENCE STUDIES OF NUCLEOPROTEINS 121 m #• Fig. 9(a). Fig. y(b). Fig. 9 — Ultraviolet absorption (a) and autofluoreseence (b) of bone-marrow cells of an irradiated rat (dose 900 r, 4 hr after irradiation). In the upper right hand corner is a micronecrotic focus. 122 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY distinguished by a highly brilHant u.v. autoflnorescence (Fig. 9). The substance responsible for this fluorescence is nucleoprotein modified in a sj)ecific manner and firmly attached to the cellular substrate from which it is separated with difficulty. We also observed enhanced fluorescence in irradiated animal cells continuing their growth and metabolism on cultivation in vitro. This was observed already after a dose of 100 r and attained a maximum at doses of 2,000 to 3,000 r (Fig. 10). Further increase in dosage led to a diminishing of the u.v. fluorescence and at 25 to 30 kr it became extinct giving place to higher wave-length emission, in the visible region. The increase in the ultraviolet fluorescence of irradiated (with moderate doses) cells continuing to metabolize parallels the accumu- lation in them of nucleotides and ribonucleic acid and these processes are presumalily intimately connected. The newly-formed RNA obvi- ously differs from the normal one. In certain types of cells (for instance HeLa cells) after irradiation marked u.v. fluorescence ajjpears in the nucleoli. It should be stressed that cells irradiated with X-rays and continuing their metabolic activities are highly susceptible to irradiation by short u.v. waves. Such irradiation very quickly leads to extinction of u.v. fluorescence and to the appearance of longer wave visible fluorescence. We observed this in cultures of monkey kidney cells in vitro irradiated with doses beginning from 100 r. In irradiated yeast cells {Endormjces magnusii) continuing to meta- bolize on a nutrient medium we observed a well-defined u.v. auto- fluorescence of the nuclei. In non-irradiated cells the nuclei as a rule do not exhibit fluorescence (Fig. 11). This is the only case we have observed of the appearance of u.v. fluorescence in the nuclei of irradi- ated cells. CONCLUSIONS 1. Vital and supravital fluorochroming followed by observation in the fluorescence microscope permits detection of the early stages of impairment in the nucleoproteins of cell nuclei beginning immediately or soon after irradiation. These are revealed as a result of the changes in the natui'e of the complex formed between diaminoacridine fluoro- chromes and structurally damaged DNA proteins, affecting the in- tensity and colour of the fluorescence of the newly-formed com])lexes. The nature of such structural impairments is not yet com])letely eluci- dated. Possibly labilization and broakdo\\ n of tlie DNA linkage with FLUORESCENCE STUDIES OF NUCLEOPROTEINS 123 » •>;. Fig. 10(a). Fig. 10(b). Fig. 10. — Ultraviolet autofluorescence (b) and absorption (a) in the Ividney cells of a monkey (culture in vitro) 48 hr after irradiation (dose 2,000 r) (A = 26.5 mp). 124 M. N. MEISSEL, E B. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY % # ^^l^'j Fig. 11(a). Fig. 11(b). Fi^ 11 -Yeast cells {Endon,yces rnagnnsii) after irradiation and subsequent cultivation. ^' ■ (a) u V. absorption (A = 265 m^), (b) u.v. fluorescence. FLUORESCENCE STUDIES OF NUCLEOPROTEINS 125 the ]m)toiii (partial de])roteinizati()n) and the initial stage of DNA denaturation take ])lace witli transition of the DNx\ to a less rigid stnictnre. This in tnrn facilitates the aggregation of the cations of acridine orange bound with DNA {cf. Bradley and Felsenfeld, 11)59). Fluorescence microscopy in the visible region of the spectrum reveals initial and progressive changes in the physico-chemical state of DNA, in particular its separation from the chromatinic structures in the irradiated cell nuclei and depolymerization. The changes may be de- tected soon after irradiation in the hemopoietic organs and in the leucocytes of the peripheral blood stream. 2. By means of vital and supravital fluorochroming in hemojwietic organs of irradiated animals one may observe peculiar focal cellular degenerations — "micronecrotic foci". The nucleic acids of cells forming such foci acquire enhanced affinity for diaminoacridines and brilliantly fluoresce both in the visible (on fluorochroming) as well as in the ultra- violet (primary fluorescence) regions of the spectrum. The physico- chemical proi3erties of the nucleic acids and the nature of their bonds with the cellular structures evidently differ significantly from normal. 3. In the cytoplasm of irradiated cells considerable accumulation of nucleotides and nucleic acids takes place, some of them evidently differing from the normal. These substances possess a clearly defined ultraviolet autofluorescence, as a i-esult of which in irradiated cells that continue their metabolic activities there is a marked intensification of ultraviolet fluorescence. The fluorochrome acridine orange forms com- plexes with nucleotides and RNA that separate out in the form of granules with a red fluorescence. The content of such granules increases to a great degi-ee in irradiated cells. They fluoresce (autofluorescence) also in the ultraviolet region. Some of the granules in irradiated cells are characterized by increased stability. On the other hand the majority of the cytoplasmic nucleoproteins of irradiated cells become very labile and sensitive to the short wave ultraviolet spectrum. 4. Ultraviolet autofluorescence of animal cells and organs and of blood plasma quickly diminishes under the influence of intensive ultra- violet (A = 250 to 280 m/ii) and X-ray irradiation owmg to the occur- rence of photochemical and X-ray processes. In parallel this fluor- escence appears in the longer wave-length, visible part of the spectrum. The appearance of this fluorescence is a sign of major changes in the structure and state of the nucleoproteins and proteins. 5. Ultraviolet fluorescence microscopy (Brumberg, 1956) first applied to the study of radiation damage to cells yields particularly in com- bination with ultraviolet absorption microscopy and fluorescence 126 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY microscopy in the visible region, new facts on the state of celhilar nncleoproteins, and on their early and later changes due to radiation. It has been found that the brightness and spectrum of ultraviolet autotiuorescence of the cells of radiosensitive elements change signifi- cantly after X-ray irradiation. The changes are most liliely connected with alterations in the state of the cellular nncleoproteins. REFERENCES Agboskin, L. S., Korolev, N. V., Kulaev, I. S., Meissel, M. N., and Pomoshchni- KOVA, N. A. (1960). C.R. Acad. Sci. U.R.S.S. 131, 1440. Armstrong, J. A. (1956). Exj). Cell. Res. 11, 640. Barsky, I. J., Brumberg, E. M., Bitkhman, M. P.. Va.silevskaja. V. K., and Pluzhni- KOVA, G. F., (1959). Bot. Zh. 44, 639. Bertalanffy, L., and Bickis, J. (1956). .7. Histocliem. Cytoclie)/). 4, 4.S1. BiEBL. R. {Idi2). Proloplasma, 36, 491. Bradley, D. F., and Felsexfeld G. (1959). Nature, Lond. 184, 1920. Brumberg, B. M. (1955)../. gen. Biol. Moscow, 16, 222. Brumberg, E. M. (1956)../. gen. Biol., Moscow, 17, 401. Brumberg, E. M., and B.a.rsky, I. J. (1960). Cytologia (Leningrad) 2, 318. Brumberg, E. M., Mkissel, M. X., Barsky. I. ,J.. and Bukhman. M. P. (1958). •/. geti. Biol, Moscow, 19, 99. Brumberg, E. M.. Barsky, I. J., Kondratjeva, T. M., Chernogriadskaya, N. A., and SH^TDEL. M. S. (1960a). C.R. Acad. Sci. U.R.S.S. 135, 1521. Brumberg, E. M., Barsky, I. J. and Shudel, M. S. (1960b). Cytologia (Leningrad) 2, 5. Bukhman, M. P., and Kondb.^tjeva, T. M. (1959). Biophtisic.^ (Riiss.) 4, 454. Haselmann, H., and Wittekind, D. (1957). Z. wiss. Mikr. 63, 216. Hercik, F. (l939).Protoplasma, 32, 527. Kh.4N-M.\gometova, Sh. D., Gutkina, a. V., Meissel, M. N., Agroskin, L. S., and Korolev, N. V. (1960). Biojjhysics (Russ.) 5, 446. Kondratjeva, T. M. (1956). C.R. Acad. Sci. U.R.S.S. Ill, 89. Kondratjeva, T. M., and Bukhman, M. P. (1960). Cytologia (Leningrad) 2, 309. Kondratjeva, T. M., and Pinto, R. I. (1961). Cytologia (Leningrad) 3, 10(i. Krebs, a. (1947). Naturivissenschaften 34, 59. Krebs, a., and Gierlach, Z. S. (1957). Amer. J. Roentgenol. 65, 93. Meissel, M. X. (1957). "Ionizing Radiations and Cell Metabolism". Reports of tlie AU- Union Scientific and Technical Conference on tlie Uses of Isotopes and Radiations. Moscow. Meissel, M. N. and Sondak, V. A. (1955). C.R. Acad. Sci. U.R.S.S. 105, 1221. Meissel, M. N. and Sondak, V. A. (1956). Biophysics (Russ.). 1, 262. Meissel, M. N., and Z.warzina, N. B. (1947). Mikrobiology, Moscow, 16, 394. Meissel, M. N., Kondratjeva, T. M., Sondak, V. A., and Gutkina, A. V. (1958). RadnRes.9, 151. Meisel, M. N., and Korchagin, V. B. (1952). Bull. Biol. Med. e.rp. U.R.S.S. 33, 49. Meissel, M. N., Larionov, L. F., and Kondratjeva, T. M. (1951). C.R. Acad. Sci. U.R.S.S. 76, 723. Schummelfeder, N., Ebschner, K. J., and Krogh, E. (1957). Naturwissenschaften 44, 467. Sondak, V. A. (1957). Biophysics (Russ.) 2, 495. Strugger, S. ( 1940). /ena. Z. Naturw. 73, 97. Strugger, S. p., Krebs, A. T., and Gierlach, Z. S. (1953). Amer. J. Roentgenol . 70, 365. Wels, p. (1938). Arch. exp. Path. Pharmak. 189, 115. Zanker, V. (1952). Z. phys. Chem. 199, 225; 200, 250. DISCUSSION ERRERA: I am interested in this fluorochrominc; of the cells. Were they living cells which were stained? FLUORESCENCE STUDIES OF NUCLEOPROTEINS 127 MEissEL: In all the expoi'inicnts on fluoniclifotiiina: sliowii Iu-it \ital or siipra\i(ivl staining was performed. marcovich: What was the concentration of the acridine orange? Did the staining of the medium occur? How many generations of the yeast cells did you observe at the concentration used? MEISSEL: Acridine orange diluted 1:100,000 or 1:50,000 in physiological saline or in the medium for animal cell cultures was used. Acridine orange blocks cell structures which results in inhibition of the normal division of the yeast cells. When the concentration of the stain is not very high, the vitally fluorochromed yeast cell, being transferred to a fresh culture mefliinn, would first of all free its structures from fluorochrome, excrete the stain and only afterwards begin its mviltiplication. SHABADASH : According to your microphotographs radiation effects, as evidenced by autofluorescence. are found both in radioresistant (kidney) as well as in radio- sensitive (haemopoietic organ) cells. Can we conclude that all the cell types in the body are radiosensitive? When absoriDtion and fluorescence in the ultraviolet light of the kidney culture cells is studied mitochondria are discernible which also show autofluorescence. There is an increase of this effect following irradiation. How should we consider the part which ribonucleoproteins play in the overall cytoplasmic reaction? MEISSEL : Tissue culture cells are somewhat more radiosensitive than similar cells in the body. Here we showed cells that had received doses of 2,000 r. Strictly speaking all cells are radiosensitive, but they differ as to the degree of their sensitivity. I believe that among those cell nucleoproteins which begin to show bright fluorescence in the ultraviolet region following irradiation, there are mito- chondrial ribonucleoproteins. but probably also some other types of the cyto- plasmic RNA as well as nucleotides. TOBIAS : Did you observe any changes in these same cells under the microscope some time after exposure? Did not the photodynamic effect of the acridine orange manifest itself in the cells ? What changes occur there when you expose them to light for a long time while taking the photographs. MEISSEL: In our work we tried to achieve vital fluorescence of the cell. For this purpose fluorescence of the cells fluorochromed with acridine orange was excited not by ultraviolet, but by blue-violet light, which is much less dangerous than ultraviolet. Apart from this, much depends on the exposure during observation and taking j^hotographs. ExjDosure in our work was short, and no distinguishable changes caused by induced fluorescence were observed. W^hen observation was carried out for a longer time distinct photodynamic reactions manifested them- selves in the cells. ZEiTLiN: Did any changes occur in the absorption in the short wave ultraviolet region following formation of the complex acridine orange +nucleic acid? MEISSEL : The fact is that acridine orange absorbs ultraviolet light in the same region as nucleic acids. As to whether there are changes in the absorption and fluorescence due to complex formation: nucleic acid — diaminoacridine — this is a problem we are studying at present. Important data on this problem have been 128 M. N. MEISSEL, E. M. BRUMBERG, T. M. KONDRATJEVA AND I. J. BARSKY obtained by De Bruyn and co-workers (1953) Morthland et ah. (19o4), Bradley and Felsenfeld (1959) and others.! KUZiN : How soon after the irradiation could the shift of the fluorescence maxi- niiun be shown? MEISSEL: Practically immediately following exposure. GRAY : I would like to ask about the shift of the ultraviolet autofluorescence maximiun. which occurred following irradiation with 25,000 r. In order to obtain this effect is it necessary to use so high a dose, or could you observe it at lower dose levels? Meissel: Unfortvmately we have not yet studied the dose dependance of the effect. But just after irradiation beginning with doses of 25,000 r this effect was observed, while at the doses of the order of several thousand roentgens we did not observe it. PASSYNSKY: What is the relative role of the chemical and physical alteration of the nucleic acids or nucleojiroteins in the ultraviolet fluorescence changes? Do similar changes occiu- in piu-e RNxV and nucleoprotein preparations or do they become manifest only at the eel hilar level? MEISSEL: First may I answer the second question? At present we have at our disposal only preliniinary observations regarding ifltraviolet fluorescence of the isolated nucleic acids, including RNA. Ribonucleic acid isolated from irradiated micro-organisms shows earlier changes in the pattern of its ultraviolet fluor- escence, induced by short wave viltraviolet light, than does RNA from non- irradiated cells. As to the first cjuestion. Here, probal)Iy, both types of change occm-. It is difficvilt to say which are prevalent, but i:)robably owing to configuration changes of DNA molecules during its denaturation. an additional capacity for binding acridine orange appears. Initial depolymerization processes are also evidently liberating some free bases which are capable of binding acridine orange; this is one possibility; another possibility lies in the fact that a changed DNA configura- tion or the transition from two-helix to one-helix form brings about more active formation of dimer and trimer complexes of the acridine orange. PASSYNSKY: But these structural changes require some time, while in your experiments they were observ^ed inunediately. MEISSEL: "Immediately" is but relative. Since it is difficult to make obsei'vations directly under the beam, there was a lapse of several minutes and sometimes 10, 20 or even more minutes between irradiation and the recording of the effect. TUMERMAN : You have pointed out that most probably the physical cause for the changes in the fluorescence spectrum of the acridine orange consisted in differ- ences in its concentration. A phenomenon is known of a fluorescence spectrum change in the long wave range occurring with the increase of the concentration, i.e. with the increased interaction between molecules of a substrate and the stain. Could the possibility be ruled out that configuration changes allow the develop- ment of trij^let long term luminescence? In the preliminary exiDeriment we t De Bruyn, P. P. H., Farr, R. C, Banks, H.. and Morthland. F. W. (1953). Exp. Cell Res. 4, 174. Morthland. F. W., De Bruyn, P. P. H., and Smith. X. H. (1954). Exp. Cell Res. 7, 201. Bradley, D. F., and Felsenfeld. G. (1959). Nalure, LoixL 184, 1920. FLUORESCENCE STUDIES OF NUCLEOPROTEINS 120 earrieil out to<;etlier witli you tliis jiossihility was evidently not confirmed. The spectrum of the orangt^ Uuninescence of (he acri(hne orange adsorbed to bio- logical substrate is identical with that of a very concentrated water solution, while the spectrum of the green fluorescence coincifles with that of the diluted water solution of this stain. There is at present also no indietion of the change of the duration of the excited states. Thus the physical mechanisui of the observed changes consists probably in a greater or lesser proximity between the adsorbed stain molecules, i.e. a greater or lesser degree of interaction between molecules of the stain adsorbed by the biological substrates, and a greater or lesser amount of the stain. gray: In relating the changes to the time, should it not be measured starting from the begirming not from the end of the exposure. What was the irradiation dose -rate in yovir experiments? MEissEL: In the case of the total body irradiation the dose-rate was about 74 to 75 r/min, i.e. all tlie dose was administered to the animal during about 15 minutes. When tissue cultures were irradiated it was possible to use irradiation of a very high dose-rate of the order of 20,000 to 25,000 r per njinute. A DISCUSSION OF SOME IMMEDIATE EFFECTS OF X-IRRADIATION ON LIVING CELLS BARBARA E. HOLMES DejKirtment nf Badiotherapeutics, University of Cambridge, Emjlnnd SUMMARY Attention is drawn to a few of the immediate bioeliemical effects of X-rays mentioned in the hterature. Some of these may be due to radical or direct attack on enzyme systems or cell strvictm*es. One difRciilty in discussing the initial effect of irradiation on cells is to decide what we mean by this. Our target is liable to be that event in which we are most interested at the time and, in any case, our ideas are usually conditioned and limited by the ease of detection of the effect ; in fact, by its manifest importance to the cell. Normally, also, we are considering effects which are irreversible or only slowly reversible. By using special techniques of killing cells during the course of irradiation, rapidly reversible changes can sometimes be seen, for instance in the proportions of oxidized to reduced co-enzyme. One is inclined to think that such changes and many reparable cyto- plasmic injuries are unimportant. It seems possible that, though no immediate dramatic effect is usually to be seen, part of the phenomenon of ageing by irradiation may be the result of such causes. An immediate dramatic effect can be shown in the result of the irradiation of the sporangiophore of Phycomyces. Forssberg (1960) found that the elongation of the sporangiophore could be inhibited temporarily by doses as low as 0-0005 r, a maximal effect being pro- duced by 1 -0 r. The inhibition is quickly reversible when irradiation is stopped. The mechanism of elongation is not fully known and it is difficult to picture in what way it could be affected by such tiny doses. If an enzyme or specific chemical substance is concerned, then it must surely be directly connected with the elongating cell wall or the water- imbibing membrane and some energy-transporting mechanism must be imagined. Forssberg has found an accumulation of lactic acid during this period of inhibition of elongation and also an abnormal distribution 131 132 BARBARA E. HOLMES of organic phosphates. This could be a very interesting system for studying an initial reaction and its final visible effect. The manifest effect of irradiation on the cell nucleus and the con- sequences to cell life have made us regard this as the main target and think of initial effects as being within the nucleus; occasionally, how- ever, events caused by simple enzyme inhibitions, probably uncon- nected with the nucleus, obtrude themselves upon our notice. A striking example is given by the work of Gordon (1956) who found that the growth of young mung bean plants coukl ])e stimted for one or more days by low doses of X-rays. He traced this effect to an immediate fall in the auxin content of the irradiated plant. This in turn was found to be due to a considerable decrease in the usual formation of auxin from trytophan, probaljly because of an inhibition of the enzyme re- sponsible for the last stage of the transformation. Gordon considered that the enzyme responsible was that causing the oxidation of indole- acetaldehyde to auxin and could show that extracts made from bean plants immediately after irradiation showed a lower enzyme activity than extracts from normal plants. The initial action of the X-rays. l)io- logically speaking, was apparently the inactivation of this enzyme, but it is not yet possible to say what, in its structure or surroundings, made it particularly sensitive. Another interesting case of the obvious manifestation of the result of irradiating an enzyme system is given by Ungar et al. (1955) who studied the irradiation of an adrenal gland during its perfusion with blood containing A.C.T.H. At the end of the experiment, when the total dose had reached 2,000 r, it was found that the usual production of adrenal steroids had been much reduced. It was possible to show that definite steps in the formation of these steroids had been inhibited and the effect must have been on specific enzymes or on the formation of these enzymes. To come at last to events in the nucleus, it has, of course, struck all workers that the huge nucleoprotein molecides offer a target that cannot be missed and that some of the initial effects of irradiation must be within them. This idea will be discussed later, hut it is worth ])ointing out that it has proved easier to produce changes in other mechanisms connected with the mitotic process than to show any such effect. For instance, it was deduced by Pelc and Howard (1953) from auto- radiographs and later demonstrated directly by ourselves on the re- generating rat liver (Holmes and Mee, 1955) that fairly small doses of X-rays given before the synthesis of deoxyribonucleic acid had started, were effective in sto])ping this synthesis. Im])ortant work by Bollum and Potter (1960), supported by the indejDendent evidence of Ord and IMMEDIATE EFFECTS OF X-RAYS ON LIVING CELLS 133 Stocken (1900), showed tliat the real effect was an inhibition of the formation of the enzymes needed to produce the synthesis of the nucleic acid. If this effect on the formation of adaptive enzymes is likely to be a usual point of attack by X-rays, it must be further studied. It is unfortimately not yet known whether, when such en- zymes appear in the tissue in response to a new need for them, they represent a new or much increased formation or actually an uncovering of enzymes already present. That this type of uncovering of enzymes or removal of enzyme inhibitors can occur in tissues has been demonstrated in various cases. Schneider (1960), however, has been able to cause an inhibition of nucleic acid synthesis in regenerating liver by giving in- hibitors of protein synthesis at this early stage. It may be that, in his experiments also, the appearance of new enzyme is being prevented, and therefore, that this new appearance is indeed a new formation of protein. Another system of synthesis, this time actually in the nucleus is the phosphorylation of nucleosides demonstrated by Osawa et al. (1951) in isolated thymus nuclei and shown in Dr. Stocken's department to be extremely easily inhibited by previous irradiation of the animal. It was difficult to demonstrate the existence of the phosphorylating enzyme in various other tissue nuclei but recent work by Crathorne and Shooter (1960) on the whole animal suggests that a phosphorylating enzyme exists in the nuclei of ascites tumour cells and of regenerating liver cells at some early stages of regeneration. When thymidine is injected into the animal, mono-, di- and triphosphate derivatives are found in the nucleus in much greater amounts than in the cytoplasm. It will l)e most interesting to know whether this system is also a primary target of X-ray action. Osawa et al. found that, as would be expected, the exis- tence of a phosphorylating system is necessary for the formation of protein in the isolated nuclei. We have lately been following the uptake of amino acid into one fraction of the nuclear protein and attempting to trace the effect of irradiation upon this and upon nucleic acid synthesis at the same time. For this we have done experiments in vivo with rat liver in the condition of regeneration after partial hepatectomy. The animals were taken at a time after hepatectomy when nucleic acid synthesis was already occurring actively and when a large X-ray dose (2,000 to 3,000 r) was necessary to reduce it to 50 per cent of the normal rate. In our laboratory, Looney et al. (1960) demonstrated that this inhil)ition represented a slowing of synthesis only and that the irradiated cells eventually formed their full complement of DNA. To do this, they needed about 13 hr instead of the usual 8. 134 BARBARA E. HOLMES The figures are taken from Dr. Looney's paper. He gave 3(K)0 r to the regenerating Uver lobe (a dose which had been shown to reduce 32p uptake into the DNA to about half the normal amount) the rest of the body being sliielded as well as possible. Tritiated thymidine was given immediately after irradiation and the animal killed 3 hr later. Figure 1 is a histogram showing grain count distribution. The irradiated cells HISTOGRAM OF LOG OF GRAIN COUNT DISTRIBUTION OF NUCLEI (Figure l) WITH SUPERIMPOSED NORMAL CURVE. f//^i IRRADIATED CONTROL 0-80 l-OO 120 I 40 I-60 I-80 200 2 20 LOG GRAIN COUNTS PER NUCLEUS 2-40 Fig. 1. — Histogram of the logaritliin of grain count distiil)utioii of nuc curve superimposed. with tlie normal have a smaller grain count than those from an unirradiated but otlier- wise exactly comparable animal but there is no decrease in the number of cells in synthesis. At this time after hepatectomy no mitosis has yet occurred in either cell population. The reduction in grain counts in the irradiated cells might indicate a lower rate of DNA synthesis. In order to prove that this was the correct interpretation, measurements were made of the content of DNA in individual cells by a light absorption method using Feulgen-stained cells. Figure 2 shows the results. Un- labelled, that is to say non-synthesising, cells indicated the 2n, 4n and Sn amounts of DNA. Most hepatocytes in these animals contained a 4n amount which increased to an 8w amount before cell division. Cells which were synthesising DNA at 17 hr after hepatectoirry become IMMEDIATE EFFECTS OF X-RAYS ON LIVING CELLS 13( labelled and are indicated here by black dots. The figure shows that the normal cells had all attained the full 8/i amount of DNA G hr after labelling whereas the irradiated cells had only completed about half their synthesis. • Labelled nuclei n Unlabelled nuclei c =] O u O 60 50 40 30 20 10 J 4) 2N 4N PBN^ o 2 200 600 1,000 1,400 1,800 DNA (abltrary units) 3 _V u 13 C V a. (/> 60 50 40 30 § 20 2 10 o 2N (Radiation dose 3,000r) • Labelled nuclei A Labelled divided nuclei n Unlabelled nuclei 200 600 1,000 1,400 1.800 DNA (arbitrary units) Fig. 2. Plot of grain counts against DNA content of the same hepatic nuclei of a rat given tritiated thymidine at 1 7 hr after hepatectoniy and sacrificed (5 hr later. In absolutely similar material we have now made a simultaneous study of the synthesis of nucleic acid and of the residual protein to which the nucleic acid is found to be attached after removal of the histones. The synthesis both of this protein and the histones normally seems to take place at much the same time as the synthesis of the DNA in regenerating liver, since the uptake of lysine and of arginine into the proteins is then three or four times higher than that in resting liver tissue. 136 BARBARA E. HOLMES We have not nearly enongh data to make any statement abont the radiosensitivity of the formation of this ]iarticular protein. We can, however, say that in many individnal animals, the X-rays have been sufficient to cause a sharp inhilntion of nucleic acid synthesis without causing any inhibition of synthesis of this particular protein fraction, which in normal cells apparently occurs at the same time as the nucleic acid synthesis. The effect of X-rays appears therefore, to cause some dislocation in the usual relationship between the two. The work of Richards (personal communication), who has made measurements on individual cells, is in agreement with the idea that the rate of ])rotein formation is not depressed together with the rate of deoxy nucleic acid formation. After these diversions one must, however, return to a considera- tion of the effect of X-rays on various nuclear structures, since there is no doubt of the great importance of these effects in dividing cells or resting cells that may later come into division. The nucleolus may, perhaps be considered as the prime target for the inhibition of synthesis in the nucleus at some stages of the mitotic cycle. Seed, (1900), in our laboratory, irradiated the three nucleoli in the nuclei of mouse heart fibroblasts with about 500 rad X-rays. The X-rays were collimated into a thin pencil 1// in diameter. Two daughter cells were chosen from a previous division; one of them was used as a control and the other irradiated about 2 hr after the division. The pro- gress of DNA synthesis in these nuclei was followed by taking an ultra- violet photograph after the chosen period of time had elapsed. It was found that synthesis had occurred in the control nucleus but not in the irradiated nucleus at times of 3 to 7 hr after the irradiation. If we wish to speak of an initial effect we should obviously have grounds for considering it to occur immediately after irradiation ; in a technique such as Dr. Seed's this cannot be demonstrated. However, Gaulden and Perry (1958), working at Oak Ridge, have been able to demonstrate immediate cessation of mitotic progress after nucleolar irradiation with u.v. light in very early pro])hase. Later stages of mitosis were not so sensitive to nuclear irradiation so that here we may consider oui- effective target to have changed. The nature of the X-ray injury to the nucleoprotein complex of the cell has not proved easy to detect. The extraction of nucleoproteins from irradiated and unirradiated cells for comparison of their physico- chemical properties is not al)solutely satisfactory because such small changes in technique cause variations in the extracted products. The control sam])le may show different viscosities, different abilities to form gels and so forth if the speed or duration of homogenization of the IMMEDIATE EFFECTS OF X-RAYS ON LIVING CELLS 137 tissue, for instance, is varied. Dr. Ivutli Itzliaki has been carj-ying out work of this sort, and has found that the descri])tion of extraction methods given in the literature are often quite inadequate because such details are not given. For instance, when homogenizing mouse spleen at fairly high speed in normal saline, she found that insoluble and prob- ably denatured nucleoprotein fibres were formed, while at slow speeds whole cells were still present and no fibres. Reproducibility was difficult because the resistance of the homogenate reduced the speed of the homogenizer by an unknow^n amount. These facts are mentioned merely to illustrate the difficulties of such work with nucleoprotein complexes. Many experiments reported in the literature give no information about the initial lesion produced by the irradiation, since the nucleo- protein extraction is not made immediately after irradiation. Perhaps the most definite evidence of damage to this structure by irradiation has been given by the work of Ord and Stocken (1960), who used the Bendich method of separating different fractions of the DNA and found that irradiation caused an increase in the amount of the more soluble fractions at the expense of the less soluble. Irradiation in vitro of already extracted nucleoprotein must give different results according to the method of extraction used and one is then faced with the difficulty of deciding which preparation most nearly resembles the substance as it occurs in the living cell. To give a rather extreme example Anderson and Fisher (1960) found that the extraction of rat thymus brei with 2 volumes of 1 -4 M sodium chloride produced a sample of very high viscosity. The authors considered that linear aggregates of DNA were present, other linkages having been broken by the strong salt. As expected by Anderson, these complexes were very sensitive to irradiation and a definite fall in viscosity was noted after a dose of 50 r. The kindness of Professor J. S. Mitchell allows me to present to you some partly unpublished data of his own (Mitchell, 1959) which deal wdth a true initial effect and possibly one in which the deoxyribo- nucleic acid is involved. The Walker rat carcinoma has been irradiated in vivo in an anaesthetized animal with 2,000 r given in 2 min 16 sec. By a special device, liquid nitrogen is applied to the tumour less than 1 sec after irradiation. On w^arming the excised tissue high energy u.v. quanta may be released mainly at - 5°C and 50 to 70°C. In 35 recent experiments definite results have been observed in 5 cases and possible ones in a further 6 cases. The long-lived excited states must last for some seconds or even minutes at body temperature and might be associated with reactive chemical intermediates, but the experimental results are not inconsistent with an excitor mechanism. If the DNA is con- 138 BARBARA E. HOLMES cerned in this the energy conld probably be utihzed in some part of the DNA or be available for release in the protein closely associated with it. The secondary effects of snch an initial process conld be different according to the exact condition of the DNA. The irregnlar chromosome structures sometime found in tumours, virus-infected nuclei, chromo- somes with puff formation, DNA during the process of reduplication, and so on, might suffer changes rather different from the usual. Pro- fessor Mitchell himself is trying to determine whether the findings can l)e related to the radio-curability of the tumour. At all events, there is here a chance of seeing an absolutely initial effect and of investigating its real meaning and relating it to the results which follow later. ACKNOWLEDGEMENTS 1 am most grateful to the Wellcome Foundation for providing my travelling expenses to this meeting. REFERENCES Andersox, N. G.. and Fisher, W. D. (1960). In "The Cell Nucleus", p. I'J.J. (J. S. Mitchell, ed.) Butterwoi-ths, London. BoLLUM, F., and Potter, V. R. (1960). Caticer lies. 20, 138. Crathorne, a. R., and Shooter, K. V. (1960). Nature, Lond. 187, 614. FoRSSBERG, A. (1960). Congress of Pliotobiology, Copenhagen. In press. Gaulden. M. E.. and Perry, R. P. (lO.lS). Proc. not. Acad. Sci., Wash. 44, r)."i3. Gordon, S. (19.")6). In "Progress in Radiobiology". (Mitchell, Holmes and Smith, ed.) Oliver and Boyd, Edinburgh. Holmes, Barbara E., and Mee, L. (1955). In "Proceedings of the Radiobiology Sym- posium, Liege, 1954". (Z. M. Bacq, ed.). Butterworths. London. LooNEY, W.. Campbell, R. C, and Holmes. Barbara E. (1960). Proc. nat. Acad. Sci., Wa.sh. 46, 690. Mitchell, J. S. (1959). Eep. Brit. Emp. Cancer Campgn. Ord, M. G., and Stocken, L. A. (1960). In "The Cell Nucleus", p. 157. (J. S. Mitchell, ed.) Butterworths, London. OsAWA, S., Allfrey, V. G., and Mirsky, A. E. (1951). ./. gen. Physiol. 40, 491. Pelc, S.. and Howard, A. (1953). Acta Radiol. Sup]>l. 1 1(>. Richards. Seed, J. (1960). In "The Cell Nucleus", p. 49. (J. S. Mitcliell, ed.), Butterworths, London. Schneider, J. (1960). J. biol. Chem. 235, 1437. Ungar, F., Rosenfeld, G.. Dorfman. R. I., and Pincus, G. (1955). Endocrinologi/, 56, 30. DISCUSSION TliMERMAN: I am very interested in the information on Prof. Mitchell's discovery of nltraviolet emission during warming of frozen tissues. I would like to know whether the temperature dependence of the intensity of this luminescence was studied and what is Prof. Mitchell's or your opinion on the physical mechanisms of the origin of this i-)henomenon. Are you inclined to consider this luminescence as a chemiduminescence attendant upon a free radical recombination j)rocess, or some oxidation reaction, or as a phenomenon of thermoluminescence resemb- ling the known therniohiminescence of chloroplasts discovered by Arnold and Sherwood? IMMEDIATE EFFECTS OF X-RAYS ON LIVING CELLS 139 HOLMJis: I know that the emission occm-rcd at different teini)eratiiics. hotli at 5° and 50°C. Prof. Mitchell's hypothesis is that one of tliese pi-ocesses is associ- ated with the presence of long-hved int(>nnechary cheinical products, for instance hydrogen peroxide. The other one in his opinion may be (hie to DNA molecules; the energy released is passed either into the surroumhng medium or to other DNA molecules. His opinion is not uiore definite than that. This is all that T can t(^l] about these experiments. MOUTON : Is radiation damage to the cell accompanied by an increase in the lactic acid content? HOLMES : In Dr. Forssberg's experiments a temporary rise of the lactic acid con- tent was observed at a time when increase in length of the sporangiophores was inhibited. Afterwards the lactic acid was removed. errera: I am interested in Prof. Mitchell's study. I would like to ask whether you know what the emission spectrum was? holmes: No, I do not know that. Prof. Mitchell told me only that it was in the short wave ultraviolet with unexpectedly high energies. powers: Which cells have you studied when nucleoli were irradiated by nucro- beams of X-rays? HOLMES : Fibroblasts of the mouse heart. Every one of the three nucleoli in these cells was irradiated. This study was made by Dr. Seed. barendsen: What was the irradiation applied to the nucleoli? HOLMES: Approximately 500 r per nucleolus. I myself have never used this equipment and carmot give an exact description of it here. manilov : You have told us that if, after the radiation exposure, mononucleotides are added, DNA synthesis continues but is delayed by 13 hr. Could you tell us which nucleotides should be added and how in your opinion these nucleotides penetrate into living cells and are taken up by the nucleus? holmes: We have never attempted experiments with nucleotides. We do not know what is the cause for this delay of synthesis, but according to some in- vestigators it is due to damage to the template or to some part of it in any case. It is possible that the damaged part is less protected than the nucleoprotein mole- cule as a whole. My personal opinion is that the delay may also be due to some interference with the phosphorylation process coimected with nucleotides. Dr. Looney did not administer nucleotides but only thymidine. Addendum to Discussion by Dr. Barbara E. Holmes It is known that mitosis can be inhibited by mechanisms other than the in- hibition of DNA synthesis, since a fairly small dose of X-rays will prevent cells from passing into mitosis even when the dose is given after DNA synthesis has been completed in those cells. We can illustrate this point with regenerating rat liver, since a dose of 450 r given to the dividing tissue immediately prevented any more cells from passing into mitosis— but I do not know how long this effect lasts. At the other extreme 450 r given to cells before DNA synthesis had started 140 BARBARA E. HOLMES caused a 12-hr delay in the onset of synthesis without aUerinp; the shape of the DNA synthesis curve, and without altering the time relationship between the peak of synthesis and the beginning of actual mitosis, in fact, there was no further delay in mitosis. This means that DNA synthesis and mitosis were merely post- poned for 12 hr and it does not mean that there was no damage to DNA, because a large percentage of these cells coming into division showed chromosome abnormalities (as detected by Professor Roller). R ADIAT rON-TNDUCED DISTUR P> ANCES OF T H E LIPIDS OF CELLULAR MICR08TRUCTURES N. N. DOEMIN AND V. D. BLOKHINA Parlor Ixstitate of Physiol ogij and Institute of BiopJiysics, U.iS.S.B. Aaideiinj of Sciences, Leningrad, U.S.S.R. SUMMARY The content of different lipid fractions has been determined in celhilar micro- structures of the rabbit liver and intestinal mucosa under normal conditions and after total exposure to 1,000 r y-rays of cobalt-60. Determinations were made on summary joreparations of cytojalasmic organelles and of hyaloplasm of liver cells 24 and 72 hr after exposure and in mitochondria, microsomes and hyaloplasm of intestinal mucosa cells 2, 24 and 72 hr after exposvire. Post-irradiation changes were studied in the following lipid fractions: "free" — extractable by petrolevim ether; "loosely-bound" — extractable by methanol -chloi'oform mixture (minus the "free" lipids); and "firmly -bound" — which can be extracted after alkaline hydrolysis from the residue after methanol-chloroform treatment. Considerable changes in the content of individual lipid fractions and their quantitative interrelation in various micromorphological cellular components may occur within 2 hr of radiation injury. Later on these changes proceeded in various directions. The radiation-induced changes in the content of the various lipid fractions of sub -cellular comj^onents of irradiated intestinal mucosa differ from those of liver. The simultaneous changes of the lipid fractions of microsomes, mitochondria and hyaloplasm may be variously directed. The changes in the composition of li]:)id complexes of cellular organelles pro- duced by ionizing radiation may be a cause of many subsec£uent disturbances in the cellular metabolism of damaged tissues. Recent advances in cytology enable ns to visnalize clearly the great role of individual cellular micro- and ultramicrostructiu*es in the bio- logical activity of cells. The present-day achievements of biochemistry have made available many concrete facts characterizing the partici- pation of cell micromorphological components in the metabolic pro- cesses and their regulation. The biochemical turnover in cells proceeds in a complex multiphase organized system, and its individual links are localized and intimately connected with definite structures. It may be assumed that these structures composed of biochemically active sub- stances are not merely ^^assive plastic formations, but represent spatially oriented participants of many-sided reactions which "endow"' them with high-rate arrangement. Heterogeneity of the cell contents 141 142 N. N. DOEMIN AND V. D. BLOKHINA leads, in particular, to the possibility of a counter-directed develop- ment of some definite reactions in various cell compartments, within and upon its various structures. Hence, the highly important role of topochemical investigations is quite evident. It is noteworthy that the functional peculiarities of the cell organelles are determined to a great extent by the lipid-protein complexes which are their components. Therefore, various changes of these complexes as a whole or of their single components, i.e. li])ids or proteins, ought to effect the course of biochemical reactions dependent upon such sub- stances (e.g. by the changes of adsorptive bonds with the enzymes and their substrates and also by the changes of the distribution of charges, and likewise by the changes of the distance between active groups). Natm'ally, great attention should also be attached to those shifts observed in the lipid-protein complexes of cell organelles which de- velop as a result of the action of ionizing radiations. The action of radiation is accompanied by a series of disturl)ances observed in various biochemical complexes within the cell. First there are changes in the nucleoproteins. In the course of radiation-bio- chemical investigations the more or less pronounced disintegration of complexes was emphasized. Sissakian (1055) ])ointed out that the disturl)ance in the co-ordination and in the coupling of the enzymatic processes is one of the primary consequences of radiation injury. It may be basically a result of dis- turbances of the biochemical complexes in the cell microstructures. Kuzin (1055) indicated that irradiation initiated the depolymerization of high-polymer substances constituting such structures. He noted also the disintegration of lipoproteins following irradiation. Possibly various types of alteration in respect of composition and of the dy- namics of the chemical components of tissue and their dislocations at the micromo])hological level are indis])ensibly involved in the mechan- ism of the initial and verv early effects of radiation. At our laboratory Ilyina e^ al. (1957) showed that soon after a single X-ray ex})osure (800 r) rats display a transient reduction of the relative lipoprotein content in mitochondria and microsomes of the liver cells. Blokhina (1050a) later established that, in the course of the develop- ment of the radiation disease, a progressive decrease of the lipid content may be observed in the lipoproteins of the liver mitochondria. This evidence pointed also to a certain degree of disintegration of the lipo- protein complexes of the cytoplasmic organelles in the liver cells and to disturl)ances of their resynthesis leading to an altered composition. According to other data obtained l)y Blokhina (1950b) the total lipid content of the mitochondria and microsomes showed an initial increase CHANGES IN LIPIDS OF CELLULAR MICROSTRUCTURES 143 following irradiation and subsequently decreased; the phospholipid content of the same celhilar niicrostructures di-opped slightly; at this point the total lipid content of the hyaloplasm is increased. With the aim of ])romoting the study of disturbances initiated by radiation injury in the lipid complexes of cellular organelles and in their lipid metabolism in general, we investigated the quantitative ratio between various lipid fractions of the entire cytoplasm and the hyaloplasm of the liver cells in rabbits exposed to the action of y-rays of 60Co at a dose of 1,000 r and a dosage rate of 500 r/min (Blokhina and Doemin, 1959). This radiation dose is sufficient to give an acute form of the radiation disease in rabbits. Most of the animals died within 5 to 7 days of exposure. As a rule in every experiment one of two rabbits (of equal weight) was irradiated ; the other was used as a control ; 16 hr before the experiment both animals were deprived of food and after irradiation both animals were killed with an air embolism. The livers of the rabbits were washed with isotonic saline and homogenized with a ten-fold amount of isotonic sucrose. Cytoplasm and hyaloplasm preparations of the rabbit liver cells were made as described in detail earlier (Ilyina et al., 1957). The dry weight of the preparations (5 ml) was established after elimination of sucrose by dialysis for 48 hr against distilled water. The contents of the dialysis bag w^ere transferred to a weighing bottle and dried at 60°C to constant weight. The content of three lipid fractions was measured in the cytoplasm and hyaloplasm preparations. These fractions were called "free", "loosely bound" and "firmly bound" lipids. The determination of the "free" lipids was made:-10 ml of the cyto- plasm or hyaloplasm preparations was shaken with 25 ml of petroleum ether at room temperature for 3min; after centrifugation at 2,000 r.p.m. for 5 min the top ether layer w^as decanted into a separate flask. A similar extraction with new portions of ether was repeated 4 times. The combined lipid ether extracts were evaporated at room temperature to minimal volume and the lipid contents determined in an aliquot by a modification of Bloor's technique (1947). The total lipid content was estimated by utilizing another 10 ml of sample of the cytoplasm or hyaloplasm preparations. At first the lipids were extracted according to the method of Folch et al. (1951). After numerous extractions with the aid of methanol-chloroform mixtures, the residue was subjected to alkali hydrolysis with a 30-fold quantity of 8 per cent alcoholic alkali solution for 40 hr; the fatty acids were extracted from the hydrolysate by Romantzev's method (1952). The content of total lipids was determined by adding the amount of lipids 144 N. N. DOEMIN AND V. D. BLOKHINA extracted according to Folch and of the fatty acids released by snl)se- qnent alkali hydrolysis (the fraction of the "firndy bound'" lipids). The (juantity of the "loosely bomid' li})ids was ascertained by esti- mating the difference between the cinantity of li]>ids extracted with the niethanol-chloroform mixture and that soliil)le in petroleum ether ("free" lipids). The hyaloplasm comprises about 40 per cent of the total dry weight of the liver cell cytoplasm. The a])proximate data with regard to the content of the lipid fractions in the cytoplasmatic microstructures (in round figures) were calculated on the basis of this value and the results of the determination of li])id fractions in the entire cytoplasm and in the hyaloplasm separately. The determinations were performed on control animals and at 24 and 72 hr following radiation injury; under the conditions of our experiments these times corresponded to the beginning and the highest point of the radiation disease. Under normal conditions the liver tissue in i*abbits is relativelv very rich in lipids ; the total content of the latter in the cytoplasmic organ- elles and in the hyaloplasm is approximately equal and comprises about 30 per cent of the dry weight. It was established that under normal conditions according to the ratio between the separate lipid fractions the cytoplasmic organelles and the hyalo]3lasm of the liver cells differ greatly. In the cytoplasmic organelles all the lipids are actually in a o 60 1 — '■*-' u o i_ ,--^ -TJ 50 - / N CL / x ■-= /' N — 40 - -t-» 5 30 - — " ^ \ 4-> -v^ Vl V 20 / ^>^ o ^^-n u ^„^ D c 10 ^^"■^■--^ u N orma 1 24 72 Hours after irradiation Fig. 1. — The content (percentage of dry weight) of various li])id fractions in the cyto- plasmic organelles of the rabbit liver cell imder conditions of acute radiation disease. I. Total lipid content ; II. "Loosely bound" lipids; III. "Firmly bound" lipids IV. "Free" lipids. CHANGES IN LIPIDS OF CELLULAR MICROSTRUCTURES 145 bound state, mostly in the "loosely bound" form. The hyaloplasm re- veals a rather different ])ieturc: about 40 per cent of its lipids are in the "free" state, and the bound lipids are found in the "firmly bound" fraction (Figs. 1 and 2). 5 1) -o ■^ EL D .9- a. 60 50- 40 30 20 10 — -I /" 11 'IV Normal 24 Hours after irradiation 72 Fig. 2. — The content (percentage of drj' weight) of various hpid fractions in the liyalo- plasm of rabbit Uver cells under conditions of acute radiation disease ; curves as in Fig. 1. Twenty-four hours after irradiation (in accordance with the investi- gations carried out earlier) an increase in the total lipid content was found especially in the mitochondria and microsomes (Table I). In the hyaloplasm these increases were a consequence of an increase in the bound (mostly "firmly bound") lipids; the portion of the "free" lipids decreased (up to 30 per cent). In the organelles an even greater increase in the content of bound lijsids occm^red with a pronounced tendency to the accumulation of "loosely bound" lipids. It should be noted that a small (in terms of absolute values), but relatively sub- stantial increase of the "free" lipids up to values surpassing the normal by ten-fold (up to 2 per cent of the total lipid content) was observed. Seventy -two hours later the ensuing decrease of the total lipids to an apparently almost normal value was accompanied by further dis- turbances in the ratio between the different lipid fractions. The hyalo- plasm showed a considerable reduction of the bound fractions along with an increase of the content of "free" lipids; such a situation led to a certain normalizing of the relations between the lipid fractions. At this ])oint the content of bound lipids in the cytoplasmic organelles dropped below the normal values; and the "firmly bound" fraction was particularly lower in this case. The content of the "free" lipids in cytoplasmic organelles kept increasing and exceeded the normal values 146 N. N. DOEMIN AND V. D. BLOKHINA by 30-fold. This resulted in a pronounced qualitative shift of lipid composition in the cytoplasmic organelles. Possibly at the beginning of the development of the radiation sickness lipoprotein disintegration was retarded both in the cytoplasmic organ- elles and in the hyaloplasm of the rabbit liver cells; later on this state in the cytoplasmic organelles changes ; and an intensified disintegra- tion begins in them (of the more stable components, especially). They also disi)lay a reduced capacity for binding "free" lipids. In the hyalo- plasm "tirmly bound" lipids proved to be the most stable ones. en a 10 8 6 4 2 / / / 0) Normal 2 24 Hours after irradiation pjg 3 xiie content (percentage of dry weight) of various lii>iil fractions in the mito- chondria of intestinal nuicosa cells of rabbits under conditions of acute radiation disease; curves as in Fig. 1 . Similarly, investigations of the intestinal mucosa in rabbit were con- ducted somewhat later under similar conditions of the radiation action ; unlike the previous series these experiments did not deal with prepara- tions of the entire cytoplasm, but with the mitochondria and micro- somes separately. The mitochondria and microsome prejmrations as well as the preparations of the liver cell cytoplasm were produced from homogenates of the intestinal mucosa by differential centrifugation usinff a modified high-speed centrifuges ASL-1 and ASL-2. Additional determinations were made 2 hr following exposure. The results obtained are shown in Figs. 3 to 5 and Table II. In total lipid content and that of the separate fractions determined in our experiments, the cells of the intestinal mucosa of the rabbit differ considerably from liver cells. The total lipid content, as per cent dry weight, is significantly less in the intestinal cells. It is noteworthy that, although under normal conditions the "free" CHANGES IN LIPIDS OF CELLULAR MICROSTRUCTURES 147 1 •^ o ^c 1| ^1 O CO CC S. ^ ^;^ '^^ ■^ =*-, g o" e ,* o o CO lO 1 -+ ^^ O - !r> 1 - I ; t— lU t/y -phosphate or i4(^;-formate and eventually with a nucleotide, the bone-marrow suspension was washed with Hanks' solution, then with acetic acid and smears were prepared. After exposure with a sensitive emulsion and staining with MGG-stain, the number of cells that had significantly more grains than the corres- ponding area of the background was determined. In the presence of deoxycytidylic acid, the incorporation of ^ap.phosphate into the reticular cells increased 2-5 times and 3 times in non-ii'radiated and irradiated marrow res])ectively (Table II). Control experiments carried out concurrently showed that under the same conditions two thirds of the incorijoration of 32p into the bone-marrow could be accounted for FREE DEOXYRIBONUCLEOTIDES IN RADIATION DAMAGE 157 Table II. The effect of dCMP ^^P -phosphate and ^"^C -formate incorpora- tion into bone-marrow cells of irradiated and non-irradiated guinea-pigs studied by autoradiography. 32p. phosphate ; incorpoi •ation lie- formate incorporation Type of non-irradiated irradiated non-irradiated irradiated cell control dCMP control dCMP control dCMP control dCMP All cells 37-0 Reticular cells 12-3 Myeloblasts and proerythro- blasts 70-9 Erythroblasts 91-5 Promyelocytes 47-9 Myelocytes 51-0 -48-(> 30-9 61-5 96-4 44-5 51-8 23-3 4-7 3S0 75-7 24-2 39-3 29 0 13-9 33-3 82-8 171 41-2 3;-) -8 9-4 73-5 83-2 64-3 46-3 41-4 31-3 57-5 95-9 51 -9 42-5 24-6 4-5 30-7 74-4 22 0 32-5 25-4 9-9 3()-() 84-3 22-6 34-8 Mean numbers of cells showing significantly liigher grain counts than the background, expressed per hundred cells of that cell type. by incorporation into DNA. Similarly, the incorporation of formate, which is to a considerable degree a specific precursor of DNA-thymine in bone marrow in vitro, indicated a threefold and two-fold increase of DNA-synthesis in non-irradiated and irradiated reticular cells respec- tively. The results thus show that the increased DNA-synthesis in the j)resence of deoxycytidylic acid is caused for a great part by increased incorporation into the reticular cells. At the same time, the presence of free deoxycytidylic acid may be a limiting factor for DNA-synthesis in the reticular cells of bone-marrow. Under similar conditions, the effect of thymidylate was also studied. 32P-phosphate incorporation into DNA, determined in the entire bone- marrow suspension after fractionation according to Schmidt and Thannhauser without autoradiography has indicated increased DNA- synthesis in the presence of thymidylate. Formate incorporation was reduced at the same time, which reflects the dilution of the newly syn- thesized i4C-thymidylate by added thymidylate. The first results obtained by autoradiography have also shown increased 32p incorpora- tion into irradiated reticular and "blast" cells in the presence of thymidylate, but paradoxically they have also revealed an increase of formate incorporation into both irradiated and non-irradiated reticular cells. In "blast" cells, the expected reduction of formate incorporation was found. Deoxycytidylic and thymidylic acid had also an effect on the purine/ pyrimidine ratio in the spleen of irradiated rats (Palecek and Soska, 1960). This ratio is increased to more than 1 on the fifth day after irra- 158 J. SOSKA, L, BENES, V. DRASIL, Z. KARPFEL, E. PALECEK AND M. SKALKA diation (Berenbom and Peters, 1956). The injection of deoxycytidylic acid after irradiation has modified the giianine/cytosine and adenine/ thymine ratios towards the normal vah;e, while thymidylic acid modi- fied only the ratio G/C and not A/T (Fig. 2). 2-0 1-5 fc f m fi= K 400r I ; ; 400r + TMP 4dOr-:-d-CMP Fig. 2. — The molar ratio of guanine/cj'tosine and adenine/thymine in the spleen DXA of rats, on the tifth day after iri'adiation with a dose of 400 r on tlie fourth day after admin- istration of 1 mg deoxycytidyhc or thyniidyhe acid. The height of the columns corres- ponds to the molar ratio, standard deviations of the means are also indicated. K ^ control, non-irradiated rats. If the mechanism of the recovery effect of deoxynucleotides or of the embryo extract lies in snpplying the missing DNA-precursors, it was expected that after irradiation the content of free deoxyribonucleotides would decrease in the tissue. We carried out the hrst experimental series with spleens of rats irradiated with a dose of 600 r. However, in the first hours after irradiation no decrease of deoxynucleotides was observed in the tissue (Soska and Soskova, 1959). On the contrary, their great increase was noticed. Only from about the 24th hr follow- ing irradiation did the content of these substances fall below the initial level. The level of free deoxyribosides rose somewhat more slowly and the following decrease was also slower. The interpretation of results obtained with the spleen is, however, complicated by the fact that the cell population of this tissue is not homogeneous, that the contribution of the particular cell types changes after irradiation, and the fact of extensive cell death. For this reason, regenerating rat liver was used for oiu" next experi- ments. This material has the advantage that its cell population is FREE DEOXYRIBONUCLEOTIDES IN RADIATION DAMAGE 159 relatively homogeneous and does not change after irradiation. Accord- ing to Holmes (11)56) DNA-synthesis progresses most intensively 24 hr after ])artial he])atectomy and the ince]ition of the synthetic period is delayed by \'2 hr by irradiation in the presynthetic period. The deoxyribosides were detected by the microbiological method of Hoft'-Jorgensen (1952), the nncleotides were separated from nncleo- sides on a colnmn of strong anion-exchanger at pH 7-(). Nucleotides were converted into nucleosides by means of an enzymatic preparation from snake venom before microbiological determinations. In the first experiment, the rats were irradiated with a dose of 600 r 1 hr after partial hej^atectomy. The deoxyribose compounds were de- termined 25 hr following hepatectomy. Four groups of rats were in- vestigated concurrently viz. hepatectomized irradiated rats, only hepatectomized rats, only irradiated rats and control rats. Irradiation and hepatectomy resulted in an increase in the level of free deoxyribo- sides (Table III). The highest level was observed in irradiated hepatec- tomized rats. On the other hand, the deoxynucleotide content was highest in non -irradiated hepatectomized rats. The deoxyribonucleo- ticle content was the lowest in the control rats. Irradiation itself thus resulted in a certain rise of free deoxyribonucleoticles, but the further rise produced by hepatectomy was blocked by irradiation. Table III. The effects of partial hepatectomy and of total-body irradiation on the content of free deoxyribosides and deoxyribotides in liver Group No. of Deoxynucleosides Deoxynucleotides animals (/^g/g tissue) {/xg/g tissue) Control Irradiated Hepatectomized Hepatectomi zed + irradiated 7 9 6 9 5-49 + 0-34 6 o + 0-43 7 -So + 0-41 8-40 + 0-68 1-44 + 0-41 4o5 -1- 0-25 9-55 + 1-80 5-60 + 0-60 The irradiation followed 1 hr after hepatectomj', the rats were killed 25 hr after hepatectomy and/or 24 hr after irradiation. In the next similar experiment (Table IV), the rats were already irradiated 24 hr before hepatectomy. In this experiment, the nucleo- tides were not separated from nucleosides and all deoxynucleotidic compounds were determined together after enzymatic conversion to deoxyiuicleosides. The total level of all deoxyribosidic substances in the individual experimental and control groups Avas similar to that in the preceding experiment. It seems thus, that deoxyribotide synthesis 160 J. SOSKA, L. BENES, V. DRASIL, Z. KARPFEL, E. PALECEK AND M. SKALKA Table IV. The effect of partial hepatectomy and of totalbody irradiation on the content of free deoxyribosides + deoxyribotides in liver Group No. of Deoxynucleosides animals + deoxynucleo- tides (/lig/g tissue) Control 9 8-7 + 0-74 Iiradiated 10 121 + 105 Hepatectomi zed 10 14-7 + 1-08 Hepatectomized 8 11-2 + 0-51 -}- irradiated The hepatectomy followed 24 hr after irradiation, the rats were killed 48 hr after irradiation and/or 24 hr after hepatectomy. following partial hepatectomy is delayed even by irradiation before hepatectomy. In the third experiment (Table V), the rats were irradiated 2 hr after hepatectomy and sacrificed later, 28 hr following hepatectomy. The difference in the level of free deoxyribonucleotides in the hejjatecto- mized irradiated and hepatectomized non-irradiated rat livers was still higher than in the preceding experiments, which again indicates a lack of DNA-precnrsors after irradiation. The differences in the content of polymerized DNA in the respective gi'oups of rat-livers were small (Table V, column 5). At the same time, the content of free "ribonucleo- tides-1- ribonucleosides" (all acid-soluble material, absorbing light at 260 m/x) was almost constant in all groups (column 6) and it was only the "ribonucleoside" content (column 7); (the part of this material, passing a Dowex 1 column, acetate form, at pH 7,0) that changed more conspicuously. These results agree in general with those of Jafife et al. (1959). An attempt was made to find the differences in the content of the individual substances by means of pajier chromatography. In all experimental groups, a substance was found in the liver, corresponding by mobility in l)utanol-ammonia system and by microbiological activity to deoxycytidine, in accordance with the results of Schneider and Brownell (1957). Purine deoxynucleotides were not found in any group. But in the livers of non-irradiated rats, a distinct spot of an additional deoxyriboside was found. So far it is only known that this substance contains a pyrimidine, but it is probably neither deoxyuridine nor thymidine, but most probably deoxymethylcytidine. Deoxyril)onucleotides were chromatographed in the isobutyric acid- ammonia system. Because of the complex pattern of deoxynucleotides detected on the chromatogram, the individual substances could not be identified. It could only be seen that the number and intensity of the FREE DEOXYRIBONUCLEOTIDES IK RADIATION DAMAGE 161 to 03 r^ •^ ',T 3 • O O -n 1. o +1 +1 +1 +1 lO 10 (M -H Oi CO r^ CO '-§ >» lO 6 6 00 o3 ^ 1 !» 5^ f_, ,V. ^ • ^H '+-^ s> lO ?H ^ '-^ '■ G '^^ CO *i cp t;- ® •H-^ f~o >.2 % ,4^ o^ -^ c-i '^^ iM t*-l 5S 3 +^ s c6 "S •'^ - &■- +1 +1 +1 +1 ^ cp O O CO CO H-^- -tH (M CO rt (M o o o o r— 1 ^H p^ . — 1 o s-^ 'a:) <% '^ f^ rO ti ^ ;s « ^ ^ 'w' CO --1 CO t^ >. d o ^ 3 r-H -^ O O c 5S-S <] re 6 6 6 6 o 4^ 1^ Q bc "bC a. +1 +1 +1 +1 CO CO CO C5 ot ^ I- 'rS ^§ ^^ ^ §1 d CO IQ CO CO -H CO 'T' lO CO CO 5si e C 0 +i 6 6 6 6 ® Is ^<-^ bC § O bC Q 3 +1 +1 +1 +1 lO o o -t =? °? -r 6 -P g 1^ CC C^ Gi >— ' O -P §^^ 0 -P ■^ „ '^ i—i 'M O d © •?? -is 6.S 05 o o o 1 — ^ ^H 1 — 1 .^ part ribot ^i s ^ ?? r^ o^ « (M o CO « •ts ts -^ 1 ^1 r-H O s t<-^ ^ C ^SS r^ ni _o ^ 1>3 N 01 '43 c6 > 1 ■g 'g ^* 1 -S ^ 1 -^ 1 (K C cS ft & + 5 fH 0) 03 O hH ffi HH H 162 J. SOSKA, L. BENES, V. DEASIL, Z. KARPFEL, E. PALECEK AND M. SKALKA spots containing deoxyribosidic comiDoiinds increased in the sequence from non-irradiated control to irradiated control, irradiated hepatecto- mized to hepatectomized non-irradiated livers. Since Sugino and Potter (1960) have recently established that the activity of deoxycyti- dylate-deaminase is reduced after irradiation, it seemed probable that the nucleotides accumulating after irradiation are derivatives of de- oxycytidine whereas the others appearing after hepatectomy in non- irradiated animals might perhaps be derivatives of deoxyuridine and thymidine. The nucleotides were dephosphorylated by a snake-venom preparation and subjected then to paper chromatography as nucleo- sides: preliminary results have, however, indicated the presence of deoxyuridine and thymidine derivatives in all groups except in non- irradiated controls, i.e. also in irradiated groups. The interval of 28 hr after hepatectomy was perhaps too long and the appearance of thymi- dine derivatives may be the result of recovery. Summing uj), Ave should like to stress some of the results. The effect of deoxynucleotides on mitosis, the possibihty of increasing radiation- inhibited DNA-synthesis by means of deoxynucleotides or by the nucle- otide fraction of embryo -extract, further the changes of the content of free deoxynucleotides and deoxynucleosides in regenerating liver after irradiation. They suggest that the effect of radiation on processes leading to DNA-synthesis in animals may be more important than the effect on the process of polymerization or on the macromolecular DNA itself. This is in accordance with the results of Sugino and Potter (1960) who have found that radiation blocks the enzymatic reactions that re- sult in the synthesis of thymidylic acid. Even then there remains the unsolved question why these enzymes are inactivated, since in the case of other enzymes, no inactivation as a result of irradiation in vivo has been so far observed. Quite on the contrary, a raised enzymatic activity has been often noted. Consequently, it appears that the second possi- bility is more probable, that is to say that after irradiation there takes place disintegration of large structural subcellular units or entire specialized cells that are carriers of the respective synthetic activity. Experiments with regenerating liver would support the interpretation that irradiation blocks some inductive process resulting in the synthesis of enzymes that make themselves apparent in the preparation of DNA- SA^nthesis, in particular in the synthesis of some deoxyribotides. REFERENCES Berenbom, M., and Peters, E. R. (1956). Radn iJes. 5, ol."). CoHN, W. E., and Carter, C. E. (1950). J. Amer. rhein. Soc 72, 4273. FREE DEOXYRIBONUCLEOTIDES IN RADIATION DAMAGE 103 Drasil. v., SoSka, J., and BkxkS, L. (li)")!*). Folid Biol. 5, 334. H(iFF-JoRf!KNSKN, E. (l»r)2). Blocliem. J. 50, UH). Hoi.MKs. H. E., (llt.Ki). 7h "Ionizing Kadintion and Cell Mct.iholism", p. 225. Ciba Eoundation. l^Diidon. Jaffe, L. J., Lajtha, L. G., Lascelles, J., and Ord. M. (!. (IStf)!)). hit. J. Rudn Biol. 1, 241. PALEfEK, E., and Soska. J. (llKiO). Folia Biol. 6, 168. Schneider, W. C, and Brownell. L. W. (1!).")7). ./. nut. Cancer fn.'^t. 18, TiTO. Soska, J., and Soskova, L. (1959). Folia Biol. 5, 425. SosKa, J., DraSil, v., and Karpfel, Z. (195S). Second U.N. Int. Conf. "Peaceful Uses of Atomic Energy". A/Conf. 15/P/2121, Geneva. Soska, J.. Karpfel, Z.. and Drasil, V. (1959). Folia Biol., 5, 190. SuGiNO, Y., Potter, R. L. (1960). Radn Res. 12, 477. DISCUSSION MANOiLOV : Did yoii administer nucleotides isolated from different organs and tissues or are the phenomena observed characteristic for spleen and bone -marrow alone ? Did the deoxyribonucleotides achiiinistered subcutaneously incorporate into spleen DNA as such, or did they imdergo preliminary splitting up? SOSKA: The effect of deoxyribonucleotides on bone-marrow alone has been studied. We have not studied as yet, whether the nucleotides administered are broken down in tissues or not. xjtkin: How did you determine the mitotic index? SOSKA: A smear of Ijone-marrow from the sacrificed mouse was prepared and stained according to Feulgen. The mitotic index was determined in a count of 8.000 to 10,000 nucleated cells. The proportion of the individual mitotic phases was recorded. Dr. Karpfel found that, following exposure, the ratio of meta- phases to prophases was changed. If, in the unirradiated mice, the ratio of meta- phases to projDhases was 0-56, in animals irradiated with 500 r this ratio rose to 1-0 or 1-5. We found that administration of pyrimidine deoyxribonucleotides following exposure brought changes in this picture, the ratio of metaphases to prophases returning partially to normal. BIOC^HIMTE ET RADTOBTOLOGTE DU NOYAU C^ELLULAIRE M. ERRERA Laboratoire de Biophysiqm et de Radiobiologie, Universite Libre de Bruxelles, Bruxelles, Belgium RESUME Les radiolesions tin noyau cellulaire sont envisagees du point de vue bio- chimique. Certaines fonctions du noyau sont imrticulierement sensibles: les phosphorylations (Creasy et Stocken, 1958) et la stabilite des ions Na+ et K+ (Creasy, 1960) sont fortement alterees par des doses de I'ordre de 100 rad. Comme ces ions jouent un role important dans la fixation des petits nucleotides dans le noyau, ces desequilibres produits par I'irradiation pourraient etre lies aux efi'ets des rayons X sur le metabolisuie des acides nucleiques. Quand on etudie I'incorporation du 32P dans I'acide desoxyribonucleique du thymus on observe, que pour des doses croissantes, I'inhibition augmente d'abord rapidement, puis plus lentement (Ord et Stocken, 1958). II en est de meme quand on etudie I'incor- poration de Tadenine dans lacide ribonucleique des noyaux isoles (Logan et al., 1959), ou rincorporation de la phenylalanine dans les proteines. Dans les trois types de metabolisme, on observe que des doses depassant 300 rad n'augmentent que tres peu I'effet observe. II est encore premature d'interpreter ces resultats en fonction des effets biochimiques decrits sur la cellule entiere ou en fonction des effets cytogenetiques. Un grand nombre de donnees experimentales indiquent clairement qne les radiolesions nucleaires ont des consequences pins graves pour la cellnle qne les radiolesions cytoplasmiqnes. Celles-ci semblent en efFet etre snsceptibles d'etre restanrees dans nne certaine mesnre a condition qn'existe nn noyan snffisamment fonctionnel (Errera et al., 1959). II est done logiqne d'essayer de comprendre les mecanismes physicochimiqnes on biochimiqnes des radiolesions nncleaires que Ton commence a con- naitre dn point de vue cytologiqne. C'est pourquoi 11 nous a paru in- teressant de rassembler dans cette breve communication les donnees existantes concernant les processus biochimique normaux qui se deroulent an sein des noyaux et la maniere dont ceux-ci sont affectes par I'irradiation ; nos propres observations seront integrees a 1' image plus generale que Ton pent degager de la litterature. PROCESSUS BIOCHIMIQUES NORMAUX On salt qu'au cours du cycle cellulaire normal la division est pre- 165 166 M. ERRERA cedee d'importants phenomenes de synthese dont ceiix qui concern- ent I'acide desoxyribonucleique (ADN) sont certainement les mieiix conniis. Mais a cote de lADN les chromosomes contieiment des acides ribonucleiqiies, des proteines. des phospholipides, et il est vraisembl- able que ceux-ci se synthetisent au niveau on dans le voisinage immediat des chromosomes. D'autre part les nucleoles. riches en acide ribo- nucleique, en proteines, et en phosjiholijiides se reforment apres chaque mitose. et il est vraisemblable egalement que les organisateurs nucleolaires des chromosomes sont le siege de syntheses actives. D'ailleurs de nombreux travaux utilisant les techniques cytochimiques quantitatives (autoradiographie, microspectro})hotometrie) montrent clairement que des precurseurs d'acides ribo- et desoxyribonucleiques, de meme que des precurseurs de proteines s'incori)orent dans ces structures cellulaires et temoignent vraisemblablement de Texistence de syntheses nettes d'acides nucleiques et des proteines (voir Brachet, 1957). D'un point de vue biochimique plus precis ces mechanismes de syntheses sont moins l)ien connus, mais ils impliquent vraisemblable- ment I'existence de systemes enzymatiques particuliers, soit fournis- sant I'energie indispensable soit effectuant les syntheses elles-meme. Nous nous preoccuperons principalement des systemes enzymatiques des noyaux de cellules lymphoides on de foie de manmifere, tout en mentionnant a Toccasion ce que Ton sait i)our d"autres organes ou meme d'autres types d'organismes. Nous ne nous etendrons pas sur ce que Ton sait ces sources nucleaires d'energie et sur la synthese de I'ADN, des aspects du x^robleme ayant deja ete envisages dans d'autres communications. SYNTHESE DES ACIDES RIBONUCLEIQUES 11 semble de plus en ])lus certain que la grande majorite de TARN des cellules qui se multii)lient est synthetise dans le noyau cellulaire: il a meme pu etre demontre dans les cellules de HeLa (Perry, 1960; Perry, Hell et Errera, 19()()) qu'environ 60 pour cent de I'ARN cyto- plasmique provient du nucleole, et qu'environ 30 pour cent provient du reste du noyau. II n'est meme pas certain qu'il existe de synthese cytoplasmique d'ARN dans les cellules qui se multiplient; d'oii Tim- portance de rechercher dans le noyau cellulaire des radio-lesions qui determinant des alterations du metabolisme de lARN. On connait encore mal les mecanismes biochimiques de la synthese de I'ARN des cellules animales. Chez les microorganismes, par contre, Grunberg Manago et Ochoa ont isole mi systeme enzymatique qui utilise des nucleotides diphosphates. Cependant, T etude de divers BIOCIIIMIE ET JiAD101iH)L0UlE DU NOYAU CELLULAIRE 167 tissiis aiiimaiix, bien (lue beancoiq) moins avancee que celiii des micro- organismes, indiqne rexistence de certains mecanismes de synthese utilisant les ribomicleosides triphosphates (Caiinelakis et Herbert, 1960), mais dans ce cas il ne se fixe que de I'acide cytidylique et adc'nyli- qiie en fin d'nne chaine d'acide ril)onncleiqiie de ])oids moleculaire rela- tivement faible et qui jouerait un role ini])ortant dans le transfert des acides amines. Un systeme capable de synthetiser de I'acide poly- adenylique pur a partir de I'ATP a ete decrit par Edmons et Abranis (1960) dans les noyaux des thymocytes. Nous sommes, done, malheureusement loin de connaitre les mecan- ismes de la synthese de TARN, et rien ne nous permet de prevoir que I'ARN des chromosomes et des nucleoles serait synthetise selon le meme mecanisme. De nombreuses observations, moins directes toutefois, ont ete effectuees sur des noyaux isoles. Logan (1957) a, par example, observe que Tadenine s'incorpore beaucoup plus rapidement et sans phase de latence dans la fraction d'ARN de noyaux des thymocytes, soluble dans du phosphate 0,1 M a pH 7,0, alors que la fraction insoluble n'incorpore que beaucoup plus lentement et apres une certaine latence. Cette fraction qui incorpore rapidement est constituee de particules ribonucleoproteiques de 25 A de diametre dont la localisation dans le noyau n'a pas encore ete etablie avec certitude (Frenster, et al. 1960). L'incorporation de I'adenine dans I'ARN des noyaux de thymocytes in vitro est notablement diminuee par Tirradiation et des experiences radioautographiques sur des noyaux de thymocytes de veau montrent qu'on atteint deja un effet maximum (environ 30 pour cent d'inhibition) apres des doses de 50 rad. Le travail a ete repris sur des noyaux de thymus de jeunes rats, et les methodes biochimiques cette fois, ont montre que la diminution de Tincorporation qui depasse rarement 20 pour cent chez le rat est deja maxinunn apres 300 rad et n'augmente pas apres desirradiations plus prolongees. Des fractionnements de I'ARN selon la technique utilise par Allfrey et Mirsky (1958) ont revele au cours d'experiences preliminaires que I'ARN soluble en milieu salin dilue (ARN I) conserve une activite normale, tandis que la fraction qui n'est pas extraite dans ce milieu (ARN II) incorpore moins d'adenine que celle des cellules non irradiees (Faures et Errera, sous presse). II serait tentant de penser qu'il s'agit la d'une fraction d'ARN dependant plus etroitement de I'ADN et des phosphorylations nucleajres, puisqu'ils sont inhibes simultanement. Dans le cas du foie de rat, I'incorporation de I'adenine dans les noyaux isoles est sensiblement accrue d'une part par 1" addition des microsomes, et d'autre part 48 ou 72 heures apres une hepatectomie 168 M. ERRERA partielle. Le role des microsomes dans ce processus n'est pas ehicide, mais ils poiirraient intervenir dans la conversion de I'adenine en un precnrsenr niienx assimilable, on en fournissant d'autres nucleotides qui pourraient etre necessaire a la formation de I'ARN. Les mecanismes de synthese de TARN nucleaire offre certainement un vaste champ d' investigation. Syntheses de profeines On ignore encore tout des mecanismes de la synthese des proteines dans le noyau cellulaire; ce que Ton connait des systemes purifies de syntheses proteiques s'applique a des fractions subcellulaires que Ton a pas encore localise avec certitude dans les cellules vivantes. II semlile toutefois. d'apres des experiences radioautographiques que dans des cellules en croissance la synthese des proteines se passe de maniere independante dans le noyau et dans le cytoplasme, et ces donnees ne font que confirmer celles de Brachet (1957) qui a demontre une synthese nette des proteines dans des fragments anuclees d' Acefabularia mediter- ranea. Les travaux sur les noyaux de thymus isoles (Mirsky et al., 11)56; Allfrey et «/., 1957) out montre d'une part qu'un acide amine marque, incorpore dans les proteines des noyaux. n'est pas deplace si les noyaux sont ensuite mis en presence d'un exces de I'acide amine non marque, ce qui indique probablement un phenomene de synthese. D'autre part Allfrey (1959) a trouve dans des preparations nucleaires, des systemes enzymatiques d'activation et de transfert d'acides amines en beaucoup de points semblable a ceux decrit par Zamecnik et al. (1958), dans le cas d'extraits tissulaires (ARN soluble, ARN des ribosomes). Les mesures d'incorporation d'acides amines dans les proteines des noyaux semblent done reellement representer un metabolisme pro- teique reel. Toutefois, un point obscur reste a comprendre, ces incor- porations d'acides amines ne sont pas inhibees par la fluorophenyl- alanine on Tethionine comme le sont la plupart des syntheses proteiques. (Allfrey et al., 1957.) Mirsky, Osawa et Allfrey (1956) ont montre que rincorporation des acides amines dans les noyaux cellulaires depend- ent des phosphorylations nucleaires, puisqu'on pent les bloquer par I'addition de c\^anure. de nitrure, de dinitrophenol, mais pas par le bleu de methylene qui inhibe les phosphorylations des mitochondries seulement (Osawa, Allfrey, et Mirsky, 1957). Cependant le dicoumarol et le vert de Janus qui n'inhibent pas les phosphorylations nucleaires, inhibent T incorporation des acides amines. Les jjhosphorylations nucleaires semblent done jouer un role dans le metabolisme des pro- teines nucleaires mais ils pourraient ne pas etre la source exclusive d'energie j^our ces ]irocessus. BIOCHIMIE ET KADIOBIOLOUIE DU NOYAU CELLULAIRE 169 D'autre part il semble y avoir un lien assez etroit entre I'incorporation d'acides amines et le nietabolisme ribonncleiqne puisque le 5:6:di- chlorobenzimidazol inhibe rincorporation d'acides amines si cet agent est ajoute en meme temps qne I'acide amine. Ficq et Errera (1958, 1959) et Logan et al. (1959) ont montre que I'incoi'jDoration de la phenylalanine dans les noyanx de thymocytes de rat est moins modifiee pendant les 30 premieres minutes d'incubation apres irradiation in vitro (50 a 900 rad) que par la suite. II en est de meme pour les noyaux du foie de rat. Dans ce dernier cas, Logan et al. ont montre que des mitochondries ajoutees aux noyaux augmen- tent I'incorporation de la phenylalanine; il a aussi ete montre que si on irradie ces mitochondries avant de les additionner aux noyaux, on obtient egalement une inhibition de Tincorporation des acides amines. En ce qui concerne les relations doses-efFets etudiees sur des noyaux de thymus de rat, elles suivent une modalite tres semblable a ce qui avait ete observe dans le cas du metabolisme de I'adenine: inhibition maxima de I'ordre de 20 pour cent pour des doses de 300 rad. II est interessant de noter que I'effet d'inhibition par les rayons X pent etre jjartiellement restaure si les preparations des noyaux sont incubees en pi'esence d'acide desoxyribonucleique meme denature par chauffage. On se souvient que Mirsky et al. (1956) avaient observe une restauration 2)ar TADN du metabolisme des acides amines, inhibe par la desoxyribo- nuclease. Cet effet n'est pas specifique de I'ADN et semble etre en relation avec I'equilibre ionique des noyaux ROLE DE L'fiQITILIBRE IONIQUE DANS LE METABOLISME DES NOYAUX DE THYMOCYTES Mirsky et al. (1956) et Osawa et al. (1957) ont montre d'une part que si on traite une suspension de noyaux an moyen d'acetate a pH 5,1, on libere du potassium et des nucleotides en meme temps qu'on inhibe rincorporation des acides amines dans les proteines. D'autre part, il existe une concentration optimum de Na+ pour rincorporation des acides amines alors qu'un exces de K+ est inhibiteur. Le I'apport NaCl/KCl et la presence de nucleotides varies joue done un role im- portant dans les phosphorylations nucleaires dont depend I'incorpora- tion des acides amines dans les proteines du noyau. II est done interes- sant que Creasy (1960) ait observe que de faibles doses de rayons X liberent du sodium des noyaux de la rate de rats (pas de seuil et 80 pour cent de liberation apres 50 rad) alors que la liberation de K+ ne debute qu'apres 30 rad pour etre quasi totale vers 100 rad. Ces doses nous I'avons 170 M. ERRERA vu sont siiffisantes pour inhiber les phosphorylations nucleaires et il est vraisemblable que la pente des ions et peut etre celle des oligonucleo- tides en sont la canse. La perte de K+ et de nucleotides liees aux noyaux peuvent aussi etre a Torigine de la diminution de Tincorpora- tion des acides amines (Osawa et al. 1957). Le fait qu'il n'y ait qu'une petite partie des sytemes nucleaires responsal)les de I'incorporation des acides amines et des bases azotees qui soit diminuee apres de petites doses d'irradiation et ciu'une irradiation plus importante n'augmente pas Teffet semblerait indiquer Texistence d'une reserve de derives phosphoryles cForigine nucleaire. On devrait toutefois aussi envisager la contamination des noyaux par une quantite suffisante de mitochon- dries qui pourraient fournir les derives phosphoryles necessaires. On pourrait aussi sup])oser qu'il existe, comme nous Tavons fait x^recedem- ment pour le metabolisme de FARN, une fraction proteique plus etroitement dependante des phosphorylations nucleaires et aussi plus radiosensible. REFERENCES Allfrey, V. G. (19o9). Dans '"Tlie Cell Xurleus", Bixtterworths, London. Sous presse. Allfrey, V. G.. et Mirsky, A. E. (1958). Proc. nat. Acad. Sci., Wasli. 44, 981. Allfrey, V. G.. Mirsky. A. E., et Osawa, S. (1957). J. gen. Physiol. 40, 451. Brachet, J. (1957). "Biochemical Cytology". Academic Press, New York. Caxnellakis, E. S. et Herbert, E. (I960). Proc. nut. Acad. .SV/., Wash. 46, 170. Creasy, W. A. (1960). Biochim. biophys. Acta 38, 181. Creasy, W. A., et Stockex, L. A. (1958). Biochem. J. 69, 17, 1. Edmons, M., et Abrams, R. (1960). J. biol. Chem. 235, 1142. Errera, M., Ficq, a., Logan, R., Skreb, Y., et Vaxderhaegk, F. (1959). Exp. Cell Res. Suppl. 6, 268. Faures, a., et Errera, M. (1961). Int. J. radii Biol. Sous presse. Ficq, A., et Errera, M. (1958). Exp. Cell. Res. 14, 182. Ficq, A. et Errera, M. (1959). Exp. Cell. Res. Suppl. 7. 145. Frensteb, a. H., Allfrey. V. G., et Mirsky, A. E. (1960). Proc. nat. Acad. Sci. Wnsli. 46, 432. Logan, R. (1957). Biochim. biophys. Acta 26, 227. Logan, R., Errera, M., and Ficq, A. (1958). Exp. Cell Res. 14, 182. Logan, R., Ficq, A., et Errera, M. (1959). Biochim. biophys. Acta 32, 147. Mirsky, A. E., Osawa, S., Allfrky, V. G. (195(5). Cold Spring Harbour Symp. 21, 49. Ord, M., et Stocken, L. A. (1958). Nature, Land. 182, 1787. Osawa, S., Allfrey, V. G. et Mirsky, A. E. (1957). J. gen. Physiol. 40, 491. Perry, R. P. (1960). Exp. Cell. Res. 20, 216. Perry, R. P., Hell, A., et Errera, M. (1960). Proc. Xth Int. Cell. Biol. Conqr. Paris, p. 110. Zamecnik, P. C, Stephenson M. L., et Hecht, L. I. (1958). Proc. nat. Acad. Sci. Wash. 44, 73. DISCUSSION HUG : You were speaking of closes of 200 to 300 r. Is it possible that, at a different dose rate, the effect woulfl be greater, even if small doses were given? EREERA: It should be tested. I have not done it. hug: Where in your opinion docs the disturbance of the RNA .synthesis occur? BIOCHIMIE KT RADIOBIOLOGIE DU NOYAU CELLULAIRE 171 ekreka: My first impression is tliat tlicrc are two iiKM-liaiiisms operating: RNA synthesis may jiroeeed in the microsomes or in tlH> nucleoli. But wo failed to differentiate them. We tried, but thd not succeed hi fin( hng a difference between the corresjionding nucIeoU and microsome fractions. They were difficult to differentiate. makcovich: RNA is Hrst synthesized in the nucleus and then it comes out into the cytoplasm? ERRERA : The transfer of RNA from nucleus into cytoplasm was demonstrated by experiments with short and long term incubation of the cells. We believe that RNA is synthesized in the nucleus and that this is conunon to all dividing cells. It is the accepted view on the matter until new data appear. gray: I should very nnich like to know^ the methods for specific inhibition. It seems to me you have made it clear enough that radiation damage to the chromo- somes is dependent in some measure on total phosphorylation. It would be very interesting to study whether it is cytoplasmic or nuclear phosphorylation. Can they be differentiated? In what way is inhibition of phosphorylation connected with mitochondria? ERRERA : Inhibition of phosphorylation in the nuclei proceeds without affecting the mitrochondria. PhosjDhorylation in the nucleus and cytoplasm can be in- hibited separately. passynsky: Is it possible to regard the post-irradiation loss of Na+ and K+ ions as a result of damage to the nuclear membrane? Wliat is the absolute quantity of Na+ and K+ ions which leaked out from the nucleus following exposure, and what is its quantitative relationship to the ionization value of the medium? ERRERA : Little is Imown about this. It is possible that basic proteins are capable of binding positive ions. In the case of Na+ about 80 per cent is lost upon exposure to 50 r, whereas in the case of K+ there is no loss of ions up to doses of 30 r. When this dose is exceeded, the loss proceeds very rapidly; at 100 r the loss of this ion is almost complete. passynsky: If this constitutes damage, I should like to draw attention to the possibility of using impedance measurement techniques to study it. ERRERA: I have no experience in this field. PASSYNSKY : As far as I know, the loss of ions may be caused by a damage to the membrane or changes of the absorption. It is a problem which may be studied by means of the impedance technique. If the membrane is damaged, several centres of damage may be discovered. ERRERA: To elucidate this question, electron microscope pictures should be available, otherwise it would be difficult to get the answer. I have never taken electron micrographs of such nuclei. My belief is that the membrane is severely damaged, but I am not sure of that. For final confirmation good electron micrographs are necessary. BACQ: In the first experiment you cited you irradiated intact cells and studied synthesis of the RNA and proteins by means of autoradiography. In other experiments it was isolated nuclei you studied? 172 M. ERRERA ERRERA : On the picture given here results of the experiments using whole cells were presented. After that isolated nuclei were used. KUZIN : If I understood you correctly, in the experiments with isolated nuclei you found apjaroximately 30 per cent inhibition : further increase in the irradiation dose produced no increase in the effect studied. At the same time using whole cells and tissues one may obtain considerably more inhibition. Do you not think that the difference is accounted for by the role jolayed by the cytoplasmic elements? ERRERA: I am not sure whether we realize what is occurring in the nucleus. But it is a fact that, for protein synthesis, inhibition in intact cells occiu-s in 90 min. The purpose of this stv;dy was to determine the most sensitive process. MOUTON : May it be that the changes you have observed are caused by the changes in the pH of the medium brought about by irradiation? ERRERA: I do not think so, since strongly buffered solutions were used throughout the experiments. MOUTON : It seems possible that there may be a connection between the radiation- induced breakdown and acid autolysis. HOLLAENDER: You sjioke about inhibition of the synthesis of the nucleic acids and proteins. How is this related to mitosis? ERRERA: Under normal conditions the mitotic index amounts to 5 to 7. It seems to me that the nuclei we studied should be considered as being in a resting phase, not in mitosis. TOBIAS: You said that RNA synthesis is inhibited by 30 per cent. How is this to be understood? Does it mean that 30 per cent of the nuclei are not taking part in the synthesis, or that synthesis in all the nuclei is inhibited by 30 per cent? ERRERA: Autoradiography has shown that all the nuclei have been affected equally. ON THE MECHANISM OF LETHAL ACTION OF X-RAYS IN ESCHERICHIA COLI K12 H. MARCOVTCH [with flic technical assistcuice of Mile B. Vai/ne) Service de Kadiobiologie et de Cancerologie de Vlnstitut Pasteur, Paris, France SUMMARY Crosses have been utilized in order to elucidate the mechanism of the lethal effect of X-rays on Escherichia coli K12. These bacteria possess two to three nuclear bodies and are killed according to a one-hit dose-effect relationship. The experiments were designed to search for the existence of X-ray dominant lethal mutations in one of the nuclei, transferable to the recombinants. No svich muta- tion was detected. Among the lesions which are responsible for permanent inhibition of division in irradiated cells, those of the nucleus are the best known and the most extensively studied. Their role seems to be predominant. In higher organisms, tissue damage is essentially the consequence of chromosomal disorders, not compatible with the normal sequence of mitotic events. The high radiosensitivity of the nucleus is clearly de- monstrated in irradiation experiments of Habrobracon eggs with a-rays (Rogers and von Borstel, 1957; von Borstel and Rogers, 1958). Mam- malian somatic cells are killed by X-rays according to a sigmoidal dose- effect relationship; two events are necessary to destroy the colony forming ability (Puck and Marcus, 1956; Puck et al., 1957). In haploid yeast, the X-rays survival curves are exponential, but in diiDloids they are two-hit sigmoidal ones (Latarjet and Ephrussi, 1949). It has been possible to have direct evidence of the existence of two kinds of genetic lesion. The more frequent are recessive lethal mutations responsible for the sigmoidal shape of the diploid survival curves. The others are dominant lethal mutations (Mortimer, 1955). In these examples, the lethal effect of X-rays may chiefly be attrilni- ted to lesions of the nuclear material. In Escherichia coli, the survival curves are, in practically all in- stances, exponential. This observation does not depend on the stage of the cultures or on the composition of the growth medium. The bacterial cells possess a variable number of nuclear bodies, according 173 174 H. MARCOVICH to the strain and the physiological conditions. Since one event is sufficient to kill E. coli cells (permanent division inhibition) the process must either involve a singly existing organelle of the cell or induce some dominant lethal damage in one of many similar entities. If the affected site were one of the nuclei, the lesion might be considered some kind of dominant lethal mutation. The experiments that will be reported in this paper aim to examine the second alternative, e.g. a dominant lethal mutation in the genetic material. PRINCIPLE OF THE EXPERIMENTS The material and the genetic methods utilized are those described in the monograph by Wollman and Jacob (1960). The properties of sexual recombination in E. coli K12 which will be used in this work are the following : 1 . The characters are transmitted from the Hfr donor Ijacteria to the F~ recipient cells with a frequency directly proportional to their dis- tance from the origin ''0'" of the chromosome. Their sequence is always the same for a given Hfr strain. 2. No cytoplasmic material has yet been detected to pass from the Hfr to the F"^ bacteria. 3. During conjugation, the parent cells may be separated by shaking the suspension vigorously ; the transfer of the markers still remaining in the Hfr is then interru]ited. Let us call a the probal)ility, per unit of dose, for the induction of a dominant lethal mutation anywhere in any one of the Hfr chromo- somes. The equation of the survival curve to X-rays would be n — = e-^D (1) where w/wq is flie proportion of surviving bacteria to D rads. Let us assume that the probability for a given chromosome to be involved in a cross does not depend u})on \^'hether or not it carries a dominant X-ray induced lethal mutation. If we assume, in addition, that the transfer to the zygote of this hypothetical mutation will, sooner or later, kill all the recombinants, the expected dose-effect relationshi]) for the survival of recombinants derived from a mating between an irradiated Hfr bacterium and a normal F~ one is __ = ^-tl/N aD (2) "0 LETHAL ACTION OF X-RAYS IN E. coli K12 175 where N is the number of chromosomes, t the probability that the postu- lated dominant lethal should be transmitted to the zygote, and / the probability that it would be expressed in the recombinants. According to equation (2), the induction of a dominant lethal mutation in the Hfr chromosomes will allow some predictions which may be experi- mentally tested: 1. The survival curve of the recombinants will be exponential and have a smaller slope than the survival curve of the Hfr. 2. If the cross is interrupted, the factor / would decrease (lower probability of transfer of the lethal mutation) and the slope of the recombinants survival curve would be the more diminished, the earlier the interruption. 3. Equation (2) may apply to any kind of selected marker, regardless of the absolute frequency of transfer from the Hfr to the F~ bacteria. It is also valid for the case of non-selected plating ; if the irradiation induces, in the Hfr chromosomes, dominant lethal genes, which kill the selected recombinants to wdiich they are transmitted, they will also express themselves in a decrease of the number of the viable F~ cells when plated, after a cross, on complete agar. MATERIALS AND TECHNIQUES Bacterial strains These have kindly been supplied by Drs. Jacob and Wollman. They are: E. coli K12 Hf^H (Bi-, Ss) ; ^. coli C600B34-5 (T-, L-, B-, Gal-, Sr); and E. coli PA309 (T-, L-, Try-, H-, Ar-, B-, Lac-, Gal-, Man-, Xyl- Mai- T+, Sr) The selected marker in the crosses utilized in this work are T+ L+ Sr. In our experimental conditions, the bacterial cells have two to three Feulgen-positive bodies, the more frequent number being two. Media The growth medium is 0-3 per cent Difco Nutrient Broth, 0-5 per cent Bacto Tryptone and 0-5 per cent NaCl. The synthetic medium utilized has been described by Vogel and Bonner. For solid medium Difco Bacto Agar is added to these media at a concentration of 1-5 jjer cent. The bacteria are plated using the soft agar (0-8 per cent) technique. Vitamine Bi, and streptomycin are added in the synthetic soft agar tubes before plating. EXPERIMENTAL PROCEDURE The bacteria are grown in broth up to 2-108 cells/ml and irradiated 176 H. MARCOVICH in the same medium. O-l ml of the Hfr strain is then mixed with 0-9 ml of the F~ strain, and the mixture slowly agitated at 37°C. Unless other- wise sj)ecified, the bacteria are alloANed to stand for 1 lir before dilution and plating. The plates are incubated at 37°C for 48 hr before counting. Irradiation is performed with a Machlett tube AEC' 50 with a tungsten target operating at 40 kV. The dose is estimated by comparison with the same lethal effect on the strains observed with a ^OQo source. RESULTS 1. HfrH survival curve and T+ L+ Sr recombinants In our experimental conditions the frequency of T+ L+ Sr recom- binants formed by the unirradiated controls varies from 20 to nearly 50 per cent. The survival curves of HfrH and of T+ L+ Sr recombinants are exponential ; the slope of the latter is less than the slope of the Hfr and depends on the F~ strain (Fig. 1). Yet the same damage to the HfrH may be quantitatively expressed differently according to the recipient 100 X 10^ rods Fig. l.—E. coli HfrH and E. coU C600B34 5 are grown in broth up to 2-108 bacteria/ml. The Hfr strain is exposed to increasing doses of X-rays and mated to the F-sti'ain: 0-1 ml of the former is added to 0-!> ml of the latter. The suspensions are agitated slowly for 1 hr and then ])hited on synthetic agar medium, supplemented witli all the required growtli factoi's except T and L. The survival curve of Hfr is determined on agar sohdifietl complete medium; the slope is the same if synthetic medium is used. LETHAL ACTION OF X-RAYS IN E. coli K12 177 strain. On the other hand, the difference between the slopes of the two curves may vary sUghtly from one ex])eriment to another. 2. Survival CKires of T+ Sr, L+ Sr and T+ L+ Sr recombinants If the decrease of the frequency of the recombinants with increasing doses of X-rays was related to inactivation of the selected marker, as for instance, mutation from prototrophy to auxotrophy for the con- trolled substance, the slope of the T+ L+ Sr recombinant survival curve would be the sum of the slopes of the T+ Sr and of the L+ Sr survival curves, assuming that the inactivation of the T and of the L markers are independent events. Figure 2 shows that this is not the case. The Fig. 2. — The same conditions as for Fig. 1. The crosses are plated on synthetic agar without T and L or with one of each amino acid in order to select for one marker only, or for both. slojDcs of the three survival curves are very close : hence the transfer of the two T L markers to the recombinants and its inactivation depend on closely -linked events. This may be demonstrated directly by the genetic analysis of the surviving T+ Sr and L+ Sr recombinants. Nearly 100 per cent of them are found to carry the non-selected marker re- spectively L+ and T+. This result indicates that the loss of the recom- binants from an X-rayed Hfr is linked to some damage to the chromo- some, apart from the selected markers themselves. 178 H. MARCO VICH Then, the X-ray lesion of the Hfr expressed in the reconil)inants must concern the transfer process, being either a damage to the trans- mission of the genes from tlie Hfr to the recombinants or a letlial mutation which might kill the zygote. The latter possibility may be tested by interruption experiments. 3. Effect of inferrupfion of conjugafiofi on survival of T+ L+ Sr re- comhinants In Fig. 3 are drawn the survival curves of the T+ L+ Sr recombinants for several interruption times. No modification in the slope is observed. -O E O E Duration of conjugation • 90 mm i 60mm O 30min « 1 0 min 100 xlO-'rads Fig. 3. — The experimental conditions are the same as in Fig. 1. But the matings are allowed to stand for a different length of time and then vigorously shaken and plated on selective synthetic agar. This eliminates the possibility that a dominant lethal, induced on the chromosome, was transmitted to the zygote at the same time as the T+ L+ markers. There remains, however, the alternative that such a liy])othetical lethal mutation might exist only on the chromosome segment between the TL loci and the origin "O" and, consequently, will not apjjear with interruption experiment using these selected markers. The following experiment will answer to this question. LETHAL ACTION OF X-RAYS IN K. coll Ivl2 179 4. Cross between irradiated Tlfr and normal F', plated on non- selective medium If a letlial nuitation was induced on the Hfr chromosome near the origm "O"" it would express itself, in the cross, by the killing of the F" hacteria ])lated on broth medium. The ex]ieriment which has been ]ier- formed consisted of mating Hfr and F" cells in the proportion of 10 to 1, so that the non-conjugated F" would be very rare. The dose given to the Hfr was such that the probability was low for a mating between an F- and an Hfr with an unharmed Hfr chromosome. As the frequency of recombination for the selected markers in this experiment was 30 per cent one would expect at least a 30 per cent decrease in the number of the colonies formed by the F" crossed with the irradiated Hfr as com- pared to the control. No detectable loss in the number of colonies has l)een observed. 5. Cross between irradiated F bacteria, and normal or irradiated Hfr If F~ bacteria are irradiated and crossed with a non-irradiated Hfr it would be expected that the survival curve of the recombinants and of the F~ would coincide, on the basis of the hypothesis of a dominant lethal being induced in the F~. Many experiments have been done to examine this point. Although for unknown reasons the results lack re- producibility, some restoration of the irradiated F" by the Hfr is always observed. If the cross is made between two irradiated bacteria, Hfr and F~, the survival of the recombinants is what might be expected on the assump- tion that the radiation damages to the Hfr and to the F"" would act independently in the zygote to reduce the number of the recombinants. This result eliminates the possibility that the exponential inactivation curve for X-rayed cells is the expression of the interaction between a radiation chromosomal event with a radiation cytoplasmic one. DISCUSSION The experiments reported here have not provided any evidence for the induction of some kind of lethal dominant mutation on the chromo- somal material of the Hfr strain. With selected markers, they have shown that a rescue by the F" cells may be exerted on the characters of the irradiated male. It seems that the X-ray damage Avould be, not on the genetic information itself, but mainly on the transfer mechanism of the selected markers to the recombinants. 32p decay experiments have yielded the same conclusion (Fuerst et al., 1956). Since there exist 180 H. MARCO VICH differences in the efficiency of rescuing the same selected markers of the Hfr by mating with two different F~ strains, the slope of the sur- vival curve is of little help, if any, for giving quantitative information on the size of the inactivated target, as has been done in a recent work (Wilson, 1960). Since no dominant lethals have been found by means of recombination methods two alternative hypotheses may be considered to explain the exponential dose lethal effect relationship in E. coLi. 1. The lethal effect is not linked to a nuclear damage. Consequently, the inactivated structure should be unique or, if there ^\'ere many, the alteration of one of them would lead to a dominant lethal sequence of events for which w^e have no model to jjropose. 2. The lethal effect is linked to some action in the nuclear material, but is not transmissible to the zygote. Such effects are known. For instance, induction of phage production by lysogenic bacteria is a dominant lethal effect. The cells are killed in relation to phage synthesis and according to a one-hit dose-effect relationship (Marcovich, 1956). On the other hand, induced phage is not transmissible to the recombin- ants. But this model cannot apply without modification to non-lysogenic or non-inducible lysogenic bacteria. Yet, a starved lysogenic strain loses its aptitude for induction either by u.v. light (Jacob, 1952) or by X-rays (Marcovich, 1957), and still the survival curves are ex])o- nential. On the other hand, starvation does not modify the shape of non-lysogenic or non-inducible lysogenic strains and does not affect their intrinsic radiosensitivity. A working hypothesis to explain these results is that the lethal event in E. coli and the very early stej) in lysogenic induction, are processes of the same nature, if not identical. Experiments are now being done to test the implications of this assump- tion. REFERENCES FuERST, C. R., Jacob, F. and Wollman, E. (1956). C.B. Acad. Sci.. Paris. 243, 2102. Jacob, F. (1952). Ann. Inst. Pasteur 82, 578. Latarjet, R., and Ephrussi, B. (1949). C.R. Acad. Sci., Paris, 228, 1354. Marcovich, H. (1956). Ann. Inst. Pasteur, 90, 458. Marcovich, H. (1957). These, Faeulte des Sciences, Paris. Mortimer, R. K. (1955). Radn Res. 2, 361. Puck, T. T. and Marcus, P. T. (1!)56)../. exp. Med. 103, 653. Puck, T. T., Morkovix, D., Marcus, P. I., and Cieciura, S. J. (1957). J. exp. Med. 106, 485. Rogers, W., and von Borstel, R. C. (1957). Radn Res. 7, 484. VON BoRSTKL, li. C, and Rogers, R. W. (1958). Rudt} Res. 8, 248. Wilson, D. E. (1960). Radn Res. 12, 230. LETHAL ACTION OF X-RAYS IN E. roli K12 181 DISCUSSION ZHUKOV-VKKEZHNicov: It seems to me that additional proofs nvr ncMMlcd that you worked with multinucleate strains of the Esclnnicha coll culture. MARCOVICH: First, the method of staining according to Feulgen has shown tlie presence of two or three nuclear bodies. Secondly, if you are working with ^^^P- labelled bacteria and allow transmutation to occur, then suixival cuives to radio- active decay will correspond to nuiltiple-type inactivation, which coiTelate with the number of bodies stained positively by Feulgen. Finally, if you make crosses, as I have already said, between "male" and "female" strains, and obtain recombination and then remove the "male", you would see that this cell remains normal, because it transmits to the zygote only a part of its genetic material, whereas an identical part of this material remained in it. ZHUKOV-VEREZHNicov: What is the largest number of nuclei, which you could distinguish morphologically in one cell in the Escherichia coli sti'ain? MARCOVICH : iTp to four in one cell which is preparing to divide. Speaking gener- ally three nuclei may also be encountered, but the number most often met with is two. Experiments aimed at determining the exact shape of the survival curve are such that two nuclei are found with absolute certainty. alichanian: I listened to your very interesting and original report with great pleasure. I am interested in a question bearing on the technique of your work. When you were preparing suspensions for inoculation and obtaining recombinants did you take into account the relative proportions of the components, i.e. the parent "male" and "female" cells? MARCOVICH: Yes, we did. The best method is to introduce one "male" per ten "females", in order to be quite sure that conjugation w^ould occur for all the "males". alichanian: Did you use in your work F± and F= or Hfr = forms? MARKOViCH : It may be carried out with Hfr forms. alichanian: I want once again to emphasize the fact that your work makes it- possible to evaluate similar curves on an entirely new basis. This work allows uS to decipher dominant and lethal mutations and to determine whether there is death of whole nuclei or some cell damage in general. gray: When crossing occurs there is probably some locus in tlie chromosome which is first transferred from male to female, and if this is so, then some of the mutations arising during irradiation, would escape analysis. MARCOVICH: Yes, this is so. If you calculate the target area for this locus, it would be very small. About 1 :200 of all the combinations escape, and this is not much. GRAY : It seems to me that it is important to know what becomes of these chromo- somes, because damage may be distributed unequally throughout the chromo- some's length. MARCOVICH : X-rays did not inactivate in these experiments any chromosome locus as such. It was possible only to study the probability of the manifestation of a given locus in the recombinant. Loci for threonine and leucine are found very 182 H. MARCO VICH close to each other and during the chromosome's injection introchice them- selves together. You irradiate and pick out those recombinants which contain both these loci, and obtain that curve which I have drawn. HERCIK: If you try to take only one of the loci, you would obtain approximately the same curve. It does not mean that the locus itself is damaged but that the probability of its reapj^earance in the recombinant is decreasing. Does it mean that there is a break in tlie chromosome? MARCOViCH : It is possible. ZHUKOV-VEREZHNicov : What is the minimum radiation dose to which your sys- tem can react with sufficient statistical reliability? MARCOVICH: The lowest dose accepted in these exi:)eriments was 5,000 rad. This material is not suitable for studying smaller doses. ASTAUROV: Dominant lethals at least in the highest organisms, constitute a com- plex group of genetic changes. The greater part of them consist of chromosome lesions belonging to the type of deficiencies or breaks. Could it not be that chromosomes with such great lesions would be unable to penetrate at all, and that such disturbances should be left out in this process? MARCOVICH : It is possible that such breaks may indeed partially explain the death of recombinants. But it seems to me that they could not explain the survival curve. Most likely in bacteria the loss of chromosome material does not lead to the cell's death. In "male" bacteria the loss of chromosome material does not harm them since if crossing occurs the "males" lose part of their nucleus, but do not die. ARDASHNicov: Plcasc explain the correction factor. MARCOVICH: a is the sensitivity of the whole cell. If N is the chromosome num- ber, and by accident only one chromosome would be isolated, then the expression of this damage would be cc/N. ARDASHNicov: Why do you connect the exponential curve with the presence of dominant lethals? Other factors may account for it, for example, damage to any other unique structure, indispensable for the life of the given organism. MARCOVICH: I cjuite agree with you. The only thing I have said is that there are two alternatives, one of them testing the chromosome dainage. But it seems to me that my results sliow that it is not the cause and the damage should be sought elsewhere. DAMAGE TO THE REPRODUCTIVE CAPACITY OF HUMAN CELLS IN TISSUE CULTURE BY IONIZING RADIATIONS OF DIFFERENT LINEAR ENERGY TRANSFER G. W. BARENDSEN RadiobioJogiad Institute oj the Organization for Health Research TNO, Rijsu'ijk {Z.H.), The Netherlands SUMMARY Kidney cells of human origin, cultured by the technique developed by Puck et at., (1956) were irradiated with a, j8, 200 kV X- and 20 kV X-radiation. The cells were cultured in special dishes with a Melinex bottom G/j. thick, which per- mitted irradiation from outside with alj^ha particles from ^lopo and beta particles from SOY. The number of cells, which after irradiation had retained the capacity for clone formation, was counted. The survival curve was found to be expo- nential in the case of a-irradiation, whereas with other radiations a more compli- cated curve was obtained, which cannot be interpreted by a two hit mechanism. The RBE of a-radiation was found to range from 2-5 at high doses to 6-0 at low doses. From the experiments with a-radiation the sensitive area of these cells for the inhibition of clone formation was calculated to be 40fi^, which was found to be approximately eciual to the cross -sectional area of the nuclei. It may be inferred that clone formation is inhibited if one a-particle passes somewhere through the nucleus. Experiments with fractionated doses showed that partial repair takes p\ace after X- and /3-irradiation, but no recovery could be detected after a-irradiation. Furthermore cells were irradiated with various doses of a-radiation followed by X-irradiation and conversely. In these experiments no de^Darture from addi- tivity was observed i.e. X-radiation acts on cells surviving after a-irradiation as if they were not irradiated at all. This contrasts with the effect of a second dose of X-radiation, which depends on the amount of X-radiation the cells have re- ceived before i.e. in this case there is some cumulative effect. Finally experiments are discussed on the sensitivity of these cells to a- and X-radiation when in eciuilibrium with different mixtvires of nitrogen and oxygen. The absence of oxygen protects the cells in X-irradiation experiments but a very small decrease in sensitivity is found with a-radiation. INTRODUCTION The development by Puck et al. (1956) of a plating technique for single mammalian cells, whereby each cell grows into a separate clone of macroscopic size has stimulated a number of investigations on pro- cesses which inhibit this unlimited proliferation. The first experiments 183 184 G. W. BARENDSEN concerning the effects of ionizing radiations on this system were re- ported by Puck (1959). Using 150 kV X-rays he showed the capacity for clone formation of HeLa cells to be very sensitive to this type of ionizing radiation, with an LD37 of the order of 100 roentgens. Hood et al. (1959) obtained a similar survival curve with 25 kV X- radiation. Elkind and Sutton (1959) studied the survival of cells derived from ovarian tissue and lung tissue of a Chinese hamster, after fractionated doses of 55 kV X-radiation. They found a definite repair of accumulated damage by surviving cells before their first post-irradiation division. In the experiments to be described in this paper the effect of ionizing radiations of different linear energy transfer (LET) was studied on the capacity for clone formation by kidney cells of human origin. The survival ciu've was found to be exponential in the case of a-radiation whereas for radiations of low LET a more complicated curve was obtained. Experiments with fractionated doses showed that partial repair takes place after X- and ^-ii-radiation but no recovery could be de- tected after a -irradiation. Furthermore the effect was studied of various doses of a-radiation followed by X-irradiation and conversely. Finally it was shown that the effect of oxygen on the radiosensitivity of these cells is much smaller for a-radiation than for X-radiation. MATERIALS AND METHODS In all experiments, kidney cells of human origin (van der Veen et al., 1958) were used, subcultured many times in glass bottles. The culture medium consisted of Hank's solution with 0-5 per cent lactalbumin hydrolysate and 5 jjer cent calf serum to which 100 lU penicillin and 0-1 mg streptomycin per ml were added. The incubator, at 37°C, was continuously flushed with air, saturated with water vapour and con- taining 3 per cent CO2 to maintain the pH of the culture medium at 7-4. The cells used in irradiation exjieriments were obtained from four days old flask cultures in the proliferation ])hase. They were detached from the glass and dispersed by gentle trypsinization (Puck et al., 1956). The cell susj^ension obtained was counted in a haemocytometer. Microscopic inspection showed that not more than a few per cent of the cells were present in groups of two or three cells. The cells were plated on culture dishes, "conditioned" by l-5x 10^ "feeder cells" in 3 ml medium which were made incapal)le of multipli- cation by a dose of 4,000 rad of X-radiation (Puck et al, 1956). After DAMAGE TO REPRODUCTIVE CAPACITY OF HUMAN CELLS 185 about four hours of incubation at 37°C more tliaii li!» per cent of the cells adhered sufficiently to the bottom of the culture dishes for the experiments to be carried out. During the irradiation experiments the cells were uiaintained at room tem])erature, 18 to 22°C. The cultures were then placed in the incubator and the cells allowed to grow and multiply for U days at 37°C. Medium was replaced once every five days. After 14 days the cells were stained in situ and the number of clones of more than fifty cells was counted. The number of clones has been taken to represent the number of surviving cells. In each experiment the mean value was taken of at least three culture dishes each of which had re- ceived the same dose. Plating efficiencies of unirradiated cells ranged from 50 to 100 per cent, with an average of about 80 per cent. The fraction of cells surviving irradiation was calculated as a percentage of the unirradiated controls in the same experiment. Irradiations were carried out with a-particles from 2i0Po (LET v 170 keV///), 20 kV X-radiation, unfiltered except for a layer of culture medium 1mm thick, (HVL 0-05 mm Al, LET ^ 6keV///), 200 kV X-radiation filtered by 1-5 mm Cu (HVL 1-9 mm Cu, LET x ^-5 keV//,0 and /3-radiation from ^oy (LET x 0-3 keV//0. Because of the low penetrating power of a-particles from 'lopo, which have an energy of 5-3 MeV and a range in water of 37^, the cells were cultured in special dishes with a "Melinex"t bottom about 6/t thick. This thin melinex permitted irradiation of the cells from outside the culture dishes a few hours after plating, when the majority of the cells adhered to the bottom. These Melinex dishes were used in all experiments and were found to support reliably the growth of each surviving cell into a macroscopic clone, even after irradiation which has been reported to give rise to toxicity in plastic surfaces (Morkovin and Feldman, 1959). Dose measurements were carried out with various dosimeters. The dose of a-radiation delivered to the cells was determined by counting the number of particles passing per minute through 1 sq. nnn, by measurement of the total current in a large ioinzation chamber and with an extrapolation chamber. The dose of |8-radiation was measured w ith the extrapolation chamber. The dose of 200 kV X-radiation was measured with a Baldwin "Substandard" ionization chamber and with the extrapolation chamber. The dose of 20 kV X-radiation was measured with a Philips ionization chamber type 37483/01, suited for HVL 0-02- 1-5 mm Al. Details of the irradiation techniques and dosimetry are given else- where (Barendsen and Beusker, 1960). t Polyethylene terephthalate. Imperial Chemical Industries T.imited, Herts., England. 186 a. W. BARENDSEN RESULTS Survival curves In Fig. 1 the fraction of cells which after irradiation have retained the capacity for clone formation is plotted as a function of dose. Doses of a-, /3- and X-radiation are given in rads. The main features of the o V en a Fig. 1. — Effects of a-, j8- and X-radiation on the capacity for clone formation. 1, Curve obtained with a-radiation. 1', Curve 1 corrected for cells, not adliering to the bottom of tlie dishes (see text). 2, Curve obtained with ^-radiation. RBE S:: 0-85. 3, Curve obtained with 200 kV X-radiation. 3', Theoretical curve nino = e-""-*^ ( 1 +7>/ 135). (see text). 4, Curve obtained with 20 kV X-radiation. RBE X 1 • 1 ;">. curves are the exponentially decreasing survival for /3-radiation and the less simple shape obtained with X- and /3-radiation. At doses of a-radiation of more than 150 rad the survival curve deviates from the exponential. This may be explained by the assump- tion that a small percentage of the cells, about 0-5 per cent, has not attached projjerly to the Melinex bottom of the cidture dish. As the range of a-particles after penetrating the Melinex bottom is only about 20 fi, cells which are still in suspension at the time of irradiation will not be irradiated at all. When a constant fraction of 0-5 per cent is subtracted from the surviving fraction at all dosages, an exponential DAMAGE TO KEPKODUCTIVE CAPACITY OF HUMAN CELLS 187 survival curve is obtained up to at least 400 rad (Fig. 1, curve 1'). The mean lethal dose (LD37) is found to be 65 rad. The ex])onential survival curve may be represented by = e-.SD n "0 in which uq is the number of cells plated, li is the numlier of cells sur- viving a dose of a-radiation D and *S' is a constant, which may be inter- preted as a "sensitive area" if Z) is expressed in a-particles per unit area passing through the cells. Taking an average LET of 170 keV//x for a-particles of 3-4 MeV, one a-particle passing per square micron is equivalent to an average dose of ;2,720 rad. From the LD37 of 65 rad S can be calculated to be 42/^^, which corresponds to a circular area of 7-4jLt diameter. This very large sensitive area is about equal to the area presented to the a-radiation by the nuclei of the cells, which, by microscopic examination, were found to range between 6 and 10/t in diameter. As it is generally accepted that the nucleus of a cell is much more sensitive to ionizing radiation than the cytoplasm, it seems very unlikely that any structure in the cytoplasm can be correlated with this sensitive area of about 42^2 Thus the cross-section of the nucleus must be identical with the sensitive area calculated from the experiments. This suggests that whenever one a-particle penetrates the nucleus any- where, the cell is sufficiently damaged to become incapable of un- limited proliferation. The survival curves obtained with /8- and X-radiation are not expo- nential but the slope of the tangent increases with increasing dose. For the curve obtained with 200 kV X-radiation this slope corresponds at 100 rad to a mean lethal dose (LD37) of about 400 rad, at 700 rad to an LD37 of about 180 rad and at 1,300 rad to an LD37 of about 100 rad. It may be noted that as a result of this difference in shaj^e between the survival curves the relative biological efficiency (RBE) of a-radiation, defined as the ratio of doses of 200 kV X-radiation and a-radiation which cause the same effect, ranges from 2-5 at 0-017 per cent survival to at least 6 at 80 per cent survival. The survival curve obtained with /8-radiation has, up to doses of 900 rad, a shape which is not significantly different from the curve obtained with 200 kV X-radiation (Fig. 1, curve 2). From the results the RBE is calculated to be 0-85 ± 0-10. The same con- clusion may be drawn from curve 4 obtained with 20 kV X-radiation, for which the RBE is calculated to be 1 -15 ± 0-10. The shape of the 200 kV X-ray survival curve suggests that some sort of accumulation of damage takes place. Puck (1959) assumes for the explanation of a similar curve obtained with 150 kV X-radiation a 188 G. W. BARENDSEN two-hit mode of action on the chromosomal material. The theoretical curve exj)ected from this assumption has the form — = e-BiA (1+D/.4) where n\n^ is the relative survival after a dose D and ^4 is a constant. In Fig. 1. curve 3' is an example of this tyjje of curve in which ^4 is chosen as 135 rad in order to obtain the best fit with the experimental points. The accuracy of our experiments is sufficient to conclude that the assumption of a two-hit mode of action as proposed by Puck is too simple. It may be noted in passing that from the assumption of a two- hit mode of action it may be inferred that the slope of the survival curve at low doses a]Dproaches zero. This would imply that for this system the RBE of a-radiation at low doses would approach infinity. However, our results at low doses show an initial slope corresponding to an LD37 of about 450 rad. An hypothesis by which our survival curves may be explained starts from the consideration that the single-hit survival curve obtained with a-radiation suggests that deposition of a sufficiently large amount of energy in a small volume anywhere in a relatively large part of the nucleus inhibits clone formation. Consideration of the spatial distribution of the energy deposition by X-radiation makes plausible that at least a small part of the damage caused by ionizing radiations of low average LET will be due to the same type of locally concentrated energy deposition i.e. part of the X-ray damage may be considered to be caused by a single-event type of action. The greater part of the energy, which is deposited in less con- centrated form, may be assumed not to result in inhibition of clone formation. The damage caused by this part of the energy deposited might be reparable (see next section). At higher doses of X-radiation two or more amounts of energy deposited in small volumes, each by itself too small to inhibit clone formation, may be found sufficiently close together to cause this effect. Thus with increasing dose a greater part of the energy deposited is effective in causing the damage measured. In this way the shape of the X-ray survival curve with increasing slope at higher doses may be explained as a combined result of one-, two- and multi-event types of action. Ejfecfs of fractionated doses A immber of experiments was carried out in which doses of X-radi- ation up to 900 rad and doses of a-radiation up to 200 rad were frac- tionated in such a way that the second half of the total dose was DAMAGE TO REPRODUCTIVE CAPACITY OF HUMAN CELLS 189 administered after intervals of :2, 4 and 12 lir. Fii the interval the cells were maintained at 37 ^C-. With a-radiation no statistically significant differences were observed between the effects of single doses of respectively 50, 100 and 200 rad and of two doses of respectively 25, 50 and 100 rad administered at intervals of 2, 4 and 12 hr. At doses up to 500 rad of 200 kV X-radiation and intervals of 2 and 4 hr the effect of fractionation was very small. At higher doses and with longer intervals the effect is significant however. For example the surviving fraction after a dose of 900 rad was found to be 1-3 ± 0-2 per cent, whereas after two times 450 rad, 12 hr apart, 4-0 + 0-05 per cent of the cells had retained the capacity for clone formation (see Fig. 2). Thus more cells survive after fractionated 5 6 7 8 9 10 Dose (rod x 100) ^ Fig. 2. Effect of fractionation of doses of 200 kV X-radiation on the capacity for clone formation. Curve 1, Surviving fraction after irradiation 4 hr after plating. Curve 2. Surviving fraction after 450 rad at 4 hr after plating +250 rad and 4;>0 rad respectively, administered 12 hr after the first dose. doses i.e. part of the damage is repaired in the interval. The higher survival was not due to cell multiplication in the interval, as cells irradiated with 900 rad of 200 kV X-radiation 16 hr after plating showed a surviving fraction of 1-5 ± 0-1 per cent. These results are in agreement with the work of Elkind and Sutton (1959) except that in our system repair appears to start somewhat later. Effects of combination of doses of a- cmd 200 kV X-radiation In Fig. 3 survival curves are given from experiments in which the same cells were irradiated with X- and a-radiation. No statistically 190 G. W. BARENDSEN significant differences in effect were observed if the order in which a- and X-irradiations were given was reversed. It will be clear from curves 1 and 2 that if a certain dose of a-radiation is given first, the curve 1003 I 23456789 10 Dose (rad x 100) *- Fis 3 —Effects of combined a- and 200 kV X-radiation on the capacity for clone for- ^' ' niation. fl ^nd b Effects of OL- and 200 kV X-radiation respectively. CiSve 1, Effects of 50 rad a-radiation + 0; 100; 150; 200; 300 and 500 rad of 200 kV X-radiation. Curve 2. Effects of 100 rad a-radiation + 0 : 100; 150; 200: 300 and 500 rad of 200 kV X-radiation. Curve 3 Effects of 300 rad 200 kV X-radiation + (); 50 and 100 lad a-radiation. Curve 4'. Effects of 500 rad 200 kV X-radiation + 0 ; 50 and 100 rad a-radiation. obtained from X-irradiation of the surviving cells has the same shape as if no preceding a-irradiation had occurred, i.e. X-radiation acts on cells surviving a-irradiation as if they were not irradiated at all. This contrasts with the effect of two doses of X-radiation where the damage, at least in part, is cumulative. The dotted lines 3 and 4 parallel to the survival curve a obtained with a-radiation indicate that, within limits of error cells surviving after X-irradiation have not become more sensitive to a-radiation i.e. in this respect there is no cumulative effect. This supports the conclusions given in the first section. Effects of different oxygen concentrations on radiosensitivity A reduction in the sensitivity to ionizing radiation resulting from anoxia has been reported for many systems (Bora, 1958; Gray et al, 1958- Neary et al, 1959). In order to measure the effect of different oxygen concentrations on the capacity for clone formation, the medium was removed from the culture dishes after the cells had attached to the DAMAGE TO REPRODUCTIVE CAPACITY OF HUMAN CELLS 191 Melinex bottom. Gas mixtures containing different amounts of O2 and N2 saturated with water vapour were then i)assed over the cells for various lengths of time. No differences in reduction of the sensitivity to X-radiation were observed when the gas mixtures were passed over the cells for 5, 10, 15 and 25 min resi)ectively. It was concluded that a time of 5 min was sufficient for the cells to reach equilibrium with the gas phase. In the experiments summarized in Table I gas was passed Table 1. — Relative numbers of cells as percentage of unirradiated con- trols, ivhich have retained the capacity for clone formatio7i after irradiation in gas mixtures of various proportions of oxygen and nitrogen. X-radiation 600 rad 1,000 rad medium Gas mixture removed (10 min) + / no gas \ \passed over/ 8-0 + 0-5 9-3 + 1-5 0-6 + 0-1 0-7 + 0-4 +- air 13-3 + 1-7 1-4 + 0-6 + 100 %02 + 0 %N2 12-4 + 1-7 0-2 + 0-2 + 50 %02 + .50 %N2 12-8 + 1-8 0-7 + 0-4 + 20 %02+- 80 %N2 13-5 + 1-8 1-4 + 0-6 + 10 %02 + 00 %N2 18-8 + 2-6 0-9 + 0-5 +- 5 7o02 + 95 %N2 20-4 + 2-2 2-8 + 0-8 + 2-8% O2 + 97-2% N2 22-6 + 2-3 4-0 + 1-0 + 1-5% O2 + 98-5 %N2 24-4 + 2-4 8-4 + 1-4 + 0-9% O2 + 99-1 %N2 32-8 + 2-8 11-8 + 1-7 + 0-3% O2 + 99-7 %N2 43-9 + 31 20-8 + 2-4 + 0 %02 + 100 %N2 65-8 ± 5-6 27-6 ± 3-5 Alpha radiat] ion 100 rad 150 rad 200 rad medium Gas mixture removed (10 min) __ / no gas \ 22-0 + 1-5 10-6 + 0-8 5-0 + 0-5 + \passed over/ 24-7 + 2-3 12-4 + 1-7 3-5 + 0-9 + air 28-7 + 2-5 13-9 + 1-7 7-3 + 1-2 + <• 99-7% N2\ 331 + 2-7 17-9 + 1-9 8-6 + 1-5 + 0-3% O2/ over the cells for 10 min before the irradiation was started. In this table surviving fractions are given for oxygen concentrations in the mixture of 100, 50, 20, 10, 5, 2-8, 1-5, 0-9, 0-3 and 0 per cent. The nitrogen from the cylinder used was found to contain 0-3 per cent O2. This oxygen w^as removed with a "BTS-katalysator"t. It may be concluded that with oxygen concentrations between 10 and 100 per cent no statistically significant differences in radiosensitivity are observed. With gas mix- tures which contain less than 5 per cent O2 the radiosensitivity of the t Badische Anilin und Soda Fabrik. 192 G. W. BARENDSEN capacity for clone formation decreases ^^■itll decreasing oxygen concen- tration. It may be noted that there is a shght difference in radio- sensitivity between cells over which no gas mixture is passed at all and cells which are in equilibrium with air. An explanation of this effect cannot as yet be given (see Fig. 4). Comparison of the curves given in Fig. 4 shows that with an oxygen 6 8 10 12 ' 14 16 18 Dose (radXiOO) »- Fig. 4. — Effects of a- and 200 kV X-radiation on cells in equilibiiuui with air and nitro- gen respectively. 1 , Curve obtained for a-radiation under normal conditions with medium not re- moved (see Fig. 1). 2 , Curve obtained for a-radiation witli cells in equilibrium with air (medium re- moved). 3 . Ciu-ve obtained for a-radiation with cells in equilibrium with i)!)-7% N2 + 0-3% O2 (medimn removed). 4 , Curve obtained for 200 kV X-radiation under normal conditions with medium not removed (see Fig. 1). 5 , Curve obtained for 2(10 kV X-radiation with cells in eciuilibrium with air (medium removed). 6 , Curve obtained for 200 kV X-radiation with cells in equilil)iium with 99-7 "0X2 + 0-3% O2 (medium removed). concentration of 0-3 per cent as used in most of the experiments, a re- duction of the sensitivity was obtained by a factor of aVjout 2 as com- pared with the sensitivity observed with oxygen concentrations be- tween 10 and 100 per cent i.e. twice as high a dose is needed to produce the same effect. With purified nitrogen a reduction of the sensitivity by a factor of 2-6 was found. Expei'iments carried out with a-irradiation of cells in equilibrium with 99-7 per cent N2 + 0'3 per cent O2 showed that with this radiation of high LET only a very slight decrease in radiosensitivity by a factor of about 1-10 ± 0-05 can be achieved as compared with aerated cells (see Table I and Fig. 4). This result is in agreement with that found DAMAGE TO REPRODUCTIVE CAPACITY OF HUMAN CELLS 193 with other systems namely that the oxygen effect is smaller for radi- ations of high LET than for radiations of low LET Bora, 1958; Gray et al, 1958; Neary et al, 1959). In Fig. 5 the sensitivity of these cells to :20() kV X-radiatioii is given as a function of the oxygen concentration, whereby the sensi- tivity of cells in equilibrium with pure nitrogen is taken as unity. The 3 - 2 4-1 '■ o 10 30 50 70 90 120 160 200 382 760 mm Hg Oxygen pressure in gas mixture of oxygen + nitrogen Y\G. 5. — Sensitivity to 200 kV X-radiation of human cells in equilibrium with gas mix- tures which contain different percentages of O2 and N2. Sensitivity of cells in ec^uili- brium with pure nitrogen is taken as unity. shape of this curve is very similar to curves obtained with other systems (Gray et al., 1958; Howard-Flanders, 1958). It can be repre- sented api)roximately by 8 7v+m[02] .S'„ (A'+[02]) where S is the radiosensitivity (l/i^s?) of cells in equilibrium with oxygen concentration [O2], ^S'^ is the radiosensitivity in nitrogen and m and K are constants. The value found from our curve for K is about 7 /xmoles/1. REFERENCES Barendsen, G. W., and Beusker, T. L. J., (1960). Radn Res. 13, 841. Bora. K. C. (1958). Proc. 2nd. Inf. Conf. Peaceful Uses Atomic Energy, Geneva, 195S. 22, 88. Elkind, M. M. and Sutton. H. (1959). Nature, Lond. 184, 1293. Gray, L. H., Chase, H. B., Dkschner, E. E., Hunt, J. W., and Scott, (). C. A. (1958). Proc. '2nd. Int. Co7if. Peaceful Uses Atomic Energy, Geneva, 1958, 22, 413. Hood. S. L. and Norris, G. (1959). Biochini. hiophys. Acta.. 36, 275. Howard-Flanders, P. (1958). Advanc. biol. med. P/iys. 6, 553. MoRKOviN, D., and Feldman, A. (1959). Brit. J. Radiol. 32, 282. Neary, G. J., Tonkinson, S. M., and Williamson, F. S. (1959). Int. J. radn Biol. 1, 201, 194 G. W. BARENDSEN Puck, T. T. (1959). Rev. mod. Phijs. 31, 433. Puck, T. T., Marcus, P. I., and Cieciura, S. J. (19.56). .7. e.vp. Med. 103, 273. VAN DER Veen, J., Bots, L., and Mes, A. (1958). Arch. ges. Virusforsch. 8, 230. DISCUSSION HERCiK : What is the thickness of the cells, I mean cells lying on the dish bottom in experiments with a-particles? BARENDSEN: The residual range of the a-particles is about 25ju,. The thickness in vertical direction of the cells attached to the bottom because of flattening, ranges from 5 to lHn so that each part of the cells is irradiated. POWERS : How do you explain differences between the survival curves for X-rays and a-irradiation? BARENDSEN : As I see it the nucleus is the most sensitive site of the cells and in order to answer your cjuestion we have to consider by what mechanism the damage is produced. Alpha particles passing through the nucleus will inevitably damage one or more chromosomes, the integrity of which is indispensable for the reproduction of the cells. X-rays will produce the same damage due to local deposition of sufficient energy) less efficiently because of the lower LET and other damage (due to less than the required number of ionizations in a small volume) may be partially restored, possibly by the metabolic activity of the cells. A possible mechanism might be that a large amount of energy deposited leads to changes in DNA such that the separation of the two sti'ands of the helix is made impossible. If the amount of energy is not large enough the DNA can still separate into two strands, and by this action the lesion is repaired. At higher doses the probability that two or more electrons pass through the same DNA molecule and together produce sufficient damage to prevent strand separation increases. This produces an increasing efficiency of sparsely ionizing radiation at higher doses and leads to a survival curve of the type oljserved with X-i-adiation. Thus the primary lesions are produced at the time of irradiation, but the final damage may be influenced by the metabolic activity of the cell. PASSYNSKY: Did the result obtained for a-irradiation depend on the mitotic phase? Have any differences been found between j^rophase and metaphase? BARENDSEN: This could not be determined since the cell divisions were not synchronous. TOBIAS : Why was the presence of 50 cells in the clone chosen as a survival criterion? BARENDSEN: We tried to take 20 cells, 100 and 200 cells, and found no difference. Every surviving cell will multiply, and from it several thousand cells will arise. TOBIAS : I am very hapjoy to hear that the number of cells is of no importance. But. in the case of yeast, colonies sometimes die out, even if they contain 50 cells. BARENDSEN : We did not observe this. SOSKA: What was the composition of the medium used for the kidney cells? BARENDSEN: The medium consists of Hank's solution with 0-5 per cent lactalliumin hydrolysate and 5 per cent calf serum to which 100 lU of penicillin and 0-10 mg of streptomycin per ml were added. ARDASHNicov: How did you calculate the dose for a-irradiation? If ynu get a one-hit curve why, in this case, are doses for a-irradiation so much smaller than for X-rays? Maybe in this case you take for a hit an a-particle, and not the ionization it produces? BARENDSEN: I usccl two methods to determine the dose. First of all I counted the number of particles per sc^uare mm. Secondly, I also used an ionization chamber placed where the layer of water is situated during the irradiation. PHOSPHATE IVIETABOLISM IN THE NUCLEUS L. A. 8T0CKEN Department of Biochemistry, University of Oxford^ England SUMMARY A brief at'c-uuiit is given of the effects of X-radiation on the biochemical events in the mitotic cycle leading to the formation of nucleic acids. It is suggested that low doses j^rodnce a disorganization of binding sites in the nucleus. Preliminary data on the capacity of the nucleus to bind inorganic and organo -phosphates are provided. This paper is a brief survey of what may be the salient biochemical features associated with the synthesis of DNA in animal cells and an attempt to see how far the derangement of DNA synthesis by X-radia- tion can be explained in the light of our present knowledge. We our- selves are convinced of the lack of basic information about the nucleus and for this reason some of our recent findings which we have not yet applied in the radiation field will be reported. The early work on the inhibition of precursor uptake into DNA is due to Hevesy (1948). Most of these data were obtained at 2 hr after irradiation and have been subjected to the criticism that by this time changes in cell population or cell death have complicated the picture. From our experiments (Ord and Stocken, 1957) with thymus gland, however, it does seem that radiation produces its effect at once and there is no further change until some considerable time afterwards. A second interesting observation due to Hevesy is the fact that once the extent of inhibition has reached about 50 per cent a considerable increase in the dose is required to produce much further change. We followed up this point and correlated the dose with the inhibition of 32p uptake into thymus DNA (Ord and Stocken, 1958). It seems clear from these experiments that radiation has at least two separate functions. A similar biphasic response has been found by Lajtha et al. (1958) when bone-marrow cultures are irradiated. In order to explain the results it is necessary to seek a sensitive as well as a rather resistant locus. The first clue to differential sensitivity of cells stemmed from the work of Howard and Pelc (1953) on the radiation sensitivity of the mitotic cycle in bean root tips. These authors showed that the cycle 195 196 L. A. STOCKEN could be divided into discrete periods which they called Gi. S, G2 and D. To jiroduce the same inhibition of uptake of DNA precursors several times the dose is required if it is given in S than if the same dose is given in Gi. Similar results have been obtained in regenerating liver l^y Kelly (1957) who used carbon tetrachloride to destroy liver cells and by Holmes and Mee (1956) who surgically removed two-thirds of the liver. Barbara Holmes also showed that if 450 r was given just before partial hepatectomy there was the same inhibition and mitotic delay as when the dose was delivered at 1 2 hr after hepatectomy. So far as mitotic delay is concerned one should perhaps remember that Carlson (1948) has shown that an arrest can be caused by as little as 4r and Forssberg and Novak j(1960) that doses of 0-1 r and less affected the growth of Phycomyces BJakeshnnus. We have very little infoi-mation about the biochemical events taking place during the j^rocess of division but we now know quite a lot about the biochemistry of interjihase in mammalian cells. During the first part of the cycle there is an early stimulation of RNA and protein syn- thesis. This has been shown both in vivo and in vitro at 6 hr after partial hepatectomy. This is followed by the appearance of thy midy late kinase and DNA polymerase (Bollum et a1., 1960) and the synthesis of DNA does not start until about 15 to 18 hours post hepatectomy. It is also to be noted that at the same time as the synthesizing enzymes appear there is a reduction in the pyrimidine catabolizing enzymes. The effect of radiation on these various steps is of some interest. If the irradiation is given before the time at which the enzyme can be detected then its appearance is delayed, but when the enzyme is present the same radiation dose has veiy little effect. In view of the relationship of RNA with protein and enzyme synthesis it is of interest that Welling and Cohen (1960) have observed a decreased incorporation of 32p into nuclear RNA of regenerating rat liver if the animal is irradiated within 6 hr of partial hejmtectomy. There is also a delay in the disap]:)earance of the catabolizing enzymes. Okada and Hempel- mann (1959) have data at 12 hr post-hepatectomy and a more complete time course has been obtained by Stevens in our laboratory (Table I). This delay, of course, only applies in comparatively low doses and when lethal doses are given it is likely that these changes are made irre- versible by more fundamental damage, such as an alteration in the template. Cole and Ellis (1954) have observed changes in spleen nucleoprotein after irradiation in vivo and by means of column chromatography we have found changes in thymus DNA (Old and Stocken, 1960). Recently Mrs. Hudnik-Plevnik in our laboratory has also obtained preliminary PHOSPHATE METAHOLISM IN THE NUCLEUS 197 results with bacterial DNA after exposure to ultraviolet irradiation and shown that the newly synthesized DNA is not the same as that in the conti'ols. The consequences of severe doses of radiation are of biochemical interest but it seems unlikely that we shall in the near future be able Table I. The effect of 400 r X-rays total body irradiation on the thymine catabolizing enzymes in regenerating rat liver Hours post-partial Control Irradiated hepatectomy Non operated 2-5 (4) — 18 2-4 (4) — 24 0-8 (4) 2-2 (8) 36 0-6 (2) — 48 O-^a (2) 1-65 (8) /[iinols catabolized/mg DNAP/30 luin. to reverse the effects of supra-lethal doses except by modifications of replacement therapy. On the other hand if we knew exactly what was the biochemical lesion caused by the near-lethal doses we might be able to promote recovery without the implantation of extraneous cells. Our present opinion is that low doses produce a local disorganization of the nucleus which alters the binding sites. Some sup]iort for this idea is given by Hagen's (1960) recent work on the extractability of DNA from thymus gland homogenate post radiation, and by Creasey (1960) who found a leakage of Na+ and K+ from nuclei of rat thymus and spleen at one hour after 1,000 r given i7i vivo. Smaller doses were effective when the nuclei were irradiated in vitro. It is this question of what maintains the nucleus in an organized state which has caused us to concentrate on some of the basic properties of the nucleus. Allfrey et al. (1957a) have extensively studied protein synthesis in isolated nuclei and shown that Na+ is an essential cation. They have also discovered nuclear phosphorylation (1957b) and it may be con- jectured that this might be the energy source for intra-nuclear synthesis. We have been concerned with another aspect of phosphorus meta- bolism in thymus nuclei. When nuclei are prepared in either ionic or sucrose medium both inorganic and organo-phosphate is bound to the nucleus. These phosphates are only released by acid conditions or by such severe mechanical damage that the nuclear structure is no longer maintained. If a rat is killed and the thymus removed within a few seconds the isolated nuclei contain phosphate predominantly as ATP. If the thymus 198 L. A. STOCKEN is left in the dead rat for a short time the ATP content decUnes. This of course is not unexpected but it is of interest that the bound inorganic phosphate increases roughly in an inverse ratio. Allfrey et al. (lOSTb) have pointed out that the phosphorylation of mononucleotide is not effected by extranuclear inorganic phosphate and with this we are in agreement. We have carried out some experiments with thymus nuclei from rats killed at various times post injection of 32p. The rather surprising finding is that the bound inorganic phosphate of the nuclei has a markedly higher specific activity than any other cell fraction. This Table II. Specific activities of acid-soluble 'phosphates in red thymus after injecting 50 /