i^/\l||)||iLQ(]Y SYMPOSIUM 1954 YMBaSJUM !)E RADIOBIOLOGIE iJACQ & AUiXANDEK aa^^B^^^^^^ss^SESE D Marine Biological Laboratory Library Woods Hole, Mass. I I I I I I I I I I D */^^«''=S. Presented t»y Acadonic Press, Inc. New lork City SQ^SBSB^^^^^S^^^ /' RADIOBIOLOGY SYMPOSIUM 1954 SYMPOSIUM DE RADIOBIOLOGIE RADIOBIOLOGY SYMPOSIUM 1954 SYMPOSIUM DE RADIOBIOLOGIE Proceedings of the Comptes-Rendus Symposium held at dii Liege Symposium de Liege August-September, Aout-Septembre, 1954 1954 Z. M. BACQ^ Professeiir a V Uiiiversite de Liege Alembre Correspondant de VAcademie Royale de Medecine de Belgique and PETER ALEXANDER Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital London NEW YORK ACADEMIC PRESS INC., PUBLISHERS LONDON BUTTERWORTHS SCIENTIFIC PUBLICATIONS 1955 BUTTERWORTHS PUBLICATIONS LTD. 88 KINGSWAY, LONDON, w.c.2 U.S.A. Edition published by ACADEMIC PRESS INC., PUBLISHERS 125 EAST 23rd STREET NEW YORK 10 NEW YORK Set in Monotype Baskerville type Printed in Great Britain by Spottiswoode, Ballantyne & Co. Ltd. London and Colchester ,(y\OAt FOREWORD In December 1952 an informal discussion was held under the auspices of the Faraday Society in Cambridge on the chemistry of biological after-effects of ultraviolet and ionizing radiations. At this well-attended meeting it became apparent that the study of radiobiology, in which many scientists were now engaged, would be advanced by having informal and friendly symposia so that the workers from different fields and disciplines engaged in studying the efTects of ionizing radiation could meet and discuss. For this purpose a small committee composed of Bacq, Bonet-Maury, Hevesy, Hollaender and Mitchell was set up and the first such meeting was organized in Aarhus (Denmark) in July 1953. The great success of this meeting showed that the need existed and that the general scheme of informality limited numbers, and emphasis on discussion was sound. The second symposium on Radiobiology was held in Liege and these meetings are now planned to take place annually. The great International Congress cannot cater satisfactorily for radiobiology since this subject cuts right across the boundaries of the different sciences and it is necessary that physicists, chemists, biochemists, physiologists, cytologists, geneticists and radio- therapists should look together at the problem involved. To ensure that the different members of the symposia can make their full contribution we felt that it would be very useful to circulate preprints of the paper or at least to provide detailed abstracts. This is particularly necessary when scientists meet who research in different subjects, since the specialist reading a paper can often not be followed easily by those not working in the same field. Yet, it is the fusion of the different disciplines which these symposia are intended to promote. At the Liege meeting papers or abstracts were pre-circulated; the high level of the discussion and the large programme covered show the value of the measure. In this volume we have collected the papers which were presented and the discussion except in a few cases where authors had made arrangements for publication elsewhere. A few participants were invited to contribute reviews but the majority of the papers consist of new experimental work. We felt that it was of the utmost importance that publication should be quick in this rapidly growing field. The appearance of this volume four months after the deadline for contributions was made possible only by the authors who adhered strictly to the dates given them and above all by Messrs. Butterworth who gave us every assistance and who dealt so expeditiously with the production. Z. M. Bac^ P. Alexander Liege and London December 1954 CONTENTS PAGE FOREWORD . V Z. M. BAcq and P. Alexander THE LI£GE meeting Z. M. BAcq XIU PARTICIPANTS xiv ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES 1 Andre Chevallier et Constant Burg Discussion .......... 25 THE NATURE OF THE PEROXIDE-LIKE SUBSTANCES FORMED IN MICE BY X-RAYS 26 V. J. Horgan and J. St. L. Philpot HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION 30 M. Ebert Discussion .......... 38 BACTERIOPHAGE INACTIVATION UNDER VARYING CON- DITIONS OF IRRADIATION 39 TiKVAH AlPER AFTER-EFFECTS OF IRRADIATION OF DNA ... 46 J. A. V. Butler and (Miss) P. Simson PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS 49 Peter Alexander and Arthur Ch.\rlesby Discussion .......... 59 TWO CLASSES OF PROTECTIVE AGENTS IN THE OXIDA- DATIVE DEGRADATION BY GAMMA RAYS OF POLY- STYRENE IN CHLOROFORM 61 M. Fox ACTION PRESERVATRICE DE DERI\'ES PHENOLIQUES VIS A VIS DE LTRRADIATION LETALE DE LA SOURIS 64 A. Lacassagne, J.-F. Duplan et N. P. Buu-Hof Discussion .......... 67 INFLUENCE OF OXYGEN ON DAMAGE TO MICRO-ORGAN- ISMS BY IONIZING IRRADIATION 68 H. Laser Discussion . . . . . . . . . .69 vii CONTENTS PAGE THE ACTION OF IONIZING RADIATION ON ENZYMES AND VIRUSES 70 Ernest Pollard Discussion .......... 74 LTRRADIATION DES VIRUS 75 P. Bonet-Maury INACTIVATION, PAR LES RAYONS X, D'UN AGENT TRANS- FORMANT DU PNEUMOCOQ.UE 79 H. Ephrussi-Taylor et R. Latarjet RADIATION DEATH IN MAMMALS 81 B. Rajewsky Discussion .......... 92 ACTION OF IONIZING RADIATIONS ON CELL CON- STITUENTS 93 Maurice Errera RELATIVE BIOLOGICAL EFFICIENCY OF FAST NEUTRONS AND GAMMA RADIATION FOR CHRONIC IRRADIATION OF MICE 104 R. H. Mole, R. J. Munson and G. J. Neary Discussion . . . . . . . • • .104 REMARKS CONCERNING SULPHYDRYL PROTECTION AGAINST MAMMALIAN RADIATION INJURY . . .105 Harvey M. Patt LE METABOLISME DE LA CYSTEAMINE . . . .110 W. G. Verly, G. Koch et S. Gregoire STUDIES ON THE MECHANISM OF RADIATION PRO- TECTION AND RECOVERY WITH CYSTEAMINE AND p-MERCAPTOETHANOL 112 Alexander Hollaender and C. O. Doudney Discussion on Papers by Patt, Verly and Hollaender . .116 OBSERVATIONS ON THE EFFECT OF SPLEEN-SHIELDING AND THE INJECTION OF CELL SUSPENSIONS ON SUR- VIVAL FOLLOWING IRRADIATION . . . .122 Leon O. Jacobson, Edna K. Marks and Evelyn O. Gaston Discussion . . ... . . . . • .133 SPLEEN PROTECTION: THE CELLULAR HYPOTHESIS . 134 D. W. H. Barnes and J. F. Loutit THE MODIFICATION OF THE RADIATION RESPONSE BY SHIELDING PROCEDURES 136 L. F. Lamerton and E. H. Belcher Discussion . . . . . . . • • .140 viii CONTENTS PAGE ON THE NATURE OF THE SPLEEN-BONE MARROW RADIA- TION RECOVERY FACTOR 141 Leonard J. Cole and Marie E. Ellis Discussion .......... 147 EFFECT OF X-RAYS ON THE RESORPTION RATE OF IN- JECTED NaH^^COg IN MICE 148 A. Forssberg and G. Hevesy Discussion . . . . . • • • • .150 A SKIN TEST IN RADIOBIOLOGICAL STUDIES . .151 B. JOLLES DU MECANISME PHYSIOPATHOLOGIQUE DE LA MORT CHEZ LES RATS IRRADIES AVEC UNE DOSE MORTELLE DE R.X 154 J. Maisin, H. Maisin et A. Dunjic Discussion . . . . . • • • • .168 LABORATORY STUDIES AND CLINICAL TRIALS OF SOME CHEMICAL RADIO-SENSITIZERS 170 J. S. Mitchell Discussion . . . . . . • • • .189 AUGMENTATION DE LA SYNTHB^ DE L'HEMOGLOBINE IjY vitro PAR LES RETICULOCYTES APRES IRRADIA- TION . . ' 194 A. NizET et A. Herve Discussion . . . . . . . • • .199 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION . . . .201 D. W. VAN Bekkum Discussion ........•• 209 ACTION D'UNE DOSE UNIQUE DE RAYONS X SUR QUEL- QUES FRACTIONS DE PHOSPHORE ACIDO-SOLUBLE ET SUR LA RESPIRATION DE LA PEAU CHEZ LE RAT . .210 P. Mandel, Ch. Gros et J. Rodesch DNA SYNTHESIS IN BONE MARROW STUDIED BY AUTO- RADIOGRAPHY 216 / L. G. Lajtha, R. Oliver and F. Ellis Discussion . . . . . • • • • .219 THE INHIBITION OF DNA SYNTHESIS BY IRRADIATION WITH SPECIAL REFERENCE TO IRRADIATION IN THE EARLY STAGES OF LIVER REGENERATION . . .220 Barbara E. Holmes and Lorn.^ K. Mee Discussion ......•••• 224 ix CONTENTS PAGE INCORPORATION DU i*C DANS LE GLYCOGENE DU FOIE APRES IRRADIATION 225 Mme. Lourau-Pitres Discussion .......... 228 OBSERVATIONS MADE ON THE HUMAN RESPONSE TO A SINGLE DOSE OF X-RAYS— THE LATENT PERIOD . . 229 W. M. Court Brown and John D. Abbatt Discussion .......... 232 COMPARISON OF THE PHYSIOLOGICAL RESPONSE TO RADIATION AND TO RADIOMIMETIC CHEMICALS . 235 L. A. Elson A NEW HYPOTHESIS FOR 'CHROMATID' CHANGES . 243 S. H. Revell EFFECT OF OXYGEN TENSION ON THE PRODUCTION OF CHROMOSOME BREAKAGE BY IONIZING RADIA- TIONS: AN INTERPRETATION 254 C. P. SWANSON THE RELATIVE EFFECTIVENESS OF VARIOUS IONIZING RADIATIONS ON CHROMOSOME BREAKAGE IN TRADE- SCAJVTIA 262 J. S. Kirby-Smith, C. W. Sheppard and Doris L. Craig CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X- RAYS . 265 G. Roy Lane THE RELATIONSHIP BETWEEN CHROMOSOME FRAG- MENTATION AND REJOINING IN TRILLIUM ERECTUM FOLLOWING THERMAL NEUTRON AND X-IRRADIA- TION 275 Eleanor E. Deschner Discussion on Papers by Revell, Swanson, Kirby-Smith, Lane and Deschner ......... 277 STUDIES ON THE EXPERIMENTAL CONTROL OF THE MUTATION PROCESS 282 Ake Gustafsson Discussion .......... 284 THE INFLUENCE OF POST-RADIATION FACTORS ON EFFECTS PRODUCED IN BARLEY 285 L. Ehrenberg PATHOLOGY OF MICE IRRADIATED AFTER INJECTION OF CYSTEAMINE ([i-MERCAPTOETHYLAMINE) . . 290 M. A. Gerebtzoff and Z. M. Bacq SOME FACTORS CONTROLLING THE HAEMATOPOIETIC REGENERATION IN WHOLE BODY IRRADIATED RATS 292 E. H. Betz 298 310 CONTENTS PAGE ACTION DE LA CYSTEAMINE SUR LES TUMEURS GREF- FEES, IRRADIEES LX LOCO S. Neukomm, Mine. L. Peguiron et A. Herve ETUDE HISTOLOGIQUE DE LA REPARATION INTESTI- NALE CHEZ DES RATS IRRADIES SOUS DIVERSES CONDITIONS DE PROTECTION 304 H. Maisin et C. Fievez Discussion on papers by Gerebtzoff and Bacq, Betz, Neukomm, Peguiron and Herve, and Maisin and Fievez CYTOLOGICAL EFFECT OF CHRONIC GAMMA IRRADIA- TION AND THE PROTECTR'E PROPERTY OF CERTAIN CHEMICALS AGAINST THE RADIATION INDUCED CHROMOSOME ABERRATIONS 316 Knut Mikaelsen THE EFFECT OF RADIATION ON FROZEN TUMOUR CELLS 32 1 Ilse Lasnitzki MECHANISMS OF MUTATION PRODUCTION IN MICRO- ORGANISMS 326 Howard B. Newcombe Discussion .....••••• ■^•^-* INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2- CHLOROETHYL)AMINE SUR L'OVAIRE DE LAPINE ADULTE, AVEC OU SANS PREPARATION PAR LA BETA- MERCAPTOETHYLAMINE 340 Paul Desaive HISTOLOGICAL CHANGES IN MAN AND RABBITS AFTER PARENTERAL THORIUM ADMINISTRATION . . .358 Charles Johaxsen Discussion .....-•••• 3dU XI ,^\0A THE LIEGE MEETING The Radiobiology Meeting in Liege was held from August 29th to Sept. 1st, 1954, in the Anatomy Building of the University and was attended by scientists from twelve countries. The names of participants are given on page xiv. The Members were entertained by the Mayor to a banquet and to a re- ception by the Provincial Governor; the Congress was officially opened by the Rector of the University, Prof. M. Dubuisson. To ensure that the heavy programme could be carried out, a tight time schedule had to be adhered to; this was made possible by providing lunch and afternoon tea, served by the Ecole d'Hotellerie of the City of Liege, in a room adjacent to the lecture theatre. As a result the cost of the Meeting was great and the catering and circulation of reprints was made possible only by generous grants of the Belgian Government, of the Foundation Francqui (Brussels) and of the Centre Anticancereux of the Liege Univ-ersity (Chairman, Prof. J. Firket). I owe a great debt to my secretary, Mrs. M. Goutier, for her very efficient collaboration; considerable help was also given by Dr. J. Frederic and by Prof. Chevremont's staff. Z. M. B. XUl PARTICIPANTS Belgium Prof. BAcq, Z. M. Dr. Betz, H. Prof. Desaive, p. Dr. Duchesne, J. Dr. Dor, L. Dr. Errera, M. Dr. Fischer, P. Prof FiRKET, J. Dr. Frederick, J. Dr. pREDERicq, P. Dr. Garsou, J. Dr. Gerebtzoff, M. Dr. GouTiER, R. Mme GoUTIER-PlROTTE, M. Dr. Herve, a. Dr. Henry, J. Dr. Heusghem, C. Prof. Maisin, J. Dr. Maisin, H. Dr. NizET, A. Dr. Ramioul H. Dr. Simon (Mile) Dr. Verly, W. Prof Welsch Prof Gueben Dr. GOVAERTS, J. Dr. Lecomte, J. Dr. Vanremoortere, E. Dr. Beaumariage, M. (Mile) Dr. Closon, J. Laboratoire de Pathologie, 1 , rue des Bonnes Villes, Liege. 1, rue des Bonnes Villes, Liege. Institut de Radiotherapie, Hopital de Ba- viere, Liege. 1 , rue Professeur Mahaim, Cointe-Sclessin. 93, rue des Wallons, Liege. 1850, Chaussee de Wavre, Bruxelles. rue de Vise, Jupille. Institut de Pathologie, 1, rue des Bonnes Villes, Liege. Institut d'Anatomie, rue de Pitteurs, Liege. 1 , rue des Bonnes Villes, Liege. Institut de Radiologic, Hopital de Baviere, Liege. Institut d'Anatomie, rue de Pitteurs, Liege. Institut de Pathologie, 1, rue des Bonnes Villes, Liege. Institut de Physiologic, Place Delcour, Liege. Institute de Radiotherapie, Hopital de Baviere, Liege. Institut Jules Bordet, 1, rue Heger Bordet, Bruxelles. 81, avenue de J'Obscrvatoire, Liege. Voer des Capucins, Louvain. Voer des Capucins, Louvain. Hopital de Baviere, Cliniquc Medicale B, Liege. 8, rue du Batty, Cointe-Sclessin. Institut Jules Bordet, 1, rue Heger Bordet, Bruxelles. 26, rue Jonruellc, Liege. Centre de recherches pour antibiotiques, Boulevard dc la Constitution, Liege. 50, rue des Wallons, Liege. Institut de Physique Nucleaire, Place du XX, Aout, Liege. 11, rue Bles, Liege. 11, rue G. Truffaut, Liege. 563, rue de Herve, Bois de Breux. Centre Anticancereux de I'Universite de Liege, Hopital de Baviere. xiv PARTICIPANTS Canada Dr. Howard, B. Dr. Newcombe, H. B. Atomic Energy of Canada, Ltd., Chalk River, Ontario. Atomic Energy of Canada, Ltd., ("halk River, Ontario. Denmark Dr. Arley, N. Dr. Christensen, B. Dr. MoGENS, F. Dr. JOHANSEN, C. France Dr. Bonet-Maury Dr. Burg, C. Prof. Chevallier, A. Dr. Davydoff, S. (Mile) Dr. DuPLAN, J. F. Dr. GiAUX Dr. Latarjet, R. Dr. Lefort, M. (Mme) Dr. Lourau-Pitres (Mme) Dr. Magat, M. Dr. Chapiro Dr. Mandel, p. Dr. Sw^ngedauw^ Institute of Theoretical Physics, University of Copenhague. Finsen Memorial Hospital, Strandboule- varden 49, Copenhague. Fiseninstitutet og Radiumstationen, Strand- boulevarde, Copenhague. Finsen Laboratory, Strandboulevarden 49, Copenhague. Institut du Radium, 26, rue d'Ulm, Paris 5e. Institut de Physique Biologique, Faculte de Medecine, Strasbourg. Faculte de Medecine, Universite de Stras- bourg. Fort de Chatillon, Fontenay-aux-Roses, Paris. College de France, 3, rue d'Ulm, Paris 5e. Centre Oscar Lambret, 17, rue du Croquet, Lille, France. Institut du Radium, 26, rue d'Ulm, Paris 5e. 8, rue Pastourelle, Paris 3e. 8, rue Ceusier, Paris 5e. Laboratoire de Chimie- Physique, 1 1 , rue Pierre Curie, Paris 5e. Laboratoire de Chimie- Physique, 11, rue Pierre Curie, Paris 5e. 18, rue Lamey, Strasbourg. 140, Boulevard de la Liberte, Lille. Germany Prof. Rajewsky, B. Dr. Pauli Dr. Hug Dr. Koch, R. Great Britain Dr. Abbatt, J. D. Dr. Alexander, P. Forsthausstrasse, 70 Frankfurt a. M. Slid. Forsthausstrasse, 70 Frankfurt a.M.Siid. Forsthausstrasse, 70 Frankfurt a.M.Siid. Institut de Radiobiologie, Universite Freiburg, i.Br. de Radiotherapeutics Research Unit, Hammer- smith Hospital, Ducane Road, London, W. 12. The Chester Beatty Research Institute, Fulham Road, London, S.W.3. XV PARTICIPANTS Dr. Alper, T. (Miss) Dr. Belcher, E. H. Dr. BoAG, J. W. Dr. Britton, R. H. W. Dr. Butler, J. A. V. Dr. Conway, B. E. Dr. Court Brown, W. M. Dr. Dale, W. M. Dr. Ebert, M. Dr. Elson, L. a. Dr. Gray, L.H. Dr. Harriss, E. B. (Miss) Dr. Holmes, B. E. (Mrs.) Dr. HoRNSEY, S. (Miss) Dr. Howard, A. (Mrs.) Dr. Howard-Flanders, P. Dr. Jolles, B. Prof. Roller, P. C. Dr. Lane, G. Roy Dr. Lajtha, L. G, Dr. Lamerton, L. F. Dr. Laser, H. Dr. Lasnitzki, L (Mrs.) Dr. Laws, J. O. Experimental Radiopathology Research Unit, Hammersmith Hospital, Ducane Road London, W.12. Physics Department, Royal Marsden Hospi- tal, Fulham Road, London, S.W.3. 20 Hollycroft Avenue, London, N.W.3. Director of Research, Horlicks Ltd., Slough, Buckinghamshire. Chester Beatty Institute, Fulham Road, London, S.W.3. Chester Beatty Listitute, Fulham Road, London, S.W.3. Radiotherapeutics Research Unit, Hammer- smith Hospital, Ducane Road, London, W.12. Christie Hospital, Wilmslow Road, Man- chester 20. Experimental Radiopathology Research Unit, Hammersmith Hospital, Ducane Road, London, W.12. Chester Beatty Research Listitute, Fulham Road, London, S.W.3. Radiobiological Research Dept., Mount Vernon Hospital and The Radium Listitute, Northwood, Middlesex. Royal Marsden Hospital, Fulham Road, London, S.\\.3. Department of Radiotherapeutics, Univer- sity of Cambridge, Downing Street, Cam- bridge. Hammersmith Hospital, Ducane Road, London, S.WM2. Experimental Radiopathology ResearchUnit, Hammersmith Hospital, London, W'.12. Hammersmith Hospital, Ducane Road, London, W.12. Department of Radiotherapy, General Hos- pital, Northampton. Chester Beatty Research Institute, Fulham Road, London, S.W.3. University of Southampton. Department of Radiotherapy, Churchill Hospital, Oxford. Physics Department, Royal Marsden Hos- pital, Fulham Road, London, S.W.3. Molteno Institute, University of Cambridge, Strangeways Research Laboratory, Cam- bridge. Dept. of Experimental Pathology, Medical School, Leeds, 2. XVI PARTICIPANTS Dr. LOUTIT, J. Dr. Mee, L. K. (Mrs.) Prof. Mitchell, J. S. Dr. Mole, R. H. Dr. Oster, I. Dr. Oliver, R. Dr. Paterson, E. (Miss) Dr. Paton, J. P. J. Dr. PopjAK, G. Dr. Philpot, J. Dr. Revell, S. H. Piof. ROTBLAT, J. Dr. Scott, O.C. Dr. Suit, H. D. Dr. Smith, C. H. Dr. Stocken, L. H, Dr. Trowell, O. A. Dr. Haigh, M. V. (Mrs.) Dr. Wright, E. A. India Dr. Gopal Ayengar, A. R. Japan Dr. Nishiwaki, Y. M.R.C. Radiobiological Research Unit, Atomic Energy Research Establishment, Harwell, Berkshire. Department of Radiotherapeutics, University of Cambridge, Downing Street, Cambridge. Department of Radiotherapeutics, Downing Street, Cambridge. Radiobiological Research Unit, Atomic Energy Research Establishment, Harwell, Berkshire. Institute of Animal Genetics, West Mains Road, Edinburgh. Churchill Hospital, Oxford. Christie Hospital and Holt Radium Institute, Withington, Manchester 20. 87 Stewarton Drive, Cambuslang, Lanark- shire. Hammersmith Hospital, Ducane Road, London, W.12. M.R.C. Radiobiological Research Unit, Atomic Energy Research Establishment, Harwell, Berkshire. Chester Beatty Research Institute, Fulham Road, London, S.W.3. St. Bartholomew's Hospital, London, E.C.I. Radiobiological Research Dept., Mount Vernon Hospital and The Radium Institute, Northwood, Middlesex. Radiobiological Laboratory, Dept. of Radio- therapy, Churchill Hospital, Oxford. Dept. of Radiotherapeutics, University of Cambridge. Biochemical Laboratory, University Museum, Oxford. M.R.C. Radiobiological Research Unit, Atomic Energy Research Establishment, Harwell, Berkshire. Christie Hospital, Manchester 20. Experimental Radiobiology Research Unit, Hammersmith Hospital, Ducane Road, London, W.12. Chief Research Cytologist, Biology Div^ision, Indian Cancer Research Centre, Parel, Bombay 12. Dept. of Biophysics, Osaka City University, School of Medicine, Osaka. XVII PARTICIPANTS Norway Dr. Devik, F. Dr. Eldjarn, L. Dr. MiCHAELSEN, K. Dr. MossiGE, J. (Mrs.) Dr. Oftedal, P. (Miss) Sweden Dr. Anderson, G. Dr. Ehrenberg, L. Dr. FORSSBERG, A. Dr. Gelin, O. E. Prof. Gustafsson, A. Dr. Nybom, N. Dr. Ostergen, G. Dr. Sherman, R. S., Jr. Switzerland Prof. Neukomm, S. Dr. Mouton, R. F. Dr. Lerch, M. Dr. Peguiron The Netherlands Prof. Cohen, J. A. Dr. Davids, J. A. G. Dr. Sangster, I. Dr. SoBELS Dr. VAN Bekkum, D. W. Dr. Vos, O. United States of America Dr. Behnke, a. R. Colonel Blount, R. H. Instituut for Patologi^ Rikshospitalet, Oslo. Biochemical Department, Norsk Hydro's Institute for Cancer Research, The Nor- wegian Radium Hospital, Oslo. Institute of Genetics and Plant Breeding, Agricultural College of Norway. The Norwegian Radium Hospital, Oslo. Radiumhospitalet, Bestum-Oslo. The Gustaf Werner Inst, for Nuclear Chemistry, Uppsala. Inst, of Organic Chemistry, Sandasgat, 2, Stockholm. Institut de Radiophysics, Stockholm. Plant Breeding Institution, Weibullsholm, Landskrona. Statens Skogoforskningsinstitut Experimen- talfaltet. The Gustaf Werner Institute for Nuclear Chemistry, University of Uppsala. Institute of Genetics, Lund. Karolinska Sjukhuset, Radiulhemment c/o Radiotherapy, Stockholm. Centre Anticancereux romand, Hopital cantonal, Lausanne. Battelle Memorial Institute, 7, route de Drize, Geneve. Centre Anticancereux romand, Hopital cantonal, Lausanne. Centre Anticancereux romand, Hopital cantonal, Lausanne. Lange Kleiweg, 139 Risjwijk, Z.H. Lange Kleiweg, 139 Risjwijk, Z.H. Steffenskamp 15, Laren, N.H. Genetisch Instituut, Stationsstraat 9, Utrecht Lange Kleiweg, 1 39 Risjwijk, Z.H. Lange Kleiweg, 139 Risjwijk, Z.H. U.S. Naval Radiobiological Defense Labora- tory, San Francisco Naval Ship Yard (Hunter Point), San Francisco, 24, California. U.S.A.F., Assistant Air Attache, American Embassy, Paris, xviii PARTICIPANTS Dr. Carlson, G. J. Dr. Cole, L. J. Dr. Deschner, E. E. (Miss) Dr. Gey, O. G. Dr. Hollaender, A. Dr. HUTCHENS, J. O. Dr. Jacobson, L. O. Dr. Kirby-Smith, J. S. Dr. Kellner, A. Dr. Patt, H. M. Dr. Pollard, E. Dr. Reimer, C. H. Dr. Swanson, C. P. Dept. of Zoology and Entomology, Univer- sity of Tennessee, Oak Ridge National Laboratory. U.S. Naval Radiobiological Defense Labora- tory, San Francisco, California Brookhaven National Laboratory, Upton, Long Island, N.Y, Jolius Hoffe University, Baltimore 5. Oak Ridge National Laboratory, Biology Division, Oak Ridge, Tennessee. Scientific Liaison Officer, Embassy of the U.S.A. Office of Naval Research, Keysign House, 429 Oxford Street, London, W.l. University of Chicago, Argonne Cancer Re- search Hospital, Chicago 37, Illinois. Oak Ridge National Laboratory, Oak Ridge, Tennessee. Brandeis University, Waltham, Mass. Argonne National Laboratory, P.O. Box 299, Lemont, Illinois. Sloane Physics Laboratory, Yale University, New Haven 1 1 , Connecticut. Jolius Hoffe University, Baltimore 5. Dept. of Biology, The Johns Hopkins Uni- versity, Baltimore 18, Maryland. Yugoslavia Dr. Hajdukovic, S. Laboratoire de Biologic, Institut Boris Kidric Vintcho-Belgrade, P.O.B. 522. XIX ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES Andre Chevallier et Constant Burg Institut de Physique biologique, Faculte de Medecine de Strasbourg (France) Si les progres de la radiochimie dans le domaine des milieux aqueux et des substances hydrosolubles ont ete considerables au cours des dix dernieres annees, par contre, les connaissances au sujet de Taction des radiations sur les substances liposolubles sont encore tres sommaires. Une mise au point de la question pent paraitre actuellement prematuree, et, de fait, les travaux portant sur ce probleme precis sont tres peu nombreux et les etudes systematiques font defaut. La difficulte s'accroit encore si Ton veut se limiter aux substances lipidiques d'ordre biologique, c'est-a-dire aux acides gras et leurs esters, aux phos- pholipides, aux sterols, aux vitamines et hormones liposolubles. C'est ainsi que pour essayer d'englober tons les aspects du probleme et, en nous referant, en premier lieu, a des preoccupations d'ordre biologique, nous examinerons non seulement les resultats physico-chimiques se rap- portant a une experimentation in vitro, ou le rayonnement a ete applique directement a la substance liposoluble, mais egalement les modifications biologiques qui ont ete obtenues, comme consequences de I'irradiation d'un animal vivant. Dans ce dernier cas, il est certain que si quelques faits peuvent paraitre clairement en rapport avec une action du rayonnement sur les lipides organiques eux-memes, la plupart ne seront que I'effet indirect d'une intervention portant sur des systemes enzymatiques complexes ou sur des elements hydrosolubles presents dans les tissus vivants. Mais, dans I'incapacite ou nous nous trouvons de faire le depart entre les premiers et les seconds, nous tiendrons compte des uns et des autres. Les considerations que Ton pent envisager sur le mecanisme d'action des radiations ionisantes sur un milieu quelconque, se classent naturellement en effets primaires a consequences immediates d'un caractere plus specialement physique et en eflfets secondaires, en general, d'ordre chimique. II ne semble pas, au premier abord tout au moins, que Ton doive faire une place particuliere aux milieux lipidiques en ce qui concerne les effets primaires. Ceux-ci sont en effet pratiquement representes par les phe- nomenes d'ionisation d'une part, et d'excitation, d'autre part, et sont en relation avec les lois d'absorption de I'energie regue par le milieu. Mais il ne faut pas oublier qu'une meme molecule pent donner naissance a un grand nombre de types d'ions differents pour des conditions d'irradiation donnee. Dans le cas de Texcitation, comme dans le cas de I'ionisation, il faut s'attendre a voir intervenir la qualite du rayonnement utilise dans les lipides comme dans les milieux aqueux. Une meme quantite d'energie administree sous forme de rayonnement v, d'electrons ou de particules a, aura des effets 1 B ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES differents en raison des differences considerables de densite des paires d'ions formes le long de la trajectoire de ces diverses particules (photons, [3, a)^- ^. De ce fait, le rendement ionique, soit le rapport entre le nombre de molecules ayant reagi et le nombre des paires d'ions formes dans le milieu, sera tres different suivant le type du rayonnement et I'energie qu'il transporte. L'accroissement de la densite des paires d'ions par unite de longueur favorisant la recombinaison ionique, ce dernier phenomene perdra progres- sivement de son importance au cours de I'utilisation d'un rayonnement X plus penetrant ou d'un rayonnement y. Ce fait qui explique en partie le faible rendement obtenu dans certaines reactions radiochimiques en milieu aqueux avec le rayonnement a compare au rendement obtenu avec le rayonnement y intervient probablement de la meme maniere lorsqu'on se trouve en milieu lipidique. A partir des reactions primaires ainsi declenchees, on considere que les molecules excitees ou ionisees subissent des fragmentations ou des modifica- tions structurales avec apparition de formes fugaces (radicaux libres) ou de composes stables. Ces molecules excitees peuvent egalement subir un pro- cessus de conversion interne, c'est-a-dire un transfert de I'energie d'excitation en energie d'oscillation des atomes constituant la molecule. La molecule ainsi excitee pourra alors provoquer des reactions qu'elle ne serait normale- ment susceptible d'induire qu'a des temperatures plus elevees^. Ainsi certains des produits d'irradiation pourront avoir la possibilite de se combiner entre eux ou avec d'autres fragments moleculaires pour donner des combin- aisons stables. D'autres, au contraire, pourront etre le point de depart de reactions en chaine de differents types. Ces reactions seront caracterisees par un rendement ionique tres eleve et elles pourront etre profondement modifiees par la presence -dans le milieu de certaines substances en quantites tres faibles, substances qui ont la possibilite de reagir avec les radicaux libres presents pour donner, soit des composes stables arretant ainsi la reaction, soit au contraire former de nouveaux radicaux libres qui pourront donner naissance a un type de chaine different du precedent. Quoiqu'il en soit, et sans entrer dans I'etude generale du mecanisme intime de la reaction qui a ete faite par divers auteurs '*"**, il est evident que toutes les reactions secondaires sont etroitement liees a la constitution des molecules irradiees et a la composition du milieu dans lequel elles se trouvent. Dans le cas des substances liposolubles la meme consideration s'applique integrale- ment et chaque milieu constitue, en somme, un cas d'espece. Cependant nous pouvons deja presumer que dans les milieux lipidiques la presence d'oxygene dissout sera particulierement importante. On pent penser que la quantite d'oxygene presente soit trop faible pour que les modifications de sa propre molecule par le rayonnement tiennent une place importante. Par contre, les molecules d'oxygene pourront reagir avec les radicaux libres formes a partir des substances lipidiques. Cette reaction pent amener la constitution d'une reaction en chaine dont certains elements peroxydiques seront susceptibles d'etre deceles a I'analyse. Cependant une telle reaction est susceptible d'etre modifiee soit dans le sens de I'acceleration, soit dans le sens du ralentissement par la presence dans le milieu de substances qui, a I'etat de traces, ont la possibilite d'intervenir dans la reaction et amener ainsi la realisation d'un etat assimilable a un effet pro ou antioxygene, 2 ANDRE CHEVALI.IER ET CONSTANT BURG L'intervention de I'a-tocopherol, comme nous le verrons plus loin, illustre ce point de vue. Un second aspect important de la radiochimie des substances lipidiques se rapporte a la presence dans le milieu irradie, soit d'un solvant lipidique, soit d'une autre phase liquide, aqueuse en principe. Dans ce dernier cas, le ' cage effect ' de Franck-Rabinovitch prend peut-etre une importance plus grande que dans le cas d'une phase lipidique continue. Mais, d'autre part, l'intervention du rayonnement ionisant pourra provoquer, a partir du solvant, des radicaux libres qui ne se seraient pas formes au depens de la substance liposoluble. Le fait est tout a fait remarquable en ce qui concerne les emulsions de lipides dans I'eau et au niveau desquelles se produisent des radicaux OH formes au depens de la phase aqueuse. Bien entendu, on pent toujours penser a une intervention de ces radicaux sur les lipides presents. Les phenomenes peuvent encore se compliquer si dans la phase aqueuse se trouvent des substances hydrosolubles a grand pouvoir reactionnel. Celles- ci, en reagissant avec les radicaux hbres formes, soit dans la phase aqueuse, soit dans la phase lipidique, seront susceptibles de modifier le cours des reactions en chaine qui tendent a prendre naissance dans cette derniere. On arrive ainsi a des systemes reactionnels extremement compliques, mais malgre leur complexite et leur incertitude, de telles eventualites meritent d'etre etudiees de pres, car elles realisent precisement les conditions qui se rencontrent au niveau des tissus vivants. Pour tenir compte des considerations precedentes, nous sommes conduits a presenter les resultats experimentaux qui ont ete obtenus a propos de Taction des radiations ionisantes sur les lipides de la maniere suivante. Dans la premiere partie, consacree a I'experimentation in vitro, nous envisagerons successivement Taction des radiations : (7) sur des lipides purs en Tabsence de solvant et de phase aqueuse, en examinant successivement les reactions en Tabsence d'oxygene dissout puis en presence d'oxygene, (2) sur des lipides en presence d'oxygene dissout et d'une phase aqueuse pure ou contenant des substances hydrosolubles, et (3) sur des graisses complexes. Dans la seconde partie, nous distinguerons, a la suite de Tirradiation d'un animal ou d'un tissu vivant, les modifications qui ont ete constatees : (7) d'une maniere immediate sur les graisses du tissu adipeux, (2) de fagon indirecte sur les constituants lipidiques de differents organes ou tissus : intestin, sang, foie, cerveau. experimentation 'in vitro' Action des Radiations Ionisantes sur des Lipides en l'Absence d'Oxygene Dissout et en l'Absence d'Eau et de Solvants Lipidiques. Tres pen de travaux ont ete consacres a Tetude de la radiochimie des acides gras supe- rieurs en Tabsence d'oxygene. Cependant on peut, a partir de resultats obtenus sur des hydrocarbures en phase vapeur^ ou en phase liquide, conclure a la possibilite d'isomerisation et de fragmentation de la molecule. A ce litre, la fragilite de la liaison carbone-carbone entre la fonction 3 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES carboxyle et le carbone adjacent, permet de supposer que la reaction de decarboxylation doit etre assez aisee. Les resultats des experiences de Sheppard et Burton^ montrent ces differentes possibilites. Sheppard et Burton ont irradie avec des particules a de I'acide acetique a 130°G sous forme vapeur et les acides caprylique, laurique et palmitique sous forme solide a la temperature ambiante. Ces auteurs ont de plus observe la presence de pentadecane dans les produits d'irradiation de I'acide palmitique et de undecane dans les produits d'irradiation de I'acide laurique, c'est-a-dire des hydrocarbures, respective- ment en C^g et en C^, mettant bien en evidence I'importance des reactions de decarboxylation. II faut egalement noter la formation de petites quan- tites de monoxyde de carbone due, soit a une action directe du rayonnement, soit par action sur le dioxyde de carbone et I'hydrogene moleculaire Hg forme au cours de la reaction. On pent, en outre, supposer que la formation de radicaux libres et des atomes libres d'hydrogene determine des reactions de condensation ou de polymerisation. L'etude de Taction des radiations ionisantes sur les phos- pholipides et sur les differentes vitamines et hormones liposolubles en I'absence d'oxygene n'a pas ete faite, soit a I'etat cristallise, soit en solution. II faut cependant noter ime observation^ qui relevent une destruction du carotene et de la vitamine A en solution dans I'hexane, en I'absence d'oxy- gene, sous azote. Action des Radiations Ionisantes sur les Lipides en Presence d'Oxy- gene Dissout et en l' Absence d'Eau et de Solvants Lipidiq^ues. (7) Esters d'acides gras purs. En presence d'oxygene dissout, le probleme des modifi- cations chimiques provoquees par I'irradiation des esters d'acides gras prend un aspect un peu different. On doit avoir, dans ce cas particulier, les memes reactions c|u'en I'absence d'oxygene, c'est-a-dire formation d'atomes d'hydrogene, de radicaux hydrocarbones de longueurs diverses et de mole- cules stables d'hydrocarbures, a nombre d'atomes de carbone variable, dues a des reactions de decarboxylation ou de fragmentation de chaine. II serait interessant de rechercher systematiquement la presence de telles substances dans les produits d'irradiation en presence d'oxygene dans la limite ou techniquement leur mise en ev'idence est possible. A cote de ces reactions il faut envisager la possibilite de formation d'ions et de radicaux libres a partir de I'oxygene moleculaire dissout. La pro- babilite de cette reaction par action directe du rayonnement est cependant tres faible si Ton tient compte du fait que le nombre d'atomes ionises de chaque element, dans un milieu donne, est grossierement proportionnel au rapport en poids de cet element dans le milieu. Les radicaux libres formes directement par action des radiations ionisantes sur I'oxygene moleculaire dissout seraient done proportionnellement tres peu nombreux et leur inter- vention ulterieure dans des reactions en chaine pourrait etre consideree, en principe, comme negligeable. Une autre reaction, par contre, prend une importance des plus grandes. En effet, on pent envisager I'existence de reactions entre les radicaux libres formes au depens des molecules d'acides gras et I'oxygene moleculaire dissout entrainant la formation de reaction en chaine. ANDRE CHEVALLIER ET CONSTANT BURG Une de ces reactions, entre autres, entraine la formation de peroxydes suivant le schema : RH w«v_^ R* + H* R* ^ O2 - ROO* + RH ^ ROOH ^ R* ROO* Ces peroxydes donnant des reactions analytiques relativement tres sen- sibles ont ete recherches par de nombreux auteurs. Hannan et BoAG^'^ en utilisant I'oxydation de'J'ion Fe- " en Fe^ ^ en presence de thiocyanate, ont observe la presence de peroxydes dans des echantillons de stearate, oleate et linoleate de methyle, ainsi que dans de la tristearine irradiee. Les auteurs administrent des doses de 2.000.000 de roentgen equivalent physical (rep) sous forme d'electrons provenant d'un geneiateur de van de Graaff. lis ont releve des quantites de peroxydes de 50 .10-^ UO 30 t ^20 I ^ 10 Laurafe de methyle Oleate de methyle Linoleate de methyle _L 5.0 DO 10.000 Dose de rayons X exprimee en t Ib.OOO Figure 1. Variation du taux de peroxydes formes au cours de V irradiation par les rayons X en fonction de la dose I'ordre de 2 a 3 micromolecules par gramme d'esters irradies. Ces memes auteurs ont obtenu des resultats analogues par irradiation d'hexane et de cjc/ohexane. II est interessant de relever qu'a tres basse temperature — 70° C, le taux de peroxydes obtenu dans ces experiences a ete tres faible par rapport a celui obtenu a la temperature ambiante. Ce fait est, pent etre, lie au ralentissement de la vitesse de diffusion de I'oxygene. Des resultats analogues ont ete obtenus par Chevallier et Burg^^ en utilisant la methode de dosage de Hartman et Glavind^^^ c'est-a-dire I'oxydation de dichlorodihydroxyphenylenediamine en 2 : 6-dichlorophenol indophenol qui donne dans le xylene additionne d'acide acetique une couleur rouge intense dont I'intensite pent etre determinee par spectrophotometrie. Les experiences ont consiste dans Tadministration a des esters methyliques, des acides laurique, oleique et linoleique de doses de rayons X variant entre 2.500 r et 1 5.300 r sous 80 kV, soit a un acide sature et a des acides contenant une et deux doubles liaisons {Figure 1). 5 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES On peut constater que pour 2.500 r le laurate de methyle ne presente pas de peroxydes decelables par la methode utilisee. Mais, par centre, leur production est tres nette dans le cas de I'oleate et encore plus marquee dans le cas du linoleate de methyle. Hannan et Boag^" avaient obtenu des peroxydes a partir du stearate de methyle qui est egalement un acide gras totalement sature. Mais il faut observer que dans I'experience de Hannan et Boag les doses de rayonnement utilisees sont entre 100 et 1.000 fois superieares a celles que nous avons employees dans notre experience personnelle. Du fait que pour des doses identicjues, le linoleate de methyle presente un taux de peroxydes beaucoup plus eleve que I'oleate de methyle qui n'a qu'une seule double liaison. On peut conclure que la presence des doubles liaisons facilite considerablement la production des peroxydes. II serait interessant d'etudier de plus pres la cinetique de formation de ces peroxydes et de verifier s'ils ont la meme structure que les hydroperoxydes formes au cours de I'auto-oxydation des acides desatures. II y aurait egalement interet a savoir si les peroxydes formes peuvent etre detruits par le rayonnement lui-meme. On peut d'autre part, penser que cette production de peroxydes par reaction en chaine doit s'accompagner de la formation de produits de poly- merisation due a I'interruption des chaines. La possibilite des reactions suivantes est en effet a envisager : R* + R* ^ R— R R* + ROO* -. R— O— O— R ROO* + ROO* ^ R— O— O— R + O., La presence de polymeres, difficile a mettre en evidence, n'a pas ete recherchee. Cependant il faut relever une observation i" qui, par irra- diation d'ester de I'acide linoleicjue a — 70° C par des electrons, a obtenu une gelification de I'echantillon, traduisant sans doute un phenomene de poly- merisation ou de condensation. Cas particulier des esters d' acides polyethyleniques {isomerisalion) . Dans le cas particulier des acides polyethyleniques on pouvait se demander si la formation de peroxydes s'accompagnait d'une isomerisation des doubles liaisons qui passent en position conjuguee, isomerisation c[ue Ton constate lors de la formation d'hydroperoxydes lors de I'auto-oxydation de ces acides a Pair atmospherique. La presence de ces doubles liaisons est tres facile a deceler. Elles entrainent en effet I'apparition d'une bande d'absorption extremement importante a 2. 340 A. L'experience confirme ce point de vue : la presence de cette bande d'aJDSorption a ete en effet relevee lors de I'irradiation d'esters de I'acide linoleique par Mead^^ en utilisant des rayons X obtenus sous 250 kV et par Ghevallier et Burg^^ en utilisant des rayons X sous 80 kV. La variation de 1 'absorption a 2.340 A est proportionnelle a la variation de la concentration en peroxydes du milieu pour des conditions experi- mentales donnees, et la sensibilite de la determination de I'absorption est tellement grande qu'on peut I'utiliser pour suivre la variation du taux de peroxydes du milieu. II ne faut cependant pas ouljlier que le rapport entre le coefficient d'absorption a 2.340 A et le taux de peroxydes peut 6 ANDRE CHEVALLIER ET CONSTANT BURG varier suivant les conditions experimentales. II ne faudra done utiliser cette methode qu'avec beaucoup de prudence. La presence de peroxydes dans les graisses irradiees entraine egalement une diminution considerable de la stabilite de ces graisses en presence de I'oxy- gene atmospherique, surtout en ce qui concerne les acides desatures. En effet les peroxydes peuvent se decomposer en donnant naissance a de nouvelles reactions en chaines, par exemple suivant le schema propose par Holland et Ten Haven^^ dans le cas des hydroperoxydes de I'acide linoleique. ROOH -> R* reaction d'initiation ROOH -> ROO* R* -^ O2 -> RO2* reaction de propagation ROO* + RH -^ ROOH + R* R* -|^ R* ^ R — R reaction de fin de chaine ROO* + R* -> ROOR ROo* + ROa* -> ROOR + O 2 Ces reactions secondaires peuvent se produire sous Taction du rayonne- ment ou spontanement longtemps apres la fin de I'irradiation. Sur le plan pratique, elles posent des problemes importants pour la con- servation des aliments sterilises par les radiations ionisantes et peuvent pent etre intervenir en radiobiologie et expliquer certains eflfets tardifs du rayon nement. (2) Esters gras en presence de traces de substances reactionnelles. Une autre consequence des reactions en chaines provoquee par les reactions ionisantes au niveau des esters gras est la possilDilite d'interaction entre les radicaux fibres propagateurs de la reaction et les substances presentes a I'etat de traces dans le milieu. Ces reactions peuvent entrainer, soit des fins de chaines, soit au contraire, donner naissance a de nouvelles chaines de reac- tion d'une nature diflferente. Un exemple d'un tel phenomene nous est donne par les anti-oxygenes dans le domaine de I'auto-oxydation des esters gras desatures. Ainsi, par exemple, I'auto-oxydation de Tester ethylique de Tacide linoleique qui se propage par les deux radicaux ROO* et R* pent etre fi-einee par Thydro- quinone qui reagit avec le radical ROO* pour donner un produit stable, provoquant par la une interruption de la chaine 1*. Un point particufierement important est Tetude de Tintervention sur les reactions radiochimiques des vitamines et hormones liposolubles dont certaines, comme les tocopherols, ont des proprietes anti-oxygenes bien connues. Mead et Polister ont examine Taction d'un tres grand nombre de ces substances sur du linoleate de methyle au cours de I'irradiation par les rayons X, mais les experiences ont ete faites en presence d'eau, ce qui entraine des consequences particulieres et nous reviendrons sur ces travaux dans un chapitre suivant. L'action de la plupart des vitamines et hormones liposolubles sur les esters acides gras purs, en Tabsence de solvant, n'a pas fait Tobjet d'etude systematique. Cependant Chevallier et Burg" ont releve que la presence de a-tocopherol, en tres faible quantite, fi-eine d'une fa^on importante la formation de peroxydes par les rayons X dans le linoleate de methyle. Le 7 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES tocopherol avirait done, dans ees conditions, des proprietes analogues aux proprietes anti-oxygenes dont il fait preuve au cours de I'auto-oxydation des acides gras a I'air. Une etude cinetique approfondie du phenomene presenterait un grand interet {Figure 2). (3) Vitamines liposolubles. A cote de ces travaux siu" la radiochimie des esters gras, il faut citer un certain nombre d'etudes qui ont ete faites sur les vita- mines liposolubles. Comme la plupart de ces vitamines sont cristallisees a la temperature ambiante, la plupart des travaux ont ete effectues sur des solutions dans I'hexane. La structure de ce solvant se rapprochant de celle de la chaine hydrocarbonee, on pent penser que les radicaux libres formes par les radiations a partir de I'hexane sont de meme nature cjue ceux formes a partir des acides gras. Ainsi si on est oblige de tenir compte d'une action 50 70'^ l/'no/eafe de mefhyle pur Lmoleafe de mefhyle -t- focopherol 5.000 70.000 Dose ■ de rayons X exprimee en r 15.000 Figure 2. Variations du taux de peroxydes formes au cours de I'irradiation du linoleate de me'thjile en fonction de la dose, en presence et en V absence de tocopherol indirecte du rayonnement sur les vitamines dissoutes par I'intermediaire du solvant, on pent penser au moins que qualitativement les reactions seront les memes. II faudra cependant rester tres prudent dans I'interpretation des rendements ioniques obtenus. La destruction de I'acetate de vitamine A et du p-carotene en solution dans I'hexane sous Taction de rayons X ou d'electrons, a ete etudiee^' i"^. II est important de noter que la vitamine A, en solution dans des esters d'acides gras, semble beaucoup plus rapidement detruite par le rayonne- ment que lorsqu'elle est seule. Cette fragilite particuliere de la vitamine A en solution lipidique pent avoir son importance dans le probleme de la sterilisation des produits alimentaires par le rayonnement. L'etude radiochimique des autres vitamines liposolubles, D, K et meme E, soit a I'etat pur, soit en solution, ne semble pas avoir ete faite jusc{u'a present. 8 1 andre chevallier et constant burg Action des Radiations Ionisantes sur les Lipides en Presence d'Oxy- GENE DissouT ET d'Eau. L'etudc de Taction des radiations ionisantes sur les lipides en presence d'eau presente un grand interet en ce sens que Ton se rapproche davantage des conditions qui existent normalement dans Torganisme. Malheureusement le probleme se complique d'une fa^on considerable. II faut, en efiet, tenir compte des possibilites des reactions de la substance lipidicjue elle-meme et en presence d'oxygene tout en envisa- geant la possibilite de reactions indirectes dues aux radicaux libres formes dans la phase acjueuse au depens de I'eau. II faut envisager ici la possibilite d'actions indirectes analogues a celles que Dale^® a observees dans le domaine hydrosoluble. On pent admettie, avec Weiss^', que I'irradiation de I'eau forme essentiel- lement des radicaux H et OH, par exemple, suivant le mecanisme propose par Lea^^. L'electron pent egalement reagir avec un accepteur d'electrons^'', par exemple : H+ + e ^ H* H* + H+ ^ H2+ I'ion Hg^ pouvant a son tour fixer un electron et donner naissance a de I'hydrogene moleculaire. Si cette hypothese est exacte, I'hydrogene qui est forme en tres faible quantite pendant I'irradiation d'eau pure^*^ doit etre augmente d'une fac^on importante en solution acide^^. En presence d'oxygene, on pent egalement avoir des interactions entre les radicaux formes dans I'eau et I'oxygene moleculaire dissout. On peut en particulier avoir la reaction O2 + H- -^ HO^- En presence d'oxygene, I'irradiation de i'eau entraine egalement la forma- tion de petites cjuantites d'eau oxygenee qui ont ete decelees pour la premiere fois par Bonet-Maury^^. Get auteur a obtenu, en faisant agir les corpuscules a du radon sur de I'eau, 0,3 y de peroxyde d'hydrogene par microcuries detruit, ce qui correspond a 0,54 molecules de peroxyde d'hydrogene par paire d'ions formes dans la solution. Le taux d'eau oxygenee etait beaucoup plus abondant en presence d'oxygene dissout qu'en I'absence d'oxygene^^. Loiseleur-^ a egalement montre que le rendement en eau oxygenee etait fonction du pH, le rendement etant plus eleve aux pH acides. On con^oit aisement que ces produits (radicaux : H*, OH*, HOg*, eau oxygenee) formes au depens de Teau peuvent reagir sur les acides gras presents au niveau de I'interface eau/lipide et modifier profondement Taction des radiations ionisantes sur le substrat lipidique. (i) Acides gras purs, (a) Etude en Vabsence d'oxygene. Mead^^ a irradie de Tacide linoleique dans Teau a pH 8,5-9 en I'absence d'oxygene et en presence d'azote avec des rayons X a 250 kV et a constate une conjugaison des doubles liaisons, conjugaison qui accompagne generalement la formation de peroxydes. II est a noter cependant que le rendement ionique etait faible, voisin de 9. Dans ces conditions, si les peroxydes ne se sont pas formes au depens d'oxygene residuel, il faut admettre leur formation a partir de radicaux libres formes au depens de la phase aqueuse, fait qui presente le plus grand interet. 9 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES {b) En presence d'oxygene. Mead^^ a etudie le comportement de I'acide linoleique dans I'eau a pH 8,5-9 en presence d'oxygene. II a releve une conjugaison tres importante des doubles liaisons avec un rendement ionique assez eleve de I'ordre de 100 environ, ce qui semble bien impliquer I'existence d'une reaction en chaines. II est a noter cependant que le rendement ionique varie d'une fa^on importante suivant la dose de rayons X et suivant la vitesse a laquelle cette dose est administree. En collaboration avec le meme auteur, Polister-^ a egalement etudie le cas d'emulsions de linoleate de methyle, irradiees avec des rayons X a 250 kV. lis ont retrouve, en presence d'oxygene, le meme rendement ionique eleve en doubles liaisons conjuguees et en peroxydes, que lors de I'irradiation d'acide linoleique dans I'eau. Ces peroxydes ont ete doses suivant la methode de Lea^^. Pollster et Mead-'* se sont egalement adresses a des emulsions d'esters de I'acide ^-eleostearique qui presente trois doubles liaisons conjuguees sous I'influence du rayonnement X. lis ont constate la disparition d'une des 3-0 0 200 WO 600 600 Dosage txIO"^ Figure 3. Variations de I'absorption idtraviolette de I'acide ^-eleostearique au cours de Firradia- tion par les rayons y du cobalt [communique par J. F. Mead) • Absorption it 2-3U0 K o Adsorp/ior) 0,2-790 A 500 1500 2500 Dosage Figure 4. Variations de I'absorption ultraviolette de Vacidi [i-eleostearique au cours de Virradia- tion par les rayons X a 250 k V [communique par J. F. Mead) trois doubles liaisons conjuguees. Cette etude est assez commode a faire, une triple liaison conjuguee donnant une bande d'absorption intense a 2.790 A, il suffit de suivre I'absorption a cette longueur d'onde et de mesurer I'absorption a 2.300-2.350 A qui indique la formation de deux doubles liaisons conjuguees due a la disparition d'une des trois doubles liaisons conjuguees qui existait precedemment. II est a noter que si qualitative- ment la reaction est la meme avec des rayons X a 250 kV et le rayonnement Y du cobalt, quantitativement les deux reactions se deroulent d'une fa^on tres differente. II faut remarquer que dans toutes les experiences de Mead^^, il y avait dans I'eau de petites quantites d'alcool ethylique. II y aurait interet a etudier la possibilite de formation d'alcool secondaire ou de glycol par action du radical OH sur une double liaison, par un mecanisme analogue a celui mis en evidence par Keller et Weiss-'' au cours 10 ANDRE CHEVALLIER ET CONSTANT BURG de I'irradiation dii cholesterol et du 3-^i-hydroxypregn-5-en-20-one en solution aqueuse en presence d'oxygene dissout. (2) Vitamities liposolubles pures en emulsion aqueuse. Mead a egalement entre- pris des travaux sur le comportement des emulsions aqueuses de plusieurs vitamines liposolubles en presence d'oxygene sous Taction, soit de rayons X, soit de rayons y. La methylnaphtoquinone n'a pas presente de modifications de son spectre d'absorption apres administration de 40.000 r sous 250 kV. L'acetate de vitamine A, par contre, est rapidement detruit. L'analyse spectrale des produits d'irradiation montre la disparition progressive des cinq doubles liaisons conjuguees avec apparition de fragments de la chaine primitive, fragment a deux ou trois doubles liaisons conjuguees. II faut remarquer que ■^ 200 600 800 TxlO"^ Figure 5. uoo Dosage Variations de ['absorption ultraviolette de l'acetate de vitamine A au corns de /'irradiation {communique par J. F. Mead) I'absorption ultraviolette des produits de destruction est tout a fait semblable a celle qui a ete observee autrefois par Chevallier et Duboul.oz-^ dans le cas de I'oxydation photochimique de cette meme substance. L'ergosterol, la provitamine D, est egalement detruite par I'irradiation. Les bandes d'absorption aux environs de 2.800 A disparaissent progressive- ment pour donner naissance a une nouvelle bande d'absorption a 2.340 A {Figure 6). Une etude plus approfondie de la radiochimie de ces deux vitamines est actuellement entreprise dans le laboratoire de Mead^^. Par contre il ne semble pas que nous possedions actuellement de donnees precises sur la vitamine E, malgre I'importance qu'elle joue dans les phenomenes d'oxydation. (5) Acides gras en presence de traces de substances vitaminiques liposolubles. La formation de reaction en chaines au cours de I'irradiation d'acides gras purs en presence d'oxygene, nous permet de penser que la presence de certaines vitamines a I'etat de trace, peut modifier profondement I'aspect des reactions ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES en reagissant sur les radicaux formes par le rayonnement. C'est effective- ment ce qui a ete observe^*. Ces auteurs ont administre a des emulsions de linoleate de methyle dans I'eau des doses de rayons de 1 .000 r sous 250 kV, ceci apres avoir ajoute a I'emulsion des quantites tres faibles de differentes vitamines liposolubles et ils ont determine la vitesse de conjugaison des doubles liaisons. L'acetate de vitamine A, a concentration elevee, inhibe d'une fa^on importante la conjugaison des doubles liaisons. Le y-tocopherol fait egalement preuve d'un pouvoir inhibiteur consider- able, mais a des concentrations beaucoup plus faibles. Une concentration de 2,6 X 10-^M est encore efficace. Pour des concentrations plus elevees, le pouvoir inhibiteur du tocopherol est total et le rendement ionique tombe kj A 1 \ r 1 1 vo 1 1 1 1 1 1 1 1 > / 1 \ / 1 \ 1 1 './ 1 1 1 1 -v 1 > ' 1 1 1 1 ^ A - 0 5 "^ ■' \ \ \ 1 ^ / ,1 •-. -^ 0 1 1 1 1^-^ 1 Figure 6. Modifications spectrales de Vergosterol au cours de I' irradiation {commimiqid par J. F. Mead) £1/0 2 SO 280 Wavelength 300 miL a zero. A la dose de l.OOOr les auteurs n'ont pas releve de destruction ap- preciable du tocopherol. La vitamine D a egalement fait preuve d'un pouvoir inhibiteur, mais beaucoup moins marque. Pour des doses de 4,000 r les auteurs n'ont pu observer de modifications importantes dvi spectre de la vitamine D. Les trois vitamines A, D et E ont done toutes trois une action inhibitrice marquee, surtout la vitamine E. {4) Acides gras en presence de substances hydrosolubles. Pollster et Mead^* ont egalement examine la possibilite d'interaction de substances dissoutes dans la phase hydrosoluble sur les reactions en chaines induites par le rayonne- ment en phase hposoluble. L'acide ascorbique, la cysteine, le glutathion, suivant leur concentration, inhibent a des degres divers, la conjugaison des doubles liaisons du linoleate de methyle. Dans le cas de l'acide ascorbique, a une concentration de 9,5 mole pour cent on a une inhibition de toute conjugaison pour des doses de rayons X de l.OOOr. II faut noter que cet effet protecteur s'accompagne d'une destruction notable de la vitamine. 12 ANDRE CHEVALLIER ET CONSTANT BURG Dans le cas de la cysteine on voit apparaitre, au cours de I'irradiation, une bande d'absorption a 2. 750 A qui n'a pas pu encore etre interprctee, mais qui indique la complexite des reactions. Dans les interactions entre les substances hydrosolubles et les processus d'oxydation qui se deroulent dans la phase liposoluble on peut se demander s'il s'agit d'une interaction a I'interface lipide/eau entre les substances hydro- solubles comme I'acide ascorbique et les radicaux intervenant dans les reac- tions en chaines en phase liposoluble. II pourrait s'agir en somme d'un mecanisme analogue a celui de Taction anti-oxygene du tocopherol ou de I'hydroquinone au cours de I'auto-oxydation des acides gras. Mais on peut se demander, au contraire, s'il ne s'agit pas plutot d'un effet analogue a I'effet de protection observe par Dale. Ces substances protectrices, comme I'acide ascorbique, reagissant cette fois avec les radicaux libres formes par les radiations dans la phase aqueuse et non avec ceux formes en phase lipidique comme dans I'hypothese precedente. Mead et Pollster ont egalement etudie Taction de la catalase pour etablir si la presence d'eau oxygenee, fournie par I'irradiation dans la phase aqueuse, etait susceptible de faciliter la formation d'hydroperoxydes en phase lipi- dique. Le rendement ionic^ue de la conjugaison des doubles liaisons pour une dose de l,000r en presence de catalase est de 80,5 et en Tabsence de catalase de 72,5. II semble que dans les conditions ou se sont places ces auteurs, on ne releve pas d'influence appreciable de la catalase. Sterols et Hormones Steroides, Un certain nombre de travaux ont ete consacres a Tetude de la radiochimie des sterols et des hormones steroides en presence d'eau. Nous ne nous etendrons pas sur cette question. Elle a fait Tobjet d'un rapport recemment^'^ et nous ne rappellerons que les resultats essentiels. Les principales modifications chimiques relevees peuvent etre rapportees a la presence dans Teau soit de radicaux OH*, soit de radicaux HO-' dus a la reaction H* + O2 ~^ HO^*, Tatome libre H* etant forme par le rayonnement a partir de Teau. L'irradiation du cholesterol par les rayons X a ete etudiee^^. On a essentiellement une attaque de la double liaison en 5-6 avec formation de derives hydroxyles. On releve egalement la formation d'une fonction cetone en position 7 sans doute suivant le schema \/^ (OH) \/^ (O,) \/^ >C=C— G > >C=C— C ► >C=C— G \H ^O- (RH) l/H > > G=G— G > > G=G— C=0 + H2O 2 XO^H qui fait intervenir Toxygene dissout. L'irradiation du 3 p-hydroxypregn-5-en-20-one entraine egalement Tap- parition de derives hydroxyles par un mecanisme analogue a celui releve a propos de la double liaison du cholesterol. Keller et Weiss^^ ont egale- ment irradie des solutions aqueuses d'acide cholique; ils ont obtenu de la formation de I'acide 3a: 12 a-dihydroxy-7-ketocholanique. 13 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES On a a nouveau une attaque de la position 7 sans doute suivant le meca- nisme (OH) yOH >CHOH y >C' > >C > >C=0 + H20 \0H ^OH Les memes auteurs ont entrepris une etude de I'oestrone en solution alcaline ou en solution acetique aqueuse. lis ont obtenu la formation de lactone. On obtient dans ce cas une attaque du noyau pentagonal et non pas du noyau aromatique, ce qui laisserait penser a une sensibilite plus grande du cycle pentagonal aux radicaux OH. D'autres travaux ont ete egalement entrepris sur la progesterone et la cortisone. Mais les auteurs ont rencontre de grandes difficultes quant a I'isolement des produits d'irradiation qu'il est souvent difficile d'obtenir a I'etat cristallise. L'interet de ces travaux sur la radiochimie des steroides en solution aqueuse est considerable en ce sens qu'elle nous donne un aper^u des phenomenes qui peuvent se passer in vivo au niveau de ces molecules. II faut egalement observer que les produits obtenus par irradiation sont souvent analogues a ceux obtenus par oxydation biologique in vivo et ceci ouvre peut etre un champ de recherches interessant. Action des Radiations Ionisantes sur des Graisses Complexes. Nous n'avons rapporte jusqu'ici que les travaux qui avaient ete effectues sur des substances pures dans des conditions bien definies. D'autres travaux ont ete effectues sur des milieux naturels beaucoup plus complexes, generale- ment dans le but d'etudier le probleme de la sterilisation des aliments par les radiations ionisantes. Hannan avec Boag^" et Shepherd-^- ^^, avaien etudie le comportemeut du beurre apres administration de 2.000.000 de rep sous forme d'electrons pro- duits par un generateur de van der Graaff. lis ont tout d'abord observe une destruction rapide de la vitamine A et du carotene, observation qui avait deja ete faite par Faila et collaboratem-s, et plus tard par Chalmers et al.^ lis ont constate egalement la destruction des anti-oxygenes presents dans le beurre, essentiellement representes par le tocopherol, destruction qui se traduit par la disparition du temps de latence de la graisse au cours de son auto-oxydation spontanee a I'air. Ces auteurs ont egalement observe un fait tres curieux. lis ont procede, en presence d'air, a I'irradiation d'echantillons de beurre a — 70° C, puis ils ont conserve ces differents echantillons a des temperatures diverses allant de — 70 a +20° C, et ont determine la variation du taux de peroxydes en fonction du temps dans chaque echantillon. lis ont constate que la forma- tion ulterieure de ces peroxydes est beaucoup plus importante pendant la conservation aux environs de — 20° C qu'aux autres temperatures. D'autre part, si I'irradiation est effectuee en I'absence d'oxygene et la conservation a diverses temperatures dans les memes conditions, la mise en contact avec I'air des divers echantillons montre la formation beaucoup plus rapide des peroxydes pour I'echantillon conserve a — 20° C. De plus, un 14 ANDRE CHEVALLIER ET CONSTANT BURG sejour de 15 minutes a +20° C suffit pour faire disparaitre le phenomene. Les auteurs attribuent le phenomene a la formation a — 70°G d'un radical relativement stable a — 20°C qui se detruirait a des temperatures plus elevees et dont la formation ne necessiterait pas la presence d'oxygene. II est interessant de noter que les produits dont la vitesse de formation, apres irradiation, est plus grande a — 20°C qu'aux autres temperatures, donnent les reactions des peroxydes, c'est-a-dire oxydent KI en Ig et Fe^+ en Fe^+, mais reagissent egalement avec le reactif de Schibsed qui est un test de Schiff modifie et dont on se sert en general pour deceler la presence d'aldehyde de poids moleculaire eleve. Des travaux ont ete egalement effectues sur le lait et Kung et coUabor- ateurs^o pj-^t constate que le carotene et la vitamine A contenues dans ce liquide etaient detruits par le rayonnement y. line dose de lO^'r detruisant 85 pour cent de la vitamine A et 45 pour cent du carotene. II est a noter que la sensibilite de ces deux substances au rayonnement est beaucoup plus faible qu'a I'etat pur en solution dans I'hexane, par exemple. La vitamine A et le carotene en solution dans I'hexane, sont detruits par le rayonnement avec un rendement ionique voisin de I'unite^. Dans le serum humain le rendement ionique tombe a 0,01. La vitamine A et le carotene semblent etre proteges contre le rayonnement, aussi bien dans le sang que dans le lait et ceci s'explique assez bien a la lumiere des travaux de Pollster et Mead^^. action des radiations ionisantes sur les lipides DE l'oRGANISME ' IN VIVO ' Lorsqu'on soumet un organisme vivant a Taction des radiations ionisantes, on releve toute une serie de modifications portant sur les lipides des difTerents organes. Certaines de ces modifications semblent etre dues a une action directe du rayonnement sur les molecules lipidiques, action par consequent voisine de celles qui peuvent etre obtenues in vitro. C'est ainsi que Ton pent considerer I'apparition de substances a caracteres peroxydiques dans le tissu adipeux. D'autres modifications, au contraire, semblent relever d'une action indirecte, c'est-a-dire etre la consequence de perturbations provoquees dans d'autres systemes, sans doute en phase hydrbsoluble. Nous etudierons successivement ces deux aspects de Taction du rayonnement X sur les lipides de Torganisme. Action des Radiations Ionisantes sur les Lipides du Tissu Adipeux apres Irradiation d'un Animal Vivant. Dubouloz et a/^^ ont mis en evidence la presence de peroxydes dans un extrait acetonique de peau de rats, apres avoir administre des doses variables de rayons X allant de 3.500 r a 20.000 r. Les taux de peroxydes obtenus etant de Tordre de 1 0 ^ « molecule par centimetre cane de peau. Une partie de ces substances donnant des reactions a caractere peroxydique est soluble dans Tether de petrole, ce qui met en evidence leur caractere lipidique. Les auteurs ont egalement fait une observation curieuse : poiu^ des doses de 3.500 r et de 5.000 r le taux de peroxydes doses immediatement apres Tirradiation augmente ensuite progressivement pour atteindre un maximum vers le septieme jour. La 15 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES methode de dosage utilisee etait une methode basee sur 1 'utilisation de la thiofluoresceine mise au point par Dubouloz. Cette methode, extremement sensible, permet de doser 0,5 X 10^^ molecule de peroxydes. Bacq_^^ a/^^, en utilisant la methode de Hartman et Glavind, ont recherche des peroxydes dans des extraits au xylene de la graisse peritesticulaire de souris et de rat. Cette recherche a ete effectuee 30 minutes apres adminis- tration a I'animal de l.OOOr sous 80 kV. Ces auteurs ont releve des titres en peroxydes de I'ordre de 0,3 X 10~^ mole par gramme de graisse. Mais comme les resultats obtenus sur les animaux temoins indiquent des valeurs negatives, il faut etre assez prudent dans 1 'interpretation de ces resultats. HoRGAN et Philpot^^' ^* ont egalement entrepris de nombreux travaux sur le dosage des peroxydes organiques dans I'extrait butanolique de souris ayant re^u des doses de rayons X de 950 r. lis ont utilise tout d'abord une methode de Glavind et Hartman modifiee qui leur a permis de deceler des quantites de peroxydes correspondant a un rendement ionique de 70, mais la vitesse de reaction etant differente de celle observee avec les hydroper- oxydes habituels. Les auteurs ont alors mis au point une nouvelle methode de dosage de peroxydes faisant intervenir comme reactif le chlorure stanneux en dosant I'exces de chlorure stanneux, n'ayant pas reagi, par reduction soit du 2 : 6-dichloroindophenol, soit du bleu de phenol. Dans ces con- ditions les auteurs ont obtenu des peroxydes avec un rendement ionique voisin de celui obtenu precedemment. Pour 950 r ils ont releve un taux de peroxydes de 2,18 x 10~' mole par gramme de souris, soit vm rendement ionique de 84. Le probleme est cependant plus complique qu'il n'apparait a premiere vue et Philpot vous entretiendra lui-meme des travaux qu'il a entrepris entre autres sur la nature de ces peroxydes. On pent etre surpris d'observer un taux de peroxydes aussi faible dans les graisses du tissu adipeux apres irradiation. Cependant Chevallier et Burg^^ ont constate qu'il existait dans les graisses de reserve des substances qui freinaient la formation des peroxydes au cours de I'irradiation. En effet si Ton precede a I'irradiation, soit de fragment de tissu adipeux, soit d'extrait ethere, on ne releve, apres administration de rayons X a dose de 1.500r que des taux de peroxydes negligeables. Par contre, si on fait passer au prealable la solution etheree sur une colonne d'alumine d'activite convenablement choisie, I'irradiation de la solution lipidique entraine la formation d'une quantite considerable de peroxydes. Cette experience montre bien I'existence, dans le tissu adipeux, de substances inhibant la formation de peroxydes, substances qui sont restees sur la colonne d'alumine et qui sont sans doute du tocopherols. Modifications des Substances Lipidi^ues de Divers Tissus apres Irradiation d'Animaux Vivants. Celles-ci correspondent en principe aux effets, soit quantitatifs, soit qualitatifs constates au niveau des diverses sub- stances lipidiques appartenant aux organes ou aux tissus d'un animal soumis a Taction des radiations ionisantes, sans que Ton puisse rapporter les effets controles a une action directe des radiations. (1) Lipides totaux. Bacq et al ^- ont etudie les variations des lipides totaux de I'organisme du rat apres administration de doses lethales (l.OOOr) de rayons X a 200 kV. Pour eviter les difficultes soulevees par I'anorexie due 16 ANDRE CHEVALLIER ET CONSTANT BURG aux rayons, le travail a ete effectue sur des animaux soumis au jeune. Les graisses etant extraites suivant la methode de Kumagavva, reprises dans I'ether de petrole et pesees apres evaporation du solvant. Les graisses totales, qui s'etablissent en moyenne vers 22 grammes au depart, accusent une diminution tres marquee. Chez les temoins vers la cinquantieme heure on trouve en\iron 15 grammes, puis 10 grammes apres 80 heures, 7 grammes apres 100 heures et un chiffre voisin de 4 grammes pour des durees de jeune variant entre 120 et 1 70 heures. Bien qu'il existe des differences individuelles marquees, on obtient une courbe assez reguliere jusqu'a 120 heures, puis un palier. Chez les animaux irradies, la diminution des graisses au depart est tres accusee ; elle suit a pen pres la variation observee chez les temoins, mais elle s'attenue beaucoup plus tot, et, a partir de 70 heures jusqu'a 150 heures, elle s'etablit sur une zone en plateau avec une valeur voisine de 9 grammes. II y a done la une difference tres nette avec les animaux temoins. Cette difference pourrait s'expliquer soit par I'inhibition d"un systeme d'utilisation lipidique, soit par I'acceleration de la synthese sous I'influence du rayonne- ment. Mais il y a de plus un autre element qui apparait tres nettement et qui reside dans la tres grande irregularite du phenomene a I'echelle indi- \iduelle. La dispersion des resultats est beaucoup plus marquee que chez les temoins. Par contre, si on procede au dosage des acides gras a deux, trois et quatre doubles liaisons, on constate que leur presence ne semble pas affectee ni par le jeune, ni par le rayonnement, au moins jusqu'a la soixante- dixieme heure. La quantite totale d'acide arachidonique par animal reste constante jusqu'a la cent-trentieme heure. Elle ne commence a diminuer qu'au moment ou I'animal presente des troubles pre-mortem. Le tocopherol contenu dans le tissu adipeux ne presente guere de variations apres adminis- tration de l.OOOr. II ne semble egalement pas affecte par le jeune. (?) Resorption intestinale. On a cru pendant longtemps que la resorption des graisses par I'intestin etait modifiee par I'irradiation. Martin et RoGERS^^'^^ avaient observe une diminution de resorption chez le chien apres administration d'une dose faiblement erythemateuse. Dodds et VVebster^^ avaient egalement decrit une alteration de la resorption des graisses chez I'homme apres irradiation. Mead, Decker et Bennett^^ arrivent cependant a des conclusions tout a fait differentes. lis ont administre, a des rats, de I'oleate de methyle contenant 10 pour cent d'octadecadienoate de methyle dont les doubles liaisons, situees en 9 et 1 1, sont conjuguees et, de ce fait, facilement decelables par mesure du spectre d'absorption ultraviolet a 2.340 A. lis ont tout d'abord procede au dosage des graisses fecales de souris soumises a trois regimes differents et ayant regu des doses de rayons X de 500 r. lis n'ont releve d'augmentation de I'excretion fecale de graisse que chez les animaux soumis a un regime ne contenant pas de graisse. Les graisses fecales ne peuvent done pas etre d'origine alimentaire et doivent provenir d'une secretion intestinale, pent etre de la desquamation de la muqueuse. Ces auteurs ont egalement procede au dosage des graisses dans les differents fragments du tube digestif apres administration des lipides par une sonde stomacale. lis n'ont pas constate d'alteration de la resorption des graisses au niveau de I'intestin, mais tres souvent la resorption des lipides est retardee, 17 c ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES le bol alimentaire etant retenu beaucoup plus longtemps dans restomac par suite d'une augmentation du tonus du sphincter pylorique. Les auteurs ont egalement constate une augmentation de la motilite de I'intestin grele. CoNiGLio et aP^ ont etudie I'excretion fecale de graisses chez des rats apres irradiation, mais en compai^ant les animaux irradies a des temoins recevant la meme quantite de nourriture que celle absorbee par les animaux irradies. Dans ces conditions ces auteurs n'ont pas retrouve de difference dans I'excretion de graisse fecale entre les animaux temoins et les animaux irradies et ceci pendant 28 jours pour des doses de rayons X de 500 et 650 r. Bennett et al'^^ ont examine la resorption de la vitamine A alcool au niveau de I'intestin du rat. lis ont trouve cette absorption normale, sinon un peu acceleree, apres administration de dose de rayons X de I'ordre de 625 r. Si I'irradiation ne modifie pas la resorption des lipides, il semble cependant que Ton puisse relever un certain nombre d'alterations bio- chimiques au niveau de I'intestin. CoNiGLio et Hudson*^ ont etudie incorporation d'acetate marque au Cj^^ dans les lipides de la tunique intestinale. lis ont administre 650 et 750 r en champ general a des rats et 22 heures plus tard ils ont injecte par voie intraperitoneale de I'acetate marque dans la fonction carboxyle. Les animaux ont ete sacrifies apres 20, 40 et 60 minutes apres I'injection. Les auteurs ont observe une diminution de I'incorporation de I'acetate marque dans les lipides de I'intestin. Dans toute cette experience les temoins ont re9u la meme quantite de nourriture que celle absorbee par les animaux irradies. Hevesy et Dreyfus'*-, avaient egalement releve des modifications dans I'incorporation d'acetate marque dans les lipides de I'intestin, mais sans attribuer a leurs resultats une valeur significative. CoNARD*^ a etudie la choline-esterase de I'intestin de rats soumis a des doses de rayons X de 500 r. II a constate une diminution importante de I'activite enzymatique 15 a 20 heures apres I'irradiation. Le phenomene est maximum le quatrieme jour ou la choline-esterase voit son activite diminuee de 60 pour cent. Elle retrouve des valeuis normales vers le vingtieme jour. II semble done que la radiosensibilite bien connue de I'intestin s'accompagne de troubles affectant la motilite et de certains metabolismes biochimiques sans que cependant la resorption des lipides soit nettement afi'ectee. L'administration de doses elevees de rayons X a des animaux entraine cependant une anorexic importante et Smith et al^^ se sont demandes si cette anorexic ne pouvait etre responsable de la mort des animaux irradies. Dans ce but les auteurs ont expose a des doses lethales de rayons X des souris ordinaires et des souris rendues obeses, c'est-a-dire ayant des reserves lipidiques importantes. lis ont trouve la meme courbe de variations de poids et la meme courbe de mortalite apres irradiation dans les deux lots d'animaux, la totalite des souris irradiees, obeses ou non, etant soumise au jeune pendant toute la duree de I'experience. Les auteurs en concluent que I'anorexie n'est pas responsable de la mort des animaux irradies. (3) Constituants lipidiques du serum. L'administration de radiations ionisantes entraine des modifications de toute une serie de constituants lipidiques du serum, modifications qui portent d'une part, sur certaines lipoproteines du 18 ANDRE CHEVALLIER ET CONSTANT BURG sang, et d'autre part, siir le cholesterol, les vitamines liposoliibles et I'activite de la lipase sanguine. [a) Modifications du lau.x des lipides el phospholipides lotaux du serum. Stead- MAN*^" a etudie les modifications des lipides sanguins du lapin sous I'influence du ravonnement ; il a constate une augmentation considerable des lipides totaux du serum, une augmentation plus faible des phospholipides. Kohn et al^^^ ont observe egalement chez le rat une elevation du taux des lipides, elevation cjui etait grossierement parallelea la variation du rapport albumine- globuline. L'auteur n'a pas releve de modifications du taux des phospho- lipides apres irradiation. Buchanan et al^^ ont etudie les phospholipides plasmatiques de la rate femelle apres irradiation par des rayons X pour des doses de I'ordre de 500 r. Les auteurs n'ont pas trouve de modifications de la choline des phospholipides. Par contre 48 heures apres une irradiation comprise entre 500 et 2.000 r. Buchanan note une elevation de la choline et du phosphore lipidique plasmatique, ce qui confirme les resultats ante- rieurs deja observes par Entenman et Neve*^ qui avaient observe une aug- mentation du taux de phosphore lipidique pour les doses superieures a 500 r. [b) Modifications des Jipoproteines sanguines. Rosenthal'*^ a constate qii'apres administration d'une dose lethale de rayons X a des lapins, il observait une opalescence du serum, opalescence qui apparait 24 heures apres I'exposition aux radiations pour disparaitre completement apres 72 heures. Cette opalescence presentait le caractere particulier d'etre supprimee par agitation du serum avec I'ether. II s'agissait done d'une substance liposoluble. L'auteur a egalement pu etablir qu'il n'y avait pas de relation entre cette opalescence et la destruction, soit de globules blancs, soit de globules rouges, destruction consecutive a I'irradiation. La meme annee, Muntz et al'^^ ont etudie par electrophorese les modifications des differentes proteines sanguines apres irradiation chez le chien. Trois jours avant la mort des animaux ils ont constate une elevation des globulines a 3, mais cette eleva- tion des globulines a 3 ne parait pas liee a une modification du taux des lipides, mais probablement a un etat infectieux. GjESSiNG et Chanutin^'* ont etudie les modifications du serum sanguin des rats apres irradiation totale en administrant des doses variant de 500 a 2.000 r. lis ont procede au fi-actionnement du plasma par fractionnement alcoolique et ils ont recueilli cinq fi:-actions diflferentes. Ghaque fiaction a ete etudiee par electrophorese et les auteurs ont proced^au dosage des lipides contenus dans chacune des fractions. lis ont observe des modifications importantes dans le taux des lipides de la fi-action 4 ; apres le deuxieme, troisieme, quatrieme et cinquieme joiu', une augmentation qui, pen a peu, revient a la normale. Kohn'^'^ a examine les modifications du rapport albu- mine/globuline du serum de cobaye apres administration des doses variant entre 200 et 600 r. II a constate une elevation tres importante du rapport albumine/globuline pendant les quatre premiers jours consecutifsa I'adminis- tration du rayonnement lorsque la determination des albumines et globulines est faite par precipitation saline ou par ultracentrifugation. Par contre, lorsque le taux de globulines et d'albumines est determine par electro- phorese, on ne trouve pas de modifications du rapport albumine/globuline. De meme si le plasma est extrait a I'ether juste avant la determination du taux de globulines et d'albumines, on retrouve un taux d'albumine/globuline 19 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES normal. L'adjonction in vitro a du plasma ou a du serum normal de la sub- stance extraits a I'ether eleve egalement le rapport albumine/globuline du serum de rat normal. Kohn a examine aussi I'influence de rhypophysectomie ou de I'adrenalectomie chez le rat, apres irradiation. II a retrouve la meme variation des rapports albumine/globuline chez les animaux hypophy- sectomises et irradies que chez les temoins irradies seulement. Par contre, chez les animaux adrenalectomises I'elevation du rapport albumine/globuline est plus forte que chez les temoins. L'auteur a egalement constate que I'injection d'erythrocytes de mouton bloquait Televation du taux albumine/ globuline, ce qui laisse supposer une intervention possible du systeme reticulo-endothelial. Hewitt et al^^ ont repris I'etude des lipoproteines du serum de lapin apres irradiation par ultracentrifugation. lis ont constate 12 heures apres I'irradiation une augmentation brutale des lipoproteines de faible densite ; augmentation qui etait liee a I'apparition de I'opalescence du serum. Ces auteurs ont constate egalement que I'administration d'heparine fait dis- paraitre tres rapidement les lipoproteines de faible densite, en les trans- formant en lipoproteines de densite plus elevee. L'injection de bleu de toluidine, de sulfate de protamine ou de quinidine, produit des modifications des lipoproteines identiques a celles de I'irradiation. En consequence, les auteurs supposent que I'apparition de cette opalescence dans le serum liee a un taux eleve de lipoproteines de faible densite n'est pas due a une action directe sur les lipoproteines mais probablement a un manque d'heparine consecutif a Taction du rayonnement. (c) Modifications du cholesterol. Steadman^^"^ avail constate chez le lapin une elevation considerable du cholesterol lipidique apres irradiation par les rayons X. Kohn et al'^^^ a fait la meme observation chez le rat et le cobaye et il a observe les memes variations du cholesterol plasmatique apres irradiation d'animaux adrenalectomises. Par contre, apres hypophysectomie, I'elevation du taux de cholesterol n'est pas supprimee, mais legerement modifiee. Get auteur a constate egalement que I'administration de dibena- mine, de 2-dibenzyl-aminoethanol, d'extrait lipidique corticosurrenal et de desoxycorticosterone, supprimait I'elevation du taux de cholesterol chez le rat apres irradiation. Gjessing et Chanutin^*' apres fractionnement alcoo- lique des proteines du plasma, avait constate que I'elevation du taux de cholesterol se retrouvait surtout dans la fraction 4. Roth et al'^^ ont etudie la lipase serique chez le rat apres administration de 600 r. lis ont observe une diminution nette de I'activite enzymatique du troisieme au cinquieme jour apres I'irradiation suivie d'un retour a la normale le huitieme jour. La meme observation a ete faite par del Buono^* sur la tribvityrinase du lapin. {4) Lipides hepatiques. Le foie est generalement considere comme un organe radio-resistant. Cependant toute une serie de modifications biochimiques consecutives a I'irradiation indiquent des perturbations du metabolisme lipidique dont il est le siege. Forssberg et Hevesy^^ ont constate une modification importante de la fixation de phosphate marque dans le foie de jeunes souris a qui ils avaient administre 2.000 r, 15 minutes avant l'injec- tion. Entenman et Weinman^^ ont etudie la fixation de ^^P dans les phos- pholipides du foie de rats irradies avec des doses variant de 1.000 a 2.500 r 20 ANDRE CHEVALLIER ET CONSTANT BURG et ont egalement note une modification importante de cette fixation apres irradiation. Hevesy et Dreyfus*^ ont injecte de I'acetate, marque au C14 dans le groupe carboxyle, a des souris auxquelles ils avaient administre une dose de 880 r. Ces auteurs ont constate une modification importante de I'incorporation de I'acetate marque aux lipides hepatiques sans accorder a leurs resultats une valeur statistiquement significative. Coniglio et Hud- son"*^ ont etudie le meme probleme en 1944 chez le rat. Dubois et al^'' ont constate egalement chez le rat, apres administration d'une dose de 800 r, une perturbation importante du metabolisme de I'acide citrique au niveau du foie. Enfin, un certain nombre d'auteurs ont etudie le comportement de la choline-oxydase apres irradiation. Tous ces faits semblent bien indiquer que I'irradiation entraine une per- turbation du metabolisme lipidique et il faut s'attendre a observer des modifications dans la constitution lipidique de cet organe. Cependant apres administration de doses luiiques de rayons X on ne releve generalement aucune steatose hepatique ou tout au plus des signes de steatose extremement discrets. Pohle et Bunting^^ ont administre a des souris, des doses de rayons X variant entre 600 et 2.500 r sans relever des signes histologiques des lesions hepatiques autre qu'un oedeme cellulaire sporadique. Ariel^^ a repris I'etude des lesions hepatiques dues a I'administration des doses uniques, cette fois chez le lapin, Les rayons ont ete administres sur un champ hepatique et les doses ont ete 300, 3.000, 30.000, 50.000 et 100.000 r, doses qui ont ete administrees en vine seule fois. L'auteur a note des signes histologiques d'oedeme, d'infiltration leucocytaire et de necrose, lesions variables suivant les doses administrees, mais il n'a jamais releve de steatose. Chevallier et Burg^^ ont administre a des rats des doses lethales uniques de 800 et de 3.000 r sans relever de lesions differentes de celles observees sur le lapin^^ ou sur la souris^^. Entenman et Neve^^ ont administre egalement a des rats, en champ general, des doses de 2.500 r et ils n'ont pas releve de modifications de la concentration en lipides totaux, phospholipides ou lipides non phospholipidiques. Coniglio et aP^ ont administre des doses de 500 r a des rats et ils ont examine le taux des difTerents lipides hepatiques 28 jours plus tard. lis ont constate une elevation nette du taux de triglycerides alors que le taux de phospholipides etait normal. Pensant que cette elevation du taux de lipides hepatiques pouvait etre due au jeune consecutif a I'anorexie que presentent ces animaux, l'auteur a repris ses experiences en comparant le taux des lipides hepatiques des animaux irradies a celui d'animaux temoins recevant la meme quantite de nourriture que les animaux irradies. Dans ces conditions l'auteur a constate qu'il n'y avait pas de modifications des lipides hepatiques consecutives a I'irradiation, mais que les modifications entrevues etaient dues au jeune. Cependant Ord et Stocken^*', apres administration de I.OOOr a des cobayes, ont releve des signes discrets de steatose hepatique. L'aspect morphologique des lesions hepatiques, apres irradiation, est tres different lorsque la meme quantite de rayons X est administree en une serie de petites doses quotidiennes au lieu d'etre concentree en une dose massive. Ludin^^ a entrepris des irradiations systematiques localisees a la region hepatique chez le lapin et chez le cobaye. II a releve une steatose hepatique 21 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES nette chez la plupart des animaux qui recevaient des doses quotidiennes faibles et qui, de ce fait, survecurent pendant longtemps, respectivement 21, 24 et 39 semaines. Doub et al^- reprirent les experiences de Ludin^^, mais cette fois sur le chien. Dans ces conditions ils ol^servaient, tout comme Ludin, une steatose hepatique nette. Chevallier et al^^ en administrant, en champ general, a des rats des doses quotidiennes de 50 r sous 200 kV, ont pu constater avec regularite 200 - - 180 - - 160 - - ^ no — - k^120 t -^100 ^ so to - - 60 - i - W - i i - 20 0 1 i 1 1 - LotJ Lofn Lot is: Ic/JZ LotSI Figure 7. Taux de glycerides hepatiques en Jhnction de la dose quotidienne de rayons X administree I. Lot de 10 rats normaux provenant de Velevage 11. Lot de 6 rats ayant refu quotidiennement de V aureomycine pendant une f/eriode allant de 80 a 90 jours, sans administration de rayons X IV. Lot de 15 rats recevant de r aureomycine et une dose quotidienne de 200 r V. Lot de 9 rats recevant de l^ aureomycine et une dose quotidienne de 100 r VI. Lot de 21 rats recevant de l^ aureomycine et une dose quotidienne de rayons de 50 r. des Steatoses hepatiques extremement nettes, alors qu'avec des doses quoti- diennes de lOOr I'accumulation de lipides dans le foie est moins nette etqu'elle devient insignifiante lorsque la dose quotidienne arrive a 500 r {Figure 7). Un certain nombre de travaux ont ete consacres egalement a I'etude de la vitamine A hepatique apres irradiation. Bennett et al^^ ont administre a des rates femelles des doses de 625 r en champ general et ils ont dose la vitamine A hepatique pendant la semaine qui suivait I'irradiation. Les auteurs ont procede de la fa^on suivante : ils ont administre une close unique de vitamine A et ils ont tue les animaux soit 6 heures apres I'administration de la vitamine, soit 10 heures apres. lis ont constate une diminution tres 22 ANDRE CHEVALLIER ET CONSTANT BURG importante du taux de vitamine A hepatique chez les animaux irradies. lis ont egalement observe que 6 heures apres radministration de la vitamine, on trouve dans la carcasse un taux de vitamine A beaucoup plus eleve que chez les temoins. Mais 10 heures apres I'administration de vitamine, la carcasse montre un taux de vitamine qui est redevenu normal, comme si la vitamine A deposee dans la peripherie etait rapidement metabolisee. Coniglio^^ a dose la vitamine A du foie de rat ayant re9u une dose de rayons X variant entre 500 et 650 r et il a compare les resultats obtenus au taux de vitamine A de rats temoins ayant regu la meme quantite de nour- riture que celle absorbee par les animaux irradies. Dans ce cas, I'auteur n'a pas observe de difference entre les taux de vitamine A hepatique des animaux irradies et des temoins. Roth ('/ al'^^ ont recherche, I'activite de toute ime serie d'enzymes apres irradiation. Entre autres, ils n'ont pas i-eleve de modification, ni de la choline-oxydase, ni de la lipase hepatique. (5) Lipides du cerveau. Un certain nombre d'auteurs ont etudie egalement Taction du rayonnement X sur les lipides du cerveau. Warren et al^^ considerent qu'a dose therapeutique les rayons X ne produisent pas de modifications histologique, du moins chez I'adulte. Florsheim et al^'° ont irradie a des doses de 500 et 800 r des souris adultes et n'ont pas trouve de modification de la fixation de phosphate marque au niveau du cerveau. Des doses de 1 9.000 r egalement n'ont pas revele d'alteration du metabolisme du phosphore. Hevesy et Dreyfiis'*- ont con- state une augmentation statistiquement significative de I'incorporation d'acetate marque dans les lipides du cerveau de souris ayant regu une dose de 880 r. Sch\vartz et al^^ ont etudie par spectrographie infi-a-rouge les differentes Abactions de cerveau de foetus de rat et de rats adultes. 24 heures apres administration de 1 5.000 r en champ general, les rats adultes montrent un abaissement statistiquement significatif de la fi-action lipidique ne pre- sentant pas de fonction amide du cerveau moyen et du cerveau anterieur. L'irradiation de femelles gestantes a doses de 150r cause des modifications analogues dans differentes parties du cerveau des animaux examines a I'etat adulte. Par examen spectroscopique ces auteurs ont pu etablir que ces modifications sont dues a une diminution des phospholipides et du cholesterol (Schwartz, communication personnelle). II faut enfin noter un travail effectue par Altman et al^~ sur la synthese d'acides gras par la moelle osseuse a I'aide d'acetate marque chez le lapin. Immediatement apres l'irradiation, ces auteurs constatent une elevation considerable de la synthese, aussi bien des acides satures, que desatures. Par contre apres 48 heures, si la synthese des acides satvu'es est redevenue normale, la synthese des acides desatvu-es est tombee de 18 pour cent en- dessous de la normale. 158 heures apres, au contraire, la synthese d'acides desatures est de 283 pour cent superieure a la normale, alors que la synthese des acides satures est tombee a 58 pour cent en-dessous. REFERENCES 1 Gray, L. H. Brit. J. Radiol. y.S. 1947, Suppl. 1, 7 ; 1953, 26 609. Gray, L. H.. Conger, A. D., Ebert, M., Hornsey, S. et Scott, O. C. Brit. J. Radiol. N.S. 1953, 26 638. 23 ACTION DES RADIATIONS lONISANTES SUR LES LIPIDES 2 CoRMACK, D. V. et Johns, H. E. Brit. J. Radiol. JV.S. 1952, 25 369. 3 Franck, J. et Sponer, H. Contribution a I'etude de la structure moleculaire, p. 169. Liege, 1948. 4Fano, V. Syinp. Radiobiol., Oberlin College, (June) 1950, 13. New York, Wiley. 5 Platzman, R. L. Svmp. Radiobiol., Oberlin College, (June) 1950, 97. New York, Wiley. ^ Livingstone, R. S. Trans. 5th Conf. J. Macej Jr Fdn : Biol. Anti-oxidants, p. 17. New York, 1950. ' Wallenstein, M., Wahrhaftig, a. L., Rosenstock, H. et Eyring, H. Symp. Radiobiol., Oberlin College, (June) 1950, 70. New York, Wiley. 8 Sheppard, C. W. et Burton, V. C. J. Amer. chem. Sac. 1946, 68 1636. " Chalmers, T. A., Goodwin, T. W. et Morton, R. A. Nature, Land. 1945, 53 513. 10 Hannan, R. S. et Boag, J. W. Nature, Land. 1952, 169 152. " Chevallier, a. et Burg, C. Ann. Nutr., Paris, 1953, 7 C81. 12 Hartmann, S. et Glavind, J. Acta chem. scand. 1949, 3 954. 13 Mead, J. F. Scieiice, 1952, 115 470. 14 Bolland, J. L. et Ten Haven, P. Trans. Faraday Soc. 1947, 43 201. 15 Proctor, B. E. et Goldblith, S. A. Nucleonics, 1949, 5 56. Proctor, B. E. et Goldblith, S. A. Advanc. Food Res. 1951, 3 119. i« Dale, W. M. Brit. J. Radiol. N.S. 1947, Suppl. 1, 46 ; 1951, 24 433. Dale, W. M. Symp. Radiobiol., Oberlin College, (June) 1950, 177. New York, Wiley. 17 Weiss, J. Nature, Land. 1944, 153 748. 18 Lea, D. E. Brit. J. Radiol. N.S. 1947, Suppl. 1, 59. 13 Stein, G. Disc. Faraday Soc. 1952, 12 227. 20 Allen, A. O. J. phys. Colloid Chem. 1948, 52 479. 21 Bonet-Maury, p. Brit. J. Radiol. N.S. 1951, 24 422. 22 Loiseleur, J., L.\TARjET, R. et Caillot, T. C.R. Acad. Sci., Paris, 1941, 213 370. 23 Loiseleur, J. C.R. Acad. Sci., Paris, 1942, 214 76. 2" PoLisTER, B. H. et Mead, J. F. J. agric.food Chem. 1954, 2 199. 25 Keller, H. et Weiss, J. J. Chem. Soc. 1950 2709 ; 1951 25, 1247. 26 Chevallier, A. et Dubouloz, P. Bull. Soc. Chim. biol, Paris, 1936, 18 No. 4, 704. 27 Weiss, J. Ciba Colloq. Endocrin. 1953, 7 142. 28 Hannan, R. S. et Shepherd, H. J. Nature, Lond. 1952, 170 1021. 29 Hannan, R. S. et Shepherd, H. J. Brit. J. Radiol. N.S. 1954, 27 36. 30 KuNG, H., Gaden, E. L. et King, C. G. J. agric.food Chem. 1953, 1 142. 31 Dubouloz, P., Dumas, J. et Vigne, J. C.R. Soc. biol., Paris, 1950, 144 1080. 32 BAcq, Z., Burg, C, Chevallier, A. et Heusghem, C. J. Physiol. Path. gen. 1951, 43 640. 33 Horgan, V. J. et Philpot, J. St L. Trans. Faraday Soc. 1953, 49 Part 3. 34 Horgan, V. J. et Philpot, J. St L. Brit. J. Radiol. N.S. 1954, 27 63. 35 Martin, C. L. et Rogers, F. T. Amer. J. Roentgenol. 1923, 10 11. 3« Martin, C. L. et Rogers, F. T. Amer. J. Roentgenol. 1924, 11 280. 37 DoDDS, E. C. et Webster, J. H. Lancet, 1924, i 533. 38 Mead, J. F., Decker, A. B. et Bennett, L. R. J. Nutr. 1951, 43 485. Cf. Mead, J. F., Bennett, L. R., Decker, A. B. et Schoenberg, M. D. J. Nutr. 1951,43 477. 33 Coniglio, J. G., Darby, W. J., Wilkerson, M. C, Stewart, R., Stockell, A. et Hudson, G. W. Amer. J. Physiol. 1953, 172 86. 40 Bennett, L. R., Bennett, V. C, Shaver, A. et Grachus, Th. Proc. Soc. exp. Biol, NT. 1950, 74 439. 41 Coniglio, J. G. et Hudson, G. W. Fed. Proc. 1944, 5 13, 578. 42 Hevesy, G. et Dreyfus, G. Ark. Kemi, 1952, 4 337. 43 Conard, R. a. Radiology, 1953, 61 No. 2, 312. 44 Smith, W. W., Chapman, W. H. et Alderman, L M. Amer. J. Physiol. 1 952, 169 5 1 1 . 24 DISCUSSION ^^aSTEADMAN, L. T. Qiiart. tech. Rep. Univ. Rochester, 1949. *5^ KoHN, H. I., SwiNGLEY, N., RoBERTsox, W., KiRSLis, M., Ledford, E., Cltck, G. et Lane, J. Amer. J. Physiol. 1951, 165 27. *'' Buchanan, D. J., Darby, W. J., Bridgforth. E. B., Hudson, G. VV. et Efner, J. A. Anwr. J. Physiol. 1953, 174 336. 4" Entenm.\n, C. et Neve, R. U.S.X.R.D.L. Rep. No. AD-307{B), 1951. 48 Rosenthal, R. L. Science, 1949, 110 44. 49 Muntz, J. A., Barron, E. S. et Prosser, C. L. Arch. Biochem. {Biophys.) 1949, 23 434. 50 GjESSiNG, E. C. et Chanutix, A. Arch. Biochem. {Biophys.) 1950, 27 No. 1, 191 ; see also : J. bid. Chem. 1947, 169 657. " Kohn, H. I. Amer. J. Physiol. 1950, 162 703. 52 He\\itt, J. E., Hayes, T. L., Gofman, J. W., Jones, H. B. et Pierce, F. T. Amer. J. Physiol. 1953, 172 579. 53 Roth, J. S., Eichel, H. J., Wase, A., Alper, C. et Boyd, M. J. Arch. Biochem. {Biophys.) 1953,44 95. 54 DEL BuoNO, G. Boll. Soc. ital. Biol. sper. 1951, 27 290. 55 FoRSSBERG, A. ct Hevesy, G. Ark. Kemi, 1952, 5 93. 5« Entenman, C. et Weinman, E. O. Fed. Proc. 1952, 11 44. 57 Dubois, K. P., Cochr.a.n, K. W. et Doull, J. Proc. Soc. exp. Biol.,N.T. 1951,76 422. 58 Pohle, E. et Bunting, C. Acta radioL, Stockh. 1932, 13 117. 59 Ariel, I . M. Radiology, 1 95 1 , 57 56 1 . «" Ord, M. G. et Stocken, L. A. Physiol. Rev. 1953, 33 No. 3, 356. " LuDiN, M. Strahlenther, 1925, 19 138. «2 DouB, M., Bollinger, A., et Hartman, F. Radiology, 1927, 8 142. 63 Chevallier, a., Burg, C. et Spehler, H. C.R. Soc. bioL, Paris, 1953, 147 497. «4 Warren, S., McMillan, J. C. et Dixon, F. J. Radiology, 1950, 55 375, 557. 65 Florsheim, W., Doernbach, C. et Morton, M. E. Proc. Soc. exp. Biol., N.T. 1952,81 121. 66 Schwartz, H. P., Riggs, H. E., Glick, C, McGrath. J., Cameron, W., Beyer, E., Bew, E. et Childs, R. Proc. Soc. esp. Biol., N.T. 1952, 80 467. 6' Altman, K. I., Richmond, J. E. et Salomon, K. Biochim. biophys. Acta, 195 1 , 7 460. DISCUSSION Z. M. BAcq : La teneur a-tocopherol des lipides naturel suffit-elle a assurer une protection maximale des lipides centre roxydation par les radiations ionisantes ? Je pose cette question parce que plusieurs auteurs, dont Herve et nous-meme, ont observe que I'injection d'a-tocopherol n'augmente pas la radioresistance. Peut-etre serait-il preferable pour connaitre le role de I'a-tocopherol, de voir si les animaux carences en cette vitamine sont plus radiosensibles ? H. A. Chevallier : II est certain que Ton est frappe par le fait que les hydro- peroxydes qui ont pu etre mis en evidence apres irradiation des lipides chez un animal vivant ne correspondent qu'a des quantites extremement petites, a la limite de la sensibilite des methodes de mesure, malgre des doses importantes de rayons administrees a I'animal. II semble logique de penser que leur production est entravee par la presence de substances anti-oxygenes dans les milieux vivants, ces substances agissant la comme in vitro et la prolongation de I'irradiation n'aboutissant pent etre qu'a la destruction des peroxydes eux-memes par le rayonnement. II serait sans doute interessant d'examiner si certaines actions biologiques classique- ment connues consecutives a Taction des radiations ionisantes sur le vivant apparais- sent de la meme maniere chez des animaux soumis au regime producteur de la diathese exsudative, celui de Dam, par exemple. 25 THE NATURE OF THE PEROXIDE-LIKE SUBSTANCES FORMED IN MICE BY X-RAYS V. J. HoRGAN and J. St. L. Philpot Medical Research Council Radiobiological Research Unit, Harwell In our last publication ^ we gave evidence that a lethal dose of X-rays in a mouse caused an immediate increase in peroxide-like material (called 'peroxide' for short) with mean ionic yield about 80, suggesting that the well-known chain reaction of autoxidation could be initiated by X-rays in vivo. We noted, however, that the peroxide was not as reactive as expected in that the cuprous-catalysed reaction with aerobic leucoindo- phenol was far from complete in 10 minutes at room temperature. The necessity for air as well as peroxide in the reaction with leucoindophenol complicated the interpretation and we looked for an anaerobic reagent. Stannous chloride reacted anaerobically but required heating even with ordinary 'reactive' peroxides (hydroperoxides) of the expected sort, so was useless for distinguishing 'reactive' peroxides from the 'unreactive' ones apparently present in the mouse. Leuco-brilliant cresyl blue, catalysed by alkaline cuproiodide, was mentioned as a promising new reagent but was awaiting a proper test. EXPERIMENTAL Mice, irradiated or not with lOOOr of 250 kV X-rays, were killed and extracted with 100 ml of peroxide-free n-butanol in a Waring blender or top-drive macerator, sometimes in an atmosphere of nitrogen. After filter- ing 2ml were diluted to 9 ml and deoxygenated by disdllation of the solvent ^ or better by 3 minutes' bubbling with nitrogen in a vessel that had been heated at 100° in vacuo to remove absorbed oxygen. The peroxide reagent was then added, consisting of 1 ml of a 0-002M solution of buffered leuco- brilliant cresyl blue in «-butanol. The leuco-dye was made by running the dye through a column of copper or cadmium powder, with lithium iodide or other complexing agent in the soludon to combine with the metal ions. With the copper column an amount of cuproiodide catalyst equivalent to the dye was automatically introduced in this way. After 10 minutes at room temperature or 100° C the reaction mixture was introduced anaero- bically into the cell of a Hilger Uvispek Spectrophotometer and measured at 600 m[j. where the molar extinction coefficient of brilliant cresyl blue was found to be 18,800. RESULTS AND DISCUSSION The present communication is even more of an interim report than the last, and condnual changes in technique make it even harder to quote satisfactory experiments, but the following generalizations will give some idea of the trend of the research. 26 V. J. HORGAN AND J. ST. L. PHILPOT Anaerobic alkaline leuco-brilliant cresyl blue reacts significantly more with extracts of irradiated than of unirradiated mice ; but with cuproiodide catalyst there is little evidence of unreactive peroxide since the reaction is almost complete in 10 minutes at room temperature. Experiments with brilliant cresyl blue using less or no cuprous catalyst show the presence of unreactive peroxide which is increased by irradiation. If the maceration of the mouse is performed in nitrogen rather than in air the ionic yield is somewhat less. This suggests that some of the increased peroxide is formed dui-ing the maceration if air is present, as a result of either destruction of antioxidant by X-rays or production of some very easily autoxidizable material. One of our troubles with leuco-brilliant cresyl blue was that small quan- tities of peroxide turned it pink instead of blue. This was due to an impurity extractable with butyl acetate. Although the impurity is of unknown structure it has advantages over brilliant cresyl blue as a peroxide reagent, being more stable in alkaline solution and being apparently better at dis- tinguishing unreactive from reactive peroxide. We have searched for model substances with properties resembling 'unreactive peroxide', and the results are summarized in Table I, which shows the approximate percentage reaction in ten minutes at room tempera- ture and 100° C. With peroxides of unknown concentration contained in autoxidized substances the peixentage is arbitrarily based on the extent of reaction at 100° with aerobic leuco-2,6-dichlorophenol-indophenol by our modification of the Hartmann-Glavind method ^ We thought that 'unre- active peroxides' might be merely aldehydes, but these showed no reac- tivity whatever under the conditions used. All the hydroperoxides tried and also benzoyl peroxide and hydrogen peroxide were too reactive, going to completion in 10 minutes at room temperature. Diethyl peroxide was about right. In our previous report we excluded this type of peroxide because it does not react with stannous chloride even at 100°C ; but since the mouse extract contains reactive as well as unreactive peroxide its reac- tion with stannous chloride may have been due solely to the former, and the latter can still be classed with diethyl peroxide. Disubstituted peroxides like diethyl peroxide are not supposed to be formed when simple unsaturated fats like oleates or linoleates are autoxi- dized. Consistently with this Table /shows that the peroxide in autoxidized ethyl oleate is wholely of the reactive type. It is known that conjugated double bonds can give disubstituted peroxides by 1,4 addition, and con- sistently with this Table I shows some unreactive peroxide among the autoxi- dation products of isoprene and diacetyl. Neither of these substances has, however, been found in mammals and preliminary experiments with the polyconjugated natural substances carotene and vitamin A gave discourag- ing results. We have also found 'unreactive peroxide' in autoxidized morpholine and triethylamine (used as buffers in our peroxide reagents). The nature of these is not yet clear. Very recently our attention has been directed to Squalene (dihydro- hexaisoprene) by some work of Sobel and Marmorston-, who have shown that it induces the autoxidation of carcinogens. Bolland and Hughes^ have shown that squalene undergoes chain autoxidation whereby one 27 THE NATURE OF THE PEROXIDE-LIKE SUBSTANCES FORMED IN MICE BY X-RAYS Table I. — Extent of reaction of various peroxides, etc. with cadmium reduced brilliant cresyl blue Pure Substances Butyric Aldehyde Benzaldehyde Ethyl Hydroperoxide . . tert. Butyl Hydroperoxide Tetralin Hydroperoxide Cyclohexyl-l-Hydroxyl-1 -Hydroperoxide Benzoyl Peroxide Hydrogen Peroxide Diethyl Peroxide Percentage Theoretical R.T. 0 0 111 108 43 110 108 67 0 100° 0 0 111 108 43 110 108 67 54 Impure Substances {Autoxidized) Ethyl Oleate Isoprene Diacetyl Indophenol R.T. 100 25 25 100° (100)* (100)* (100)* Brilliant cresyl blue R.T. 95 2-1 24 100° 95 9-3 100 * Arbitrary standard. hydroperoxide group and one cyclic peroxide group are introduced per molecule. This would be approximately consistent with our findings in mouse extract if the surprising assumption were made that practically the whole of the X-ray induced autoxidation in the mouse involves squalene or similar substances (1,5 dienes) rather than oleic or linoleic acids (monoenes or 1,4 dienes). Squalene has been known for some time to occur in sebum and to be mainly responsible for skin peroxides, and although only traces have been found inside the mammalian body Langdon and Bloch* have proved that it is an intermediate in the synthesis of cholesterol from acetyl groups through co-enzyme A, with the rapid turnover time of about 30 minutes. This suggests that radiation may act through chain autoxidation of squalene and its near neighbours in the metabolic pathway, leading perhaps to the formation of abnormal sterols or to blocking of the formation of normal sterols, or to interference with some function of squalene itself, e.g. in cell membranes, or to direct toxicity of squalene peroxide. In any case, if the primary hypothesis is true, it might be expected that temporary blocking of squalene synthesis via acetyl CoA might diminish radiosensitivity by temporarily diminishing the steady-state concentration of squalene and its neighbours. Bacq has pointed out that cysteamine, probably the best known antiradiation drug, is a component of co-enzyme A, and seemed at one time to imply that it might act by helping to restore damaged co- enzyme A. The new squalene hypothesis suggests that cysteamine and other — SH compounds and amines may act in the opposite direction, e.g. by preventing the oxidation of lipoic acid to the disulphide form needed for transferring acetyl groups to co-enzyme A {cf. Gunsalus^). A good 28 V. J. HORGAN AND J. ST. L. PHILPOT anti-radiation effect should be shown by pantothenic acid analogues such as pantoyltauryl-p-anisidide, which according to Klein and Lipmann^ suppresses steroid and fatty acid synthesis in liver slices by blocking acetylation. • SUMMARY (7) Alkaline cuprous-catalysed leuco-brilliant cresyl blue is oxidized by ;z-butanol extracts of lethally X-irradiated mice significantly more than by control extracts, the mean ionic yield being about 80. (2) There is some evidence that part of the oxidizing material is less reactive than the y. unsaturated hydroperoxides resulting from the autoxidation of unsaturated lipides. (3) Similar behaviour is shown by autoxidized isoprene. (4) It is suggested that the precursor of the oxidizing material in mouse extracts may be squalene and its metabolic neighbours. REFERENCES 1 HoRGAN, V. J. and Philpot, J. St. L. Brit. J. Radiol. 1954, 27 63. 2 SoBEL, H. and Marmorston, J. Mature, Lond. 1954, 174 553. 3 Holland, J. L. and Hughes, H. J. Chem. Soc. 1949, 492. * Langdon, R. G. and Bloch, K. J. Biol. Chem. 1953, 200 129. ^ GuNSALUs, I. C. Symposium on Mechanisms of Enzyme Action, p. 545. Baltimore, Johns Hopkins Press, 1953. 6 Klein, H. P. and Lipmann, F. J. Biol. Chem. 1953, 203 101. 29 HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION M. Ebert Medical Research Council Experimental Radiopathology Research Unit, Hammersmith Hospital, London Aerated water is decomposed by ionizing radiation to molecular oxygen and molecular hydrogen, and hydrogen peroxide is formed. These reac- tions are of general interest in radiation biology for two reasons : {a) because they provide information on radical reactions and interactions in aqueous systems, and {b) because hydrogen peroxide is an important substance biologically. Aqueous chemical systems exposed to radiation are known to be affected by dissolved oxygen and other gases and often show a marked pH depend- ence^. In radiation biology the effect of oxygen has been widely demon- strated^- ^, but little is known about the effect of pH. The influence of these two variables on the formation and decomposition of hydrogen peroxide will be discussed in this paper. It has been shown^ that the concentration j of hydrogen peroxide formed in aerated water by a radiation dose d can be described by the function where a and h are constants {see Figure 2) which are characteristic for one set of experimental conditions, including radiation quality, dose rate, oxygen concentration and pH. This function reaches an equilibrium value for high doses, at which the hydrogen peroxide concentration is not altered by a further increase in dose. At these dose levels the forward and back reactions proceed at the same rate, i.e. formation and break-down of hydrogen peroxide balance each other. Effect of dissolved gases The initial yields of hydrogen peroxide in water quoted in the literature are very conflicting, the disagreement being much larger than can be ascribed to errors in dosimetry, or analytical procedures-^ Some of the difficulty may be due to the fact that molecular hydrogen and oxygen are formed in water in yields comparable to those of hydrogen peroxide, and both these gases react with the primary free radicals. In some experi- mental conditions the gases formed cannot escape from the bulk of the solution. The importance of this point is illustrated by an experiment in which varying amounts of hydrogen peroxide were added to neutral aerated water, which was then irradiated with 1 -2 MeV electrons at high dose rates. The total doses delivered were approximately ten times higher than those needed to produce the equilibrium concentration of hydrogen peroxide. As shown 30 M. EBERT in Figure 1, the final yield for a given dose was proportional to the amount of hvdi'ogen peroxide added before irradiation. The slope of the curve depended both on the dose rate and on the depth of the solution, being steeper for higher dose rates and deeper solutions. In the experiment illustrated, the irradiation vessels were Petri dishes 35 mm in diameter, and the solution was only 3-5 mm deep, conditions which would at first sight appear to favour the ready diffusion of gases. These results suggested that oxygen was formed by radiolysis of the hydrogen peroxide initially present, and, at the high dose rates used, could not escape, and therefore supersaturated the solution. To avoid this complication further experiments have been carried out under conditions in which the concentration of dissolved gases was controlled. The base of the irradiation vessel was a sintered glass filter through which a gas stream could be forced. This, breaking up into small bubbles, ensured rapid equilibration of the solution with the desired gas, and the removal of gases formed during the irradiation. Gas mixtures were pre- pared by passing the individual gases through 'Rotameter' flowmeters, and 200 The amount o/ HoOj J 7 X 10«rad as a ^ ^50 Figure 1 formed by function of the initial concentration o/H,02 JOO WO LL moles H2O2/ litre the concentrations of the dissolved gases were assumed to be proportional to the partial pressures in the gas phase. The above experiment was repeated with this technique. It was found that the hydrogen peroxide equilibrium concentration no longer depended on the initial concentration. It is clear that the bubbling technique removed the gaseous products of hydrogen peroxide decomposition. Oxygen and nitrogen^The influence of oxygen on the formation of hydrogen peroxide was studied by using mixtures of oxygen and nitrogen. Each curve in Figure 2 shows, the yield of hydrogen peroxide against dose for one oxygen- nitrogen mixture. No hydrogen peroxide was detected with pure nitrogen. Within experimental error the initial rates of formation of hydrogen peroxide were independent of the oxygen concentration, whereas the equilibrium values were directly proportional to the oxygen concentration. This result confirmed the interpretation of the experiment in which dilute hydrogen peroxide solutions were irradiated at high dose rates without bubbling. In these conditions, the oxygen formed by the radiolysis of hydrogen peroxide determined the equilibrium values, ^\•hich were therefore proportional to the initial hydrogen peroxide concentrations. The formation of hydrogen peroxide is usually ascribed to two distinct mechanisms. If two excited water molecules are close enough together, they combine directly to form hydrogen peroxide via reactions of the type A, a 31 HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION eoor Figure 2. Hydrogen peroxide formed in water, various gas treatments process which appears to be independent of chemical conditions. The second mechanism B leads to radical formation. Oxygen is then needed for the formation of hydrogen peroxide. A 2H2O -^ HgOg + H2 H + OH H + O2 2H02^ H + HO2 ->H02 H2O2 + O2 H,0, . (1) • (2) • (3) Reactions leading to the break-down of hydrogen peroxide involve all the radicals produced by irradiation in water : H2O2 + OH -> H2O + HO2 H+ + O2- or HO2 - HO2 H2O2 H2O2 + H + 0, -> OH + OH H2O + OH -OH + O2 H2O + O2- (4) (5) (6) (7) Some of the experimental evidence suggests that reaction (6) is more likely to occur than a direct interaction between hydrogen peroxide and the radical HOa*- ^ ^ The simplified scheme set out implies that hydrogen peroxide is formed by radical interactions only in the presence of oxygen. Any hydrogen peroxide formed by direct interaction of excited water molecules wovild, in the absence of oxygen, be decomposed via (4) and (7). With oxygen present reaction (1) will occur giving rise to hydrogen peroxide formation via (2) and (3), and the rate of reaction (7), decomposition of hydrogen peroxide, will become slower as the hydrogen radicals are removed by reactions (1) to (3). Hydrogen and oxygen — To study the effect of hydrogen on the formation of hydrogen peroxide^", hydrogen alone and mixtures of hydrogen and nitrogen, and of hydrogen and oxygen were bubbled through neutral water. No 32 M. EBERT hydrogen peroxide was detected with hydrogen alone or with mixtures of hydrogen and nitrogen, but with mixtures of hydrogen and oxygen, hydrogen peroxide yields were higher than those obtained with oxygen alone. Figure 3 summarizes these results. The three curves were obtained with 2-9 X lO^'rad, 10-5 X lO^rad and 115 X lO^rad of 1 -2 MeV electrons. Comparison of the curves shows that the lower the dose, the smaller the range of oxygen tension which influences the yield obtained. Thus at 2-9 X lO'^rad, the hydrogen peroxide yield no longer increased, i.e. the yield became constant with oxygen concentration in the oxygen-nitrogen mixtures above 12 per cent oxygen. With the highest dose, on the other hand, the yield of hydrogen peroxide increased with oxygen tension up to 100 per cent oxygen in the mixture. The same tendency is seen in the curves for hydrogen-oxygen mixtures. At the lowest dose, hydrogen peroxide E >5 I I 700 eoo 500 / -• — • ~X • / \ H2+N2 N2 + O2 / O2+H2 • \ ^ \ 300 .<-t^ / 1 • •• N, 200 100 ^^y "" • / 1 10- /»4 — •"" Sxio"^ r 3d. /• • /y\ --« ' ^ ^ — • \ <■ 2-9x10 raa. ^•-•■' - < > i i c I : \ H2 N2 % 100 75 % 0 % 0 25 50 75 0 - - 0 50 75 WO 25 75 50 50 - 0 75 100 25 0 25 75 50 50 75 25 100 0 0 Figure 3. Effect of gas treatment on hydrogen peroxide yield yields were constant over a range of 10 per cent to 90 per cent hydrogen or oxygen respectively. It seems justifiable to extrapolate these results to still lower doses, at which the dependence of the yield of hydrogen peroxide on the amount of oxygen present (or hydrogen and oxygen in the case of hydrogen-oxygen mixtures) would be critical over a very small range indeed. The hydrogen peroxide yields with hydrogen-oxygen mixtures may be accounted for by an increase in the concentration of hydrogen radicals arising from reaction (8)^^. H2 + OH -^ HgO + H .... (8) If hydrogen is present, but no oxygen, the break-down of hydrogen peroxide via reacdon (7) predominates, and the probability of reaction (4) will be reduced. If oxygen is present as well as hydrogen, it \vill remove hydrogen radicals by reactions (1) and (3), and the reaction rates of break-down pro- cesses, (4) and (7), will be accordingly decreased. Accordingly, not only the 33 D HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION initial rate of formation, but also the equilibrium values are higher if hydrogen and oxygen are used than with oxygen alone. TOO 0 ^700 o E ^ 500 too .'iOO J- y y T )W¥-8 70^ n i ^ y ^ 1-Ux K J^ y /^ 200 100 0 y y x^ ^ / r (1)) E 1 700 / X 600 1 pH 076 500 ¥00 300 ZOO 100 n-uB ^m^rztA CO 10 20 30 ¥0 50 60 70 80 SO 100 Figure 4. ii^O,^ yield for various Ng — Og mixtures at different pH Effect of pH and dissolved oxygen — The effect of pH on the formation and de- composition of hydrogen peroxide was studied under various oxygen tensions. The results of the experiments are summarized in Figure 4^-. 34 M. EBERT The interpretation of these curves is aided by reference to Figure 3. It can be seen that at doses which are very far below those needed for equiU- brium vakies it is only at low oxygen tensions that the )ield depends on oxygen tension. The curves of Figure 4 represent three stages with respect to equilibrium values of hydrogen peroxide. Curve 1, in the pH range 4-8-9-0, shows that the dose was high enough so that the hydrogen peroxide yield was at its equilibrium value, and therefore proportional to oxygen concentration. At pH 4-2 (curve 2) the hydrogen peroxide yield was not at equilibrium, but not far below. Curve 3, at pH 0-76, was of the form which indicates a yield very far below equilibrium value. The rate of formation of hydrogen peroxide at pH 0-76 was independent of the dissolved oxygen for all oxygen-niti'ogen mixtures containing more than 3 per cent oxygen. As the equilibrium values are attained only with very high doses at low pH, the back reaction was studied by the decomposition of hydrogen peroxide. 10,000 o 2,000 1,000 500 ZOO 100 SO k O2 bubbh "3 \ ^^^ 1 ' + Neutral o A r.iH xni solution ufinn i [ N2 bubbling \ i Acid solution \ X, pHr + |pH 6-5- t 1 1 1 i 1 10 xlO rad. Figure 5. HgOg decomposition in neutral and acid solutions X 70^ rad. Figure 5 shows that the rate of decomposition of hydrogen peroxide was independent of pH if oxygen was bubbled. When nitrogen alone was used, the decomposition of 0-01 M hydrogen peroxide was carried to completion with a dose of 4 x 10*^ rad at pH 6, whereas at pH 2 the decomposition was much slower and appeared to approach equilibrium. In the reaction scheme suggested the break-down of hydrogen peroxide into molecular oxygen goes via the reaction H2O2 + O2" ->OH + OH- + O2 .... (6) This follows the only reaction which is pH dependent H02^H+ +O2- or H02 + OH-->H20+02- .... (5) so that at low pH the rate of reaction (6) will be much reduced and molecular oxygen will therefore be lost very slowly. Even in acid solutions decomposition of hydrogen peroxide takes place via reactions (4) and (7), but the forward reactions (2) and (3) are still able 35 HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION to occur, thus avoiding the major loss of bound oxygen via reaction (6). The loss of oxygen from the sohition should be of no significance when oxygen is in excess, so that hydrogen peroxide decomposition is pH independent with oxygen bubbling. Discussion The function y = a(\ — e'^^) fits the experimental data very closely, and seems to govern the production of hydrogen peroxide for doses used in these experiments. It was shown that the initial yield was independent of oxygen tension. The yield can be derived by differentiating _>) with respect to d, and for small doses it is dy j^ = ab = c. It has been found that for large doses the hydrogen peroxide concentration was proportional to the oxygen tension /. Under these conditions the exponential term of y approaches 0, y = a = c^f Thxx^ y = cj\\ - e~ ^^f) dy -^ and^ = q. '^^^ The equilibrium value of hydrogen peroxide concentration J^' will be achieved if / -^ 0. This condition is fulfilled if — ^, i.e. ^, is very large. The hydrogen dy cj J peroxide concentration rises to within 10 per cent of the equilibrium value / 2-3.1 It follows that if the dose is low, a small change in oxygen tension leads to a large change in yield at very low but not at high oxygen tensions. It is possible to calculate for different doses d the oxygen tensions / at which the hydrogen peroxide formation becomes independent of oxygen tension. In Table I are listed these doses and the corresponding oxygen tensions. Table I. Calculated oxygen tensions for given doses, at which the yields of hydrogen peroxide have reached 90 per cent of the equilibrium values /, oxygen tension as d dose (r) per cent of 1 atm. of oxygen 10 9 X 10-3 100 9 X 10-2 1,000 9 X 10-1 10,000 9 The values in Table I reflect the relative probabilities of reactions (1) and (7) for the stated conditions. In living tissue, however, the principal 36 M. EBERT competition for H-radicals is between oxygen molecules and organic molecules. It can be expected therefore that the values in Table I will be applicable to biological systems only qualitatively. In fact, most of the observed oxygen effects in radiation biology show increasing damage with increasing oxygen tensions as long as the oxygen tension is low. For high oxygen tensions an increase does not result in an increase in damage. SUMMARY {1) At low doses and moderate oxygen tensions the yield of hydrogen peroxide is directly proportional to the dose. {2) The yield of hydrogen peroxide changes with changing oxygen tension. At high doses, which give equilibrium values, the yield is directly proportional to oxygen tension. At low doses, on the other hand, a small change in oxygen tension leads to a large change in yield, if the oxygen tension is low, but to a much smaller change if the oxygen tension is moderate or high. This effect is enhanced at low pH. [3) At small doses the admixture of small amounts of hydrogen to oxygen, or vice versa, increases the change in yield of hydrogen peroxide considerably. The addition of large quantities of hydrogen does not change the yield at low doses. The doses used in radiation chemical experiments are usually high from the radiobiological point of view, and some reluctance may be felt in applying the data to biological systems. It is undecided whether hydrogen peroxide molecules are formed in tissue via the radical mechanisms discussed, with doses used in radiobiology, and whether hydrogen peroxide can play a significant part in causing radiation lesions. However, radical reactions are bound to occur in irradiated tissue, and the investigations reported here serve to show how much some radical reactions are influenced by variation in both oxygen tension and h)'drogen ion concentration, particularly with small doses and low oxygen tensions. ACKNOWLEDGEMENTS / am greatly indebted to Miss T. Alper and Mr. P. Howard-Flanders for helpful discussions during the progress of the work. REFERENCES 1 Dewhurst, H. a., Samuel, A. H. and Magee, J. L. Rad. Res. 1954, 1 62. 2 Gray, L. H. Brit. J. Radiol. 1953, 26 608. ^ Gray, L. H., Conger, A. D., Ebert, M., Hornsey, S. and Scott, O. C. A. Brit. J.Radiol. 1953, 26 638. * Ebert, M. and Boag, J. W. Disc. Faraday Soc. 1952, 12 189. 5 Allen, A. O. Rad. Res. 1954, 1 85. « Allen, A. O. Disc. Faraday Soc. 1952, 12 79. ' Barb, W. G., Baxendale, J. H., George, P. and Hargra\e. Trans. Faraday Soc. 1951, 47 462. 8 Bray, J. J. Amer. Chem. Soc. 1938, 62 3357. * Weiss, J. Trans. Faraday Soc. 1953, 31 668. i« Ebert, M. and Alper, T. Nature, Loud. 1954, 173 987. 11 Hochanadel, C. J. J. phys. Chem. 1952, 56 587. 12 Alper, T. and Ebert, M. Science, 1954, 120 608. 37 HYDROGEN PEROXIDE PRODUCTION UNDER VARYING CONDITIONS OF IRRADIATION DISCUSSION M. Magat : II me parait hasardeux, dans I'etat actuel de nos connaissances, d'essayer d'attribuer la perturbation, par des molecules organiques, du schema cinetique de Ebert, a la reaction de ces molecules avec un type de radicaux donne. En effet, les differents radicaux sont assez peu selectifs et en general il faut tenir compte de la competition des molecules organiques pour tous les radicaux presents, H, OH, HO2, dont le dernier est probablement le moins reactif. Le schenia cinetique devient difficile a ecrire et encore plus difficile a resoudre. II va dependre de la molecule organique en question, de son pouvoir de reagir preferentiellement avec I'un ou I'autre des radicaux et de la nature de cette reaction. En absence de donnees experimentales, c'est une discussion theorique assez serree qu'il faut faire dans chaque cas. II est evident que dans une cellule, ou un grand nombre de composes chimiques sont presents, le nombre des reactions initiales possibles a priori devient tres eleve et les reactions ulterieures deviennent imprevisibles. Plusieurs annees de travail s'ecouleront avant que Taction des rayonnements sur I'eau pure soit connue en detail et que nous puissions dire avec certitude ce qui arrive dans la cellule vivante. 38 BACTERIOPHAGE INACTIVATION UNDER VARYING CONDITIONS OF IRRADIATION TiKVAH AlPER Medical Research Council Experimental Radiopathology Research Unit, Hammersmith Hospital, London Although in recent years bacteriophage has been shown to have some degree of organization i' -, it is probably still the simplest living, or self- reproducing, entity with which we can experiment. In the free state, i.e. when not in contact with bacteria, phage does not metabolize, so far as is known. A stock may retain all its properties for months, or even years. As soon as they are placed in contact with sensitive bacteria, however, active phage particles attach themselves, penetrate and reproduce. Inacti- vation of the phage may be due to an interference with any one of these steps. It is well known, for example, that after exposure to ultrax-iolet radiation, phage particles are inactivated because they do not reproduce, although their ability to adsorb to, and penetrate, bacteria may be unim- paired. On the other hand, phage may be inactive through a failure to adsorb, as is the case with the 'tryptophane-deficient' mutant of phage T4, which will not adsorb to its normal host, coli B, unless tryptophane is present^. In this paper, no attempt will be made to discuss which steps in the reproductive cycle are affected by the various inactivating agents to be described. The 'inactivation' of a phage particle will be defined as a failure to form a plaque on a confluent growth of host cells {Figure 1) (page 40). Ionizing radiations may inactivate phage through a variety of mechanisms. Lea and Salaman^ exposed the small dysentery phage S13 in the dry state to ionizing radiations of different ion densities, and concluded from their data that a single ionization taking place within a particle could inactivate it. This is usually referred to as the direct effect of radiation, and ultraviolet inactivation appears to be of the same type. When the phage is suspended in an aqueous medium, it may be affected also by the decom- position products of water. This is usually called the indirect effect of radiation. If the suspension is very dilute, the probability of inactivation by direct effect may be so small, compared with that of indirect effect, that it may be neglected. This paper will deal only with such suspensions, and direct effect will therefore not be considered. It is possible to show that indirect effect itself may proceed through one of three mechanisms : [1] If hydrogen peroxide is formed by radiation, it has an inactivating effect on the phage. (2) The phage may be changed by free radicals, so that although it is still able to form plaques if allowed to adsorb to bacteria soon after irradiation, it is rendered hypersensitive to certain inactivating 39 BACTERIOPHAGE INACTIVATION UNDER VARYING CONDITIONS OF IRRADIATION agents (hydrogen peroxide and ascorbic acid). I have called this ' part-inactivation ' of the phage. (3) The phage may be rendered inactive immediately by free radicals. To some extent it has been possible to study these mechanisms separately. The effect of irradiated suspending medium, i.e. of radiation-produced HgOg, was studied by introducing non-irradiated phage into irradiated buffer solution. Part-inactivation was studied by comparing the suscepti- bility of irradiated and non-irradiated phage to radiation-formed hydrogen peroxide, commercial hydrogen peroxide, or ascorbic acid. The immediate effects of radical action could to some extent be separated from the other two mechanisms by irradiating at a high dose rate, so that radiation was Figure 1 completed within a time which was short, compared with that required for the action of formed hydrogen peroxide. It was necessary, in these experi- ments, to sample as quickly as possible after the end of each period of irradia- tion. The immediate effects of free radicals on phage were studied in this way, in varying conditions of gas treatment and of hydrogen ion concentration. The techniques followed in the phage experiments were those used by Dr. Ebert^ in examining formation of hydrogen peroxide. Information was almost always derived by plotting survival curves, which involved taking several samples of the phage during the course of each irradiation. Such curves can of course be regarded as reciprocal yield-dose curves, since survivors are counted instead of inactivated particles. It was reported previously^' ''• ^ that the immediate indirect effects of radiation on phage appear to be reductions. This was fully confirmed l^y irradiating phage in 40 TIKVAH ALPER the presence of various gases or gas mixtures. As shown b)- Figure 2, the survival curves were steepest with hydrogen bubbhng, and flattest with hydrogen and oxygen mixtures. In the particular experiment illustrated, the inactivation doses (37 per cent doses) were 900, 1,800, 3,600 and 1 l,600rads for H2, N2, O2 and a H2 + Og mixture respectively. The relative yields (based on the curve in nitrogen) were respectively 2, 1, 0-50, and 0-16. As has been shown^, these results conform with the idea that phage particles are reduced by H radicals. When the only dissolved gas present is nitrogen, the free radicals formed are H + OH . . . . (1) H2O when h\'drogen is present, we have H2 + OH H + H2O (2) so that the probability of a phage inactivation by means of a hydrogen radical, with hydrogen present, is twice that with nitrogen present. I r s 4 0 005 0007 0-07 Figure 2. Survival curves ofSlS under various gas treatments It is usually agreed that, when oxygen is present in the suspension, the reaction H + O, HOo (3) '2 ^ ^^'^2 • • • occurs. This would explain why the presence of oxygen protects the phage against inactivation, at high dose rates. Nevertheless, inactivation does take place, and this may be due to reduction by the radical ion 0<^^ , arising from the dissociation HO, H+ +0, (4) '2 -<— ^^ ^ ^2 ... This possibility was supported by the results of irradiating in hydrogen and oxygen mixtures, in which both HO2 and H radicals are removed by the reaction HO2 + H -> H2O, .... (5) 41 BACTERIOPHAGE INACTIVATION UNDER VARYING CONDITIONS OF IRRADIATION which prevents the immediate reaction of reducing radicals with the phage. In fact the act of formation of HgOg can be regarded as a protective mech- anism against immediate action on the phage, a fact which is emphasized by comparing the effects of gas treatment on HgOa formation and on phage inactivation [Figure 3). To bring out the complementary nature of the mechanisms of phage inactivation and HgO., formation, inactivation doses [i.e. reciprocals of yields) have been plotted. The general form of the curve applying to phage follows most closely that for H2O2 production at the .0 ,X 100 75 ;7o 0 - ,% 0 25 50 50 25 0 - - - 0 25 50 7b 100 — 0 25 50 75 100 75 50 25 0 15 100 15 50 25 0 - — — 0 Figure 3. Effect of gas treatment on : — (a) Hydrogen peroxide yield (b) Inactivation dose for bacteriophage SI 3 lowest dose level used by Dr. Ebert^, which were considerably higher than the phage inactivation doses. It can be seen that the full protective effect of oxygen-hydrogen mixtures was obtained with small amounts of oxygen. Reaction 4, viz- HOo ^ H+ + O2- is clearly dependent on hydrogen ion concentration, so that oxidation yields should be greater at higher hydrogen ion concentrations, and reduc- tion yields should be smaller. pH dependence is well known in various radiation chemical systems, including formation and decomposition of HgOa^ It was found that when phage was exposed to the indirect action of 42 TIKVAH ALPER radiation in suspensions of pH about 9, 7 and 5, the inactivation yield was o-reatest in the most alkaUne suspension, and least in the most acid. Since reaction (4) occurs only in oxygenated suspensions, it is in these that pH dependence should occur, if reaction with the Og" radical ion is responsible for inactivation in the presence of oxygen. The pairs of curves presented in Figure 4 show that there was more marked pH dependence under oxygena- tion than with hydrogen bubbling. An explanation must, however, be sought for the fact that some pH dependence was found with no oxygen present, and it seems that this may lie in the reaction H + H+ H, (6) which was originally postulated by Weiss i" to account for pH dependence of the ferrous-ferric system in deaerated solutions. This pH dependent .§ TO 0-5 0-1 005 0-07 r 0-005 0001 V \ : \ t\ - \ \l E H>v[ \ 1 - ^ ) 0 5 pH 8-92 70 k ^ A \02 : "^ H > - 1 5 10 5 pH7-05 pH4-83 Figure 4. Effect o/pH on survival curves of SI 3 {Abscissa: minutes' irradiation at ISOOradjmin) 75 reaction would act as a scavenger mechanism for H radicals. It appears that Hg+is fairly stable in aqueous solutionis. It may be eliminated by reaction with a reducing substance, in a reaction of the type R- + H2+ ^ R + H2 t , or possibly by reactions which lead ultimately to water and gas molecules. From the experiments which have been described, it can be seen that bacteriophage in dilute suspensions can be vised as an indicator for radiation chemical reactions, and, since phage is inactivated by reduction, it is useful as a complementary system to those in which oxidations are studied, which at present are in the majority. It is worth remembering, however, that phage is a self-reproducing entity, and if the effectiveness of radiation is so strongly influenced by factors such as gas treatment and hydrogen ion con- centration, it seems reasonable to suppose that these factors operate also in the living cell. 43 BACTERIOPHAGE INACTIVATION UNDER VARYING CONDITIONS OF IRRADIATION It will probably have been noticed that there is an apparent paradox in the relationship between bacteriophage and hydrogen peroxide : I have described it as an inactivating agent for phage, and yet have shown that its formation acts as a protective mechanism. The paradox is resolved if the time factor is taken into account ; the formation of hydrogen peroxide pro- tects the phage only when radiation is delivered within a fairly short time, and the phage sampled immediately afterwards. At low dose rates the hydrogen peroxide can attack the phage during irradiation, the more so since part-inactivated phage particles are counted as survivors in short-term experiments, but may, with longer time lapses, be inactivated by the peroxide. There is evidence that phage is attacked by hydrogen peroxide only when it decomposes, and then only when it decomposes via reducing radicals. In fact it seems justifiable to postulate that any radicals which are removed at the time of radiation by HgOa formation may act as inactivating agents when the HgOg decomposes. The HgOg therefore acts as a bank for radicals, in any experiment in which dose rates are low, or which is of such a nature that a time lapse occurs between the end of irradiation and the measurement. One can take this argument a step further. In biological experiments in which the end product of radical action is not defined in chemical terms, it is often assumed that enhancement of radiation effects implies an oxidative mechanism. But the presence of oxygen allows of the formation of hydrogen peroxide, and therefore, in effect, of the storage of free radicals which would otherwise be lost by recombination into water or into Hg and O^ molecules. The H2O2 may decompose, either under the action of the radiation, or after irradiation ceases, and so give the radicals a further probability of reacting with sensitive systems. If the nature of a radiation experiment is such that dose rates are low, or long term effects are involved, it seems possible that an important role of oxygen may be to make radicals available for later reaction, rather than exclusively to enhance oxidative reactions. SUMMARY From high dose rate irradiations of bacteriophage under various gas treat- ments, and at various hydrogen ion concentrations, it has been concluded that phage is inactivated by H radicals, in deoxygenated suspensions, and by Og" radical ions, in oxygenated suspensions. In the former, protection may arise in acid suspensions from the reaction H + H+ :i^ Hg"^. The formation of hydrogen peroxide acts as a protective mechanism for the phage, when irradiations are completed in a short time, but the HgOa may later inactivate the phage by decomposing into the radicals which entered into its formation. It is suggested that the presence of oxygen may enhance radiation effects not exclusively through enhancement of oxidative reactions, but also because the oxygen acts as a means for 'storing' radicals which may later become available for reaction with sensitive systems. REFERENCES 1 Anderson, T. F., Rappaport, C. and Muscatine, N. A. ' Le Bacteriophage,' Ann. Inst. Pasteur, 1953, 84 No. 1, 5. 2 Hershey, a. D. and Chase, M. J. Gen. Physiol. 1952, 36 39. 3 Garen, a. and Puck, T. J. exp. Med. 1951, 94 177. 44 DISCUSSION 4 Lea, D. E. and Salaman, M. H. Proc. roy. Soc. 1946, 133 B 434. 5 Ebert, M., 1954. This Conference. * Alper, T. ' Radiation Chemistry,' General Discussions Faraday Soc. 1952, No. 12 234 ' Alper, T. Abstracts, VI. International Microbiology Congress, Rome, Riassunti delle Commmica-ione, 1953, 2 No. 528, 181. 8 Alper, T. Brit. J. Radiol. 1954, 27 50. 9 Ebert, M. and Alper, T. Nature, Lond. 1954, 173 987. 10 Weiss, J. Nature, Lond. 1950, 165 728. 11 CouLSON, C. A. Proc. roy. Soc. 1952, 211A 396. DISCUSSION L. K. Mee : Alper has mentioned the work of Anderson and McDonald on the continued inactivation of pepsin and trypsin after irradiation. They had shown that this 'after effect' is dependent on temperature. We have noticed a similar effect on the normally thermostable enzyme ribonuclease. When irradiated enzyme solutions are kept at O'^C very little further inactivation occurs, but at 25° C and higher temperatures the enzyme continues to lose activity. The continued inactivation appears to be faster at higher temperatures. No inacti- vation of the enzyme has been detected when irradiated solution is added to the unirradiated enzyme. This slow reaction appears to depend on a change in the protein during irradiation which does not affect its enzymic activity immediately but makes it unstable in some way. 45 AFTER-EFFECTS OF IRRADIATION OF DNA J. A. V. Butler and (Miss) P. Simson Chester Beatty Research Institute, Royal Cancer Hospital, London 'After-effects' of irradiation are not unusual. The irradiation initiates a sequence of eflfects which continues for some time. This is particularly the case with living organisms, which are dynamic systems, in which inter- ference with one part may produce a long sequence of changes, ending in death. After-effects can be observed in some cases in purely chemical systems such as DNA. Their interest is mainly in the opportunity they give of determining the mechanism of the processes involved. We should like to add some new observations to those previously given on the subject. The slow decrease of the viscosity of DNA which occurs after irradiation in the presence of oxygen has been ascribed^-^ to (1) the formation of substances of a peroxidic nature which are formed by processes such as RH + OH-> R- + H2O ; R* + O^ -> ROa* ; RO/ + H -> RO^H. It would follow that such peroxides would undergo a slow decomposition. (2) the formation of labile phosphate owing to the oxidation of the C4 atom of the sugar, giving a keto-sugar from which the phosphate is capable of slow hydrolysis^. We have made some further experiments in order to define the circum- stances under which the latter process occurs. Weiss and Scholes have stated that although only small amounts of phosphate are hberated in the action of X-rays, considerably greater amounts are formed by hydrolysis with acids after such treatment. We have used as a hydrolysing agent 0-5 N, H2SO4 at 70°. This produces practically no phosphate in 1 hour with unirradiated DNA, but in longer times the amount of phosphate liberated increases rapidly {Figure 1). Now acid treatment of this nature is known to liberate purines from the DNA and the initial lag period may be ascribed to the time taken for an appreci- able amount of depurination to occur since the intact nucleotide chain is apparently not susceptible to hydrolysis until the purines have been removed. Another factor is the fact, already discussed^, that two adjacent phosphate- sugar hnks have to be broken before inorganic phosphate is liberated. That is, the breakage of one phosphate-sugar link, although it breaks the nucleotide chain, can only result in the formation of a terminal phosphate group. The free phosphate produced by hydrolysis of X-ray treated DNA might then be the result of the acid hydrolysis of the terminal phosphate groups. It is found {Figure 1) that the effect of irradiation with X-rays is to shorten the initial lag period. Comparatively large doses are required to produce a marked effect. It is difficult to give an unambiguous explana- tion of this, as it might be due to {a) an increase in the susceptibility of the nucleotide chain to hydrolysis owing to loss of purines ; {b) the partial 46 y. A. V. BUTLER AND P. SIMSON oxidation of the sugar moiety, resulting in a greater ease of hydrolysis of the phosphate, or (c) the hydrolysis of terminal phosphate which has been produced by the primary action of the radiation. ; - s is to increase the hydrolysability of phosphate which is initially terminal. The actual increase of hydrolysable 47 AFTER-EFFECTS OF IRRADIATION OF DNA Table II. — Effect of X-rays on adenositie-5' -phosphate Adenosine-5' -phosphate (initial) Same after 1 0,000 r Same + 16hr standing . . Same after 1 00,000 r in Nj Same -\- 16hr standing Same after 1 hr hydrolysis with HgSO^ 0-34 0-48 0-48 2-04 1-92 phosphate is however comparatively small and of the same order as the amount of phosphate liberated during the irradiation. Similar results have been obtained after treating adenosine-5'-phosphate with photo- chemically generated hydroxyl radicals [Table III). Table III. — Effect of photochemically produced OH radicals on adenosine-5' -phosphate PO4 (y per ml.) Same after 1 hr hydrolysis with H2SO4 Adenosine-5' -phosphate After 1 hr ultraviolet treatment with HgOo Ditto after 1 6 hr standing 0-26 3-3 4.4 0-34 6-8 6-5 It can be concluded that treatment with X-rays or OH radicals does pro- duce some phosphate which is more easily hydrolysed by acids, and that this labilization is due either to the removal of the purine or to a partial oxidation of the sugar. In neutral solution the rate of the hydrolysis is rather small. IVe are indebted to Mr. E. W. Johns for assistance in some of the analyses, and to Dr. M. Ebert for the irradiations with large doses of X-rays. This investigation has been supported by grants to the Chester Beatty Research Institute from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fimdfor Medical Research, the Anna Fuller Fimd, and the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. REFERENCES 1 Butler, J. A. V. and Conway, B. E. J. Chem. Soc. 1950, 3418. 2 Conway, B. E. Brit. J. Radiol. 1954, 27 42 ; Nature, Lond. 1954, 173 579. 3 ScHOLEs, G. and Weiss, J. Nature, Lond. 1953, 171 920, 1153. 4 Butler, J. A. V. and Conway, B. E. Proc. Roy. Soc. 1953, 141 562. 48 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS Peter Alexander and Arthur Charlesby Chester Beatty Research Institute, Royal Cancer Hospital, London, and Atomic Energy Research Establishment, Harwell, respectively The primary step when a biological system is exposed to ionizing radiations is the utilization of the absorbed energy in a chemical reaction. Since the energy required to produce a biological lesion is often very small and, moreover, since it is initially deposited at random throughout the irradiated material, it would seem to be necessary that the biologically important reaction is with a macromolecule because the proportion of biologically active substances of low molecular weight (e.g. ATP or glutathione) which are changed by irradiation with a few hundred roentgens is negligibly small. We assume, therefore, that the observed lesion is the result of the chemical change of some vital macromolecules present in very limited numbers {e.g. the DNA of the chromosome threads). Since in most biological systems radiations having a high specific ioniz- ation are more effective for the same amount of energy deposited than hard X-rays or gamma rays, it seems possible that some morphological structure must be damaged by changing a number of macromolecules close together. Whatever the primary chemical events are, these cannot in general {i.e. excepting perhaps chromosome breakage) be detected in mammals because the doses necessary to produce a biological lesion, of the order of 1 ,000 r or less, cannot bring about a measurable chemical change in vivo. In principle there are three general methods of protecting against the initial chemical changes and these may be called physico-chemical, as opposed to physiological protection which interferes with the development of the primary chemical changes into biological end-effects. {a) A substance can be added which influences the conversion of the energy taken up in such a way that less chemical change occurs in the 'vital' macromolecules. {b) Repair by an added substance of the damage produced in the macro- molecule immediately after the primary reaction and before any irreversible change has occurred. In many cases this is not possible since the first re- action is irreversible, e.g. when pure stearic acid is irradiated with alpha rays^ the following chemical change occurs which cannot be 'repaired' . CH3(CH2)i5CH2COOH -> CO2 + CH3. (CH2)j5CH3. In other cases the primary action may be the loss of, for example, a hydrogen atom, to give a free radical and this can be repaired. Since successive chemical reactions of a molecule changed in this way by ionizing radiations will occur extremely rapidly, the protective substance bringing about the repair must be present before the irradiation. 49 E PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS (c) Shielding of a vulnerable group in a molecule {e.g. the prosthetic group of an enzyme) with another substance which can be removed after irradiation. The protection of an enzyme by its substrate^ is believed to function by this principle which will not be considered further here. The present paper reports an investigation on the changes produced in a number of synthetic macromolecules under a variety of conditions when protection by different mechanisms was encountered. We hope to be able to establish what type of compounds are most effective in providing pro- tection by the different mechanisms. It may then become possible to deduce by analogy the mechanism of protection in various biological systems by comparing the protective action of a number of substances in vivo with their activity in the synthetic systems. COMPETITION FOR FREE RADICALS When the action of the ionizing radiations is indirect (i.e. the energy from the radiations absorbed in the solvent produces highly reactive entities which react with the solute) a protecting substance can protect the solute by competitively removing the active entities. Dale^ was the first to find that added substances (notably thiourea) protected enzymes, by a compe- tition mechanism, when these were irradiated in dilute aqueous solution. Dainton'* established experimentally, following the original suggestion by RissE^ in 1929, that the free radicals OH* and possibly H* are formed on irradiating pure water. In the presence of dissolved oxygen the radicals formed are OH' and HOg*. In Dale's experiments the enzymes were probably inactivated by OH radicals^ and the activity of the protective agents in these systems is therefore determined by their reaction with OH radicals. The degradation of polymethacrylic acid in dilute aqueous solution by X-rays is due to HOg radicals' and the protective action of more than 100 compounds in chis system has been studied^. These compounds protect by competing for HO2 radicals and the protective action is therefore a measure of reactivity with HOg radicals. In another system, the polymerization of methacrylic acid, we have studied protection by competition for OH radicals^. The order of effectiveness of a series of compounds is not the same in the two systems. The activity of substances in protecting mice against the lethal effects of X-rays follows closely the HOg series and not the OH series^' ^. We deduce that competition for HOg radicals plays an important part in the protection of mice. protection and DIRECT ACTION At first sight it would appear that where the action of the ionizing radia- tions is direct {i.e. the energy is absorbed by the actual material undergoing change) no protection is possible. According to this view, which is widely held, once a macromolecule has absorbed sufficient energy to undergo a chemical change, the ensuing reaction is inevitable and cannot be prevented. Nevertheless an experiment carried out fifteen years ago by Svedberg and Brohult^*' pointed to the possibility that direct action was more complex. These workers found that a very specific dissociation of the giant protein molecule haemocyanin into two equal parts could be induced by irradiation 50 PETER ALEXANDER AND ARTHUR CHARLESBY with a-particles, and that the passage of one a-particle anywhere through the molecule was sufficient to produce this change. This indicated that energy absorl^ed in one part of the molecule could be transferred to those bonds responsible for holding the two halves together. If energy transfer of this kind can occur, then protection against direct action is theoretically possible. The term ' energy transfer ' does not imply any particular mechan- ism but is used as a general description of processes we do not at present understand. Thus before it was established that indirect action in aqueous solution was brought about by the intervention of free radicals the process could have been referred to as energy transfer from the solvent to the solute. Recently Milton Burton and his colleagues ^^ have found evidence for energy transport from one molecule to another when a mixture of two liquids such as benzene and cyclohexane is irradiated. In this study, transfer of charge by collision between an ionized and un-ionized molecule plays an important part. How far such a process can occur in our solid systems cannot at the moment be assessed. Protection of Polymers. — We decided to test the possibility of protecting against the direct action of ionizing radiations by admixing different chemicals with synthetic polymers and irradiating these as solids. Some polymers are crosslinked while others are degraded^- when irradiated with gamma rays * Polymethylmethacrylate, CH3 I -C1H2 — Ci — COOCH, n is degraded on irradiation and the number of breaks produced is directly proportional to the radiation dose. For every 61 eV of energy from gamma rays which is absorbed by the polymer one main-chain bond is broken. This does not mean that all the 61 eV is used to break the bond, and other chemical reactions, notably side-chain breakdown, also occur, f Films of this polymer were prepared which contained a small quantity of a low molecular weight additive and the energy required to produce one main chain bond in the polymer was again measured. Some substances exerted a truly remarkable protective action and the presence of 10 per cent of * This difference in behaviour has been explained ^^' ^^ as follows : Degradation can only occur when a chemical rearrangement of the main chain is possible to give two non- interacting products. Direct rupture of a bond into two radicals will not produce a per- manent break since the two parts, being in close proximity (cage effect), will immediately recombine. When such a rearrangement of the main chain cannot occur the predominant chemical reaction will be confined to the side chains with the result that acti\e centres capable of forming inter-chain bonds (crosslinking) will be produced. Crosslinking will not occur exclusively and some rupture of inain chains is usually also found ; this is to be ex- pected on our hypothesis since a proportion of main-chain radicals are bound to escape from the cage and result in breaks. Escape from the cage is more likely to occur with radiations of high specific ionization and these should be less effective for producing crosslinked polymers. t Much of the energy does not bring about any chemical change but is wasted in heating up the polymer by a minute amount. 51 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS dimeta-tolyl-thiourea, aniline or allylthiourea raised the energy which had to be taken up by the polymer per main-chain break to 227, 152 or 143eV respectively. Other substances such as long-chain paraffins and ethyl urea do not protect. We can see two possible interpretations : (1) the energy absorbed by the polymer is not immediately utilized to bring about a chemical change and in the interval the energy is transferred (see footnote on page 51) from the polymer to the additive. (2) The protection is brought about by repair of the polymer by the added substance which increases, for example by crosslinking, the molecular weight and thereby hides some of the main- chain breaks. We believe that the energy transfer mechanism occurs for the following reasons, (a) A detailed quantitative study by Toms (un- published) relating the protection to the concentration of the protector and to the radiation dose indicates energy transfer, [b) The list of substances which protect is so diverse that it is highly improbable that they share a chemical property such as ability to produce crosslinks, {c) Preliminary experiments indicate that the same substances which protect polymethyl methacrylate against degradation protect other polymers against crosslinking. The decisive test is to see if the energy required to modify chemically a molecule of the additive is less when incorporated in a polymer than when irradiated by itself This experiment is difficult to carry out since even when extremely large doses of radiation are used, only a very small fraction of the low molecular weight additive is changed. Preliminary experiments showed that a considerably greater proportion of aniline was changed when this was irradiated in a film with polymethylmethacrylate than by itself Instead of treating this effect as one of pi'otection the polymer can be considered to enhance the decomposition of the additive by handing on to it some of the energy absorbed {i.e. the polymer then fulfils the same function as the solvent in the case of indirect action). In this way the energy which was initially absorbed uniformly in the system* concentrate at certain points. Energy transfer within inolecides. — Two series of experiments indicate that energy transfer can occur over considerable distances within a macromolecule. The energy required to form a crosslink by irradiation with gamma rays in a straight chain hydrocarbon was independent of its molecular weight^^. We examined the energy to form a crosslink in a hydrocarbon to which an aromatic group (naphthyl) was attached to see if energy transfer to this group occurred [i.e. if some of the energy absorbed in the hydrocarbon chain was transferred to the aromatic group) . As can be seen from Table I the energy required to produce a crosslink is greater in the substituted dodecane derivatives and the protection is most marked when the naphthyl group is in the centre of the chain. We believe that a reasonable interpre- tation is that energy originally absorbed by the hydrocarbon chain is trans- ferred to the aromatic group and that the transfer is not efficient over a distance of more than a few carbon-carbon bonds. To eliminate the possi- bility that the decrease in crosslinking found was due to steric interference by the naphthyl group, a hydrocarbon was examined which was substituted * The absorption coefficients of different organic materials to hard X- and gamma rays and to particulate radiations other than neutrons do not vary by more than a few per cent. 52 PETER ALEXANDER AND ARTHUR CHARLESBY with an equally bulky group, cyclo-decalyl, but one which we knew from experiments with polymethylmethacrylate to be a very feeble energy transfer protector. Table I shows that the energy required to crosslink cyclo-decalyl-dodecane is only slightly greater than that of the pure hydro- carbon and we conclude that steric factors played at most a minor part in the protection of the substituted dodecanes. Table I. — Influence of an aromatic group substituted in different positions along the chain of the straight- cliain hydrocarbon dodecane on the energy from ionising radiations which has to be absorbed to produce one crosslink^^. Substance CH3-(CH2)io-CH3 CH2-lCH2)io-CH3 Energy per crosslink (eV) dodecane 20 naph+hyl-1- dodecane 32 CHj-iCHgjg-CH •(CH2)7CH3 naphthyl-4-dodecane 46 Cha-iCHaJ na' phthy 1-6- dodecane 49 CM, cyclo-decalyl-e-dodecane 27 * The absolute values are open to some uncertainty but the relative values are reliable. Similar results have been obtained when co-polymers of isobutylene and styrene {see Figure 1) were irradiated. Polyisobutylene is degraded^' and behaves very similarly to polymethylmethacrylate for which detailed results have been published i^. Polystyrene, on the other hand, is extremely radiation resistant but will crosslink, though the energy required is approxi- mately one hundred times that for the straight-chain hydrocarbons |. The behaviour of the co-polymers is complicated and changes as the radiation dose is increased 1^ At first they degrade in a regular manner {i.e. the t This is a case of internal protection by the phenyl side chains to vk^hich energy absorbed by the rest of the molecule is transferred. The benzene nucleus is known to be very resistant to radiation^i and can, because of its aromatic structure, dissipate a great deal of energy without chemical change. 53 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS number of main-chain bonds broken is proportional to the radiation dose) . Then fairly suddenly the average molecular weight of the material (from viscosity measurements) is no longer changed by irradiation and the main- chain breakdown in the polyisobutylene units is counterbalanced by the — CH Polysty rene CH3 CH3 CH3 C CH2 C CH2 C CHg- CH3 CH3 CH3 Pol y/sobutylene CH — Co -Polymer * The components are assumed to be distributed at random in the proportion in which they are present. Figure 1. — Composition of Polymers crosslinking of the polystyrene units. At still higher doses crosslinking pre- dominates and part of the material is converted to an insoluble gel. The energy necessary to break a main-chain band in the polyisobutylene part of the molecule can be obtained from experiments with relatively low doses and from Table II it can be seen that the polystyrene exerts a very definite Table II. — Degradation of co-polymers by ionizing radiations Polymer Energy deposited in the polymer per one main-chain break Polyisobutylene 17eV Co-polymer of 20 per cent styrene + 80 per cent isobutylene 32 eV ,50 „ „ +50 „ „ 55eV ,80 „ „ + 20 „ „ ~ lOOeV Polystyrene ~ 2,000 eV t t Polystyrene is not degraded but becomes crosslinked and the energy quoted is that required to produce one crosslink. protective effect. By making reasonable assumptions concerning the distri- bution of the isobutylene and styrene units in the macromolecule we 54 5> PETER ALEXANDER AND ARTHUR CHARLESB Y computed ^^ from the irradiation data that one styrene unit provides com- plete protection to two isobutylene units either side. This is, of course, an oversimphfication since the probabihty of protection will decrease gradually with the distance from the phenyl residue. However, this calculation gives a value for the distance over which energy transfer can occur which is very similar to that obtained from the substitution of naphthyl groups in different positions along the dodecyl chain. REPAIR OF DAMAGED MOLECULES As already mentioned on page 49 radiations can produce chemical reactions which involve the complete disruption of the molecule and are clearly not reversible. In many cases, however, the first chemical change is the loss of a hydrogen atom from an organic molecule either by direct action or as the result of abstraction by a free radical (e.g. >CH2 + OH*->>CH'4- HgO). This type of damage can be restored by what may be called a transfer reaction and this was found to occur when aqueous solutions of polyvinyl alcohol were irradiated ^''. In the absence of oxygen the molecule does not degrade but crosslinks to give a stiff gel. Some added substances, notably — SH compounds, protect by repairing the activated molecules before these can interact to give a polymer network. The general reaction may be illustrated as follows: I loss of I CHa > CH* I hydrogen I part of polymer polymer radicals which combine molecule with one another to form a gel The protective agent (PH) transfers a hydrogen atom to reconstitute the polymer before the polymer radical has had time to undergo further reactions such as crosslinking. PH + GH* -> GHa + P* 1 I reconstituted radical from the protective polymer molecule agent which is of low chemical reactivity. In the presence of oxygen the polymer radical will be converted to an unstable peroxy radical I I GH* + O2 -> GHOO* I I which will undergo further changes and may in some cases decompose in such a way as to bring about main-chain breakdown. This degradation can in principle be prevented by the protector as follows : GHOO- -f PH ^ GHOOH + P* I I In this way the polymer is changed but decomposition is prevented by the formation of a stable compound. 55 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS We have no evidence that hydrogen transfer plays a part in biological protection. Prevot-Bernas^i observed that cysteine and cysteamine act as chain terminators in polymerization processes by hydrogen transfer and postulated that the protective action of these substances in mice was due to their ability to act as transfer agents and not as competitors for free radicals. This hypothesis can be rejected for the following reasons, (a) Many pro- tective agents which are active in vivo with mice and in vitro with polymeth- acrylic acid do not act as transfer agents in polymerization reactions under conditions where the — SH compounds do.^ {b) Amines are as active as protectors in the cationic as in the un-ionized form whereas they can only transfer a hydrogen atom when un-ionized^. Crosslinking of polyvinyl alcohol is prevented by un-ionized methylamine but not by ionized methyl- amine. This further supports the view that in this system protection occurs by transfer, (c) All — SH compounds act as transfer agents while only very few are capable of protecting mice^. protection and TARGET THEORY In the earlier formulation of the target theory (see for example Lea^^) a chemical change was postulated to occur whenever an ionization had occurred ; the energy for this event plus its associated excitation was assumed to be the same as that required to ionize an atom in air {i.e. about 32 eV). Current radiochemical research has shown that this second postulate need not apply and that when pure materials are irradiated a chemical change often occurs only when very much more than 32 eV of energy has been absorbed. We have recently shown {see page 54) that for some reaction such as the breaking of main-chain bonds in polyisobutylene considerably less than 32 eV may be sufficient to produce a discrete chemical change. Thus, if radiation of low specific ionization were used to determine a mole- cular weight from the apparent ' target size ' in, for example, polystyrene (using crosslinking as a criterion), a very low value would be obtained, while with polyisobutylene the molecular weight derived would be too high. These experiments may make it necessary to re-evaluate the data obtained for the sizes of the sensitive volumes of viruses and enzymes from ' target area' calculations, since the chemical change may not be confined entirely to the track of the ionizing particle. In particular the occurrence of energy transfer throws doubt on the validity of the calculation for the number of sensitive sites in an irradiated organism from the variation in apparent ' target size' (or relative biological effectiveness, RBE) with the specific ionization of the radiation used. This problem has recently been dealt with in great detail by Zirkle and Tobias " who interpret the RBE of different radia- tions in terms of the diffusion of free radicals. Transfer of energy initially deposited at certain points may provide an alternative explanation for the observed facts. THE mechanism OF ' DIRECT- A CTION ' Largely as the result of Dale's work direct and indirect action have been clearly differentiated. The least ambiguous test for distinguishing between the two processes is to examine how the percentage inactivation, of for 56 PETER ALEXANDER AND ARTHUR CHARLESBY example an enzyme, varies with concentration in solution (i.e. if the inactivation is inversely proportional to concentration, the action is indirect). This test cannot, however, be readily applied at the cellular level since it is usually not possible to vary at will the concenti^ation of constituents within the cells. Based on the concepts that direct action is not influenced by the condition of the environment of the material, and that once sufficient energy has been absorbed (or a given number of ionizations have occurred) the subsequent damage to a molecule is unalterable, three other tests are used to distinguish between the two processes. The action of radiations of low specific ioniza- tion {e.g. hard X-rays or gamma rays*) is said to be indirect (a) if the presence Figure 2. — The ultraviolet absorption of poly - isobutene showing the effect of radiation in the presence and abserwe of oxygen {dose approx. 1 • 5 X 1 0^ r) (0-13 per cent solution in n-hexane) ^00 250 300 mu. of oxygen in the external medium enhances the biological effect ; {b) if the action of the radiation is decreased on freezing all the water in the system thereby making it impossible for free radicals to diflTuse ; (c) if the presence of other substances can reduce [i.e. protect) the irradiated system. Since protection^* and an effect of oxygen^^ is almost invariably found when living systems are irradiated with X-rays or gamma rays it has been concluded that indirect action plays an important part in bringing about the observed effects. Our work on the irradiation of dry polymers where the action must be direct shows that the last test is not decisive for indirect action, since * The oxygen effect and protection cannot be used to determine whether the action of densely ionizing radiations (e.g. neutrons or alpha rays) is direct or indirect, since they pro- duce radicals in water at ver^' high local concentration and their reacti\ity cannot therefore be influenced by the presence of oxygen or protective agents in the water. 57 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS protection is now known to be possible against direct action. In addition we have observed that the absorption spectrum {see Figure 2) of polyisobutylene is modified much more extensively after irradiation under nitrogen than under air''''. This is a clear example of an oxygen effect when the action is direct since exposure of the polymer to oxygen after irradiation does not modify the absorption spectrum. We can conclude that irradiation produces different chemical products in the presence and absence of oxygen even where the process is ' direct '. The degradation of both polyisobutylene^'' and of polymethylmethacryl- ate^^ depends on temperature. The energy required to break a main-chain bond in polyisobutylene is nearly doubled on decreasing the temperature from 70° C to — 60° C. A possible interpretation is that transfer of energy into the required bond is more efficient at higher than at lower tempera- ture {cf. fluorescence) . A similar observation has been made with biological materials. Dry enzymes^* and dry bacteriophage^^ were reported to be inactivated more readily {i.e. their target size appears to be larger) at higher temperatiu-e. This has been interpreted by Pollard-* in terms of an inherent change in sensitivity of the enzyme with temperature and by Bachofer et al^^ as due to the diffusion of an active intermediary within the dry phage. Since a temperature dependence has now been found with a completely synthetic material where neither of these explanations can apply, it may be necessary to assume an energy transfer process. Although these experiments in no way disprove that the action of ionizing radiations on living systems is indirect they show that an important contribution by direct action cannot be excluded. Since in the cell and particularly in the cell nucleus proteins and nucleic acids are present in relatively high concen- trations, it has always been difficult to understand why the action of ionizing radiation should be predominantly indirect. SUMMARY The possible ways in which the primary chemical effects of ionizing radia- tions can be reduced are reviewed. Examples are given of protection against indirect action by competition for free radicals and of repair of a damaged macromolecule by transfer agents present during the irradiation. Irradiation of solid polymers has shown that protection is possible against direct action and an undefined energy transfer process is suggested as the mechanism. Such an eflfect will require modification of the 'target theory' in its more general form. In addition to protection by added chemicals, direct action can also be influenced by the presence of oxygen and by changes in temperature. This shows that 'direct' action need not be an unalterable event and that it may play a more important part in the irradiation of living systems than is generally assumed. This work has been supported by grants to the Chester Beatty Research Institute from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. 58 DISCUSSION REFERENCES 1 Breger, I. A. J. phys. Chem. 1948, 52 551. 2 Doherty, G. D. Fed. Proc. 1952, 11 (part I) 35. 3 Dale, W. M. Biochem. J. 1942, 36 80 ; J. cell. comp. Physiol. 1952, 39 Supplement 1, 39. * Dainton, F. S. J. phys. Chem. 1948, 52 490. 5 Risse, O. Strahlentherapie, 1929, 34 578. " Holmes, B. and Dainton, F. S. Nature, Land. 1950, 165 266. ■^ Alexander, P. and Fox, M. Nature, Lond. 1952, 169 572 ; Trans. Faraday Soc. 1954,50 605. 8 Alexander, P. Brit. J. Radiol. 1953, 26 413. Alexander, P., Bac^, Z. M., Cousens, S. F., Fox, M., Herve, A. and Lazer, J. Radiation Res., in press. 9 Alexander, P. and Fox, M. J. chim. phys. 1953, 50 415. 10 Svedberg, T. and Brohult, S. Nature, Lond. 1939, 143 938. Brohult, S. Nova Acta Regiae Societatis Scientariitm Upsaliensis, 1940, 12 No. 4. 11 Manion, J. P. and Burton, M. J. phys. Chem. 1952, 56 560. Burton, M. and Patrick, W. N. J. phys. Chem. 1954, 58 421. Patrick, W. N. and Burton, M. J. phys. Chem. 1954, 58 424. 12 Charlesby, a. Nature, Lond. 1953, 17l'l67. 13 Alexander, P., Charlesby, A. and Ross, M. Proc. Roy. Soc. A. 1954, 223 392. iiBacq,, Z. M. and Alexander, P. Fundamentals of Radiobiology. London, Butter- worth, 1955. 15 Charlesby, A. Proc. Roy. Soc. A. 1954, 222 60. 1® Alexander, P. and Charlesby, A. Nature, Lond. 1954, 173 578. 1'^ Alexander, P., Black, R. M. and Charlesby, A. (to be published). 18 Charlesby, A. J. poly. Sci. 1953, 11 513. 1^ Alexander, P. and Charlesby, A. (to be published). 20 British Empire Cancer Camp. Report, 1954, 31 13. 21 Prevot-Bernas, a. J. chim. Phys. 1953, 50 445. 22 ZiRKLE, E. R. and Tobias, A. C. Arch. Biochem. Biophys. 1953, 47 282. 23 Gray, L. H. Radiation Res. 1954, 1 189. 2^ Pollard, E. C. Advanc. bid. med. Phys. 1954, 3 153. 2'* Bachofer, C. S., Ehret, C. F., Mayer, S. and Powers, E. L. Proc. Nat. Acad. Sci. 1953, 39 744. 2« Lea, D. E. Actions of Radiations on Living Cells. London, Cambridge Univ. Press, 1946. DISCUSSION M. Magat : Je voudrais faire deux remarques. D'abord au sujet de I'utilisation de la methode viscosimetrique et la perturbation eventuelle de la viscosite par les ions. La variation du pH influence, d'apres tout ce que I'on sait, uniquement la viscosite des solutions polyelectrolytes, dont DNA est un exemple. Or, a I'exception de I'acide polymethacrylique, aucun des polymeres utilises par Alexander n'est un poly- electrolyte. Sa methode experimentale est done parfaitement justifiee. Ensuite,. je voudrais preciser quelque peu le mecanisme de protection centre I'eflTet direct. Deux cas sont a distinguer — celui oii la molecule contient elle-meme un noyau phenylique et celui oia les molecules aromatiques servent de solvant. Dans le premier cas, I'excitation electronique intervenant dans n'importe quel point de la molecule, se propage a I'interieur de la molecule sous forme de ce que j'appelerai un exciton, et tombe comme dans un piege, dans le noyau phenylique. Au lieu de provoquer une rupture de liaison, I'energie sert a exciter un electron et se trouve eliminee probablement par emission d'un photon. Alexander a montre, et je 59 PHYSICO-CHEMICAL METHODS OF PROTECTION AGAINST IONIZING RADIATIONS crois que c'est un resultat tres remarquable, que la propagation de I'exciton est limitee dans I'espace a quelques 6 liaisons C— C. Remarqons que cet effet pro- tecteur du noyau, phenylique a ete pour la premiere fois signale par Burton, qui I'a appele ' effet eponge '. Dans le deuxieme cas, le mecanisme initial n'est pas bien compris. On a observe effectivement, aussi bien au laboratoire de Burton que dans le mien, qu'en presence de solvant aromatique (ou meme simplement insature) une partie plus importante de I'energie dissipee par le rayonnement est utilisee par le compose aromatique qu'il nesserait justifie, vue sa concentration. Les composes aromatiques ont un tres petit rendement en radicaux libres, une grande partie de I'energie etant reemise sous forme de luminescence (c'est le principe des compteurs a scintillation). Ageno a Rome, a pu montrer que les photons emis par une molecule sont absorbes par des molecules voisines, reemis par elles, et ainsi de suite jusqu'au moment ou ils sont reemis vers I'exterieur le point d'emission pouvant etre distant de quelques millimetres du point d'excitation. 60 TWO CLASSES OF PROTECTIVE AGENTS IN THE OXIDATIVE DEGRADATION BY GAMMA RAYS OF POLYSTYRENE IN CHLOROFORM M. Fox Laboratoire de Chimie Physique de la Faculte des Sciences de Paris* A NUMBER of compounds have been examined as possible protective agents against degradation of polystyrene dissolved in chloroform in the presence of air by gamma rays from a ^"Co source at an intensity of 23 r/min. The de- gradation was measured by the change in viscosity of the solution. Table I shows the results obtained for compounds which have been found effective in a similar investigation using an aqueous polymer system.^ Degradation is reduced by amines, hydroxy compounds and mercaptans (7), and by com- pounds having the structure — S— C or — S=C as in thiourea, mercapto- benzothiazole and dithiocarbamate derivatives [2) ; aromatic compounds are generally more effective than the aliphatic homologues. In the case of protective agents which are chelating agents, such as 8-hydroxyquinoline and dithiocarbamate, chelation does not suppress the protective action although the complex is less active. The viscosity of the polystyrene solution continues to drop for a con- siderable time after irradiation has ceased {see Table If). The effect of Co2+ ions is consistent with the view that the degradation is due to decom- position of a polymer peroxide resulting from electron transfer ^ : ROOH + Co2+ = RO* + OH- + Co3+ decomposes. The post effect is not found when the irradiation is carried out in the presence of protective agents of the thiourea type but occurs in other cases as shown in Table I. Moreover the former type of compoimd when added after irradiation reduces the post-effect, other protective agents act in the opposite sense, accelerating the degradation as shown in Table I (cf. the action of phenols on hydroperoxides^). It seems therefore that the two classes of protective agents react in a different manner. The kinetics of the reaction of ^-naphthol as a function of the degradation have been investigated in some detail*. The results show that the degradation which is initially inhibited resumes its normal course, at a dose which is proportional to the concentration of [i-naphthol. Assuming reaction of (i-naphthol with primary solvent radicals it is found that the protective agent reacts with only a small * British Council and Medical Research Council Exchange Fellow ; Present address : Chester Beatty Research Institute, Royal Cancer Hospital, London, S.W. 3. 61 OXIDATIVE DEGRADATION BY GAMMA RAYS OF POLYSTYRENE IN CHLOROFORM ^ f<< o I o r-i in in o 1 ^ 1 1 1 tH 1 o 1 ■ o o ! ^ 1 1 1 oi \ cr> CM r^ Ho X O 1—1 .— ( Th in U3 Q lO S-i R Xi S • 2 1 a ^ ^ i o 1 In 1 1 1 1 1 CO 1 CM 1 1 1 g ^.s X — " •«*» .1 c^ ^ "ts « n ■^ ^ ^ O 1 1 1 1 1 1 1 1 1 ^ 1 1 1 "« ^ r-i 1 1 1 1 1 1 1 1 1 CO 1 1 1 a Ci X 43 1-1 § ^ Si § -i3 n O 1 1 1 1 1 ai CT) o in 1 <^ 1 ■§ >~ 1 1 1 1 1 in CO r-. 1 CM 1 in 1 S fe ■« X »— I 1— C ?— 1 CM 1 t"*^ ^ K o a. ■p-j o \o s « o n -§ Tf O in « •►^ r-i I '^ 1 1 1 1 I 1 1 • 1 1 1 b- a 1 CO 1 1 1 1 1 1 1 CO 1 j 1 ^ X -*> C5 o ? o >- 2 ^ ^ o ■^ O^ 1 1 r~- ^ CM o o o I o o X lO t^ 1 1 O (^ CO 1 — 1 1— t • 2 ^ o J; 3 *~ ) ) o 1 CM 1 1 CO r^ 1 lO o o 1 o o tu R i-H 1 CO 1 1 in r^ 1 CM ^^"2 X •^ ■§ iH ■^ <-i ^3 CO •t^ 1 O r-t COOOOCOOOCM o o (^ O CT) ^ ^ § X Ci> CO CM CM — ' CM ^ >^ V ■- -s; 3 V O 0 •-■ KJ 0 P oj i 2^ 8 ^ X X Ji V 0. i: ^ u rt ni JJ O ^ 5 < ci-co CO s < 0 CO. SQ Q c^ H (U o c8 .2 rt c o .2 1- 3 a 'O lO tf) GO u . CD (S 6 "^ c o q; o \CA^ ACTION PRESERVATRICE DE DERIVES PHENOLIQUES VIS A VIS DE LTRRADIATION LETALE DE LA SOURIS* A. Lacassagne, J.-F. Duplan et N. P. Buu-Hoi Institut du Radium, Paris On sait que parmi les methodes utilisees pour diminuer chez les animaux la mortalite consecutive a une irradiation totale, certaines consistent a provoquer une anoxie anoxique ou cytotoxique, d'autres a introduire dans Torganisme des composes chimiques qui detournent des recepteurs cellulaires les peroxydes radioformes. Dans cet ordre d'idee, nous avons montre^ que le pyrogallol n'est pas doue d'activite radiopreservatrice, mais que certains de ses derives cetoniques, injectes par voie intraperitoneale avant une ii radiation totale a une dose 100 pour cent letale, etaient susceptibles de reduire la mortalite des souris. Ce pouvoir protecteur appartient tou- jours a des acylpyrogallols dont la chaine laterale possede au moins six carbones. Dans le but de preciser les configurations chimiques actives et d'etendre a d'autres phenols les resultats que nous avons obtenus avec le pyrogallol, nous avons prepare trois types de composes : (i) des 4-acyls derives du pyrogallol, du pyrocatechol et du resorcinol ; (ii) des 1-acyls derives du 2-naphtol et des 2-acyls derives du 1-naphtol; (iii) des 3-R-2- dihydroxyphenyl-indole et des 3-R-2-trihydroxyphenyl-indole, le radical R etant aromatique ou aliphatique (Figure 1). Toutes les irradiations sont pratiquees sur des souris de la lignee pure XVII, agees de 60 a 80 jours et pesant 20 a 22 g. Ces animaux, irradies par groupe de six, regoivent en une seule fois la dose de 700 r (180kV, 10 mA, 0,3 Cu, 45 cm, 65r/m) qui est, pour cette lignee, 100 pour cent letale en 12 jours. Une demi-heure a une heure avant I'irradiation, on injecte dans le peritoine de ces animaux 0,5 ml d'huile d'olive neutre dans laquelle on a fait dissoudre le produit a etudier ; seuls le pyrogallol, le resorcinol et le pyrocatechol ont ete dissous dans du serum physiologique. Les resultats que nous avons obtenus sont resumes dans le Tableau I (page 66). ^ L'activite des derives du pyrogallol se trouve confirmee mais, par contre, celle des cetones derivees des diphenols reste douteuse et demande confir- mation. Aussi bien le pyrogallol que le resorcinol et le pyrocatechol sont inactifs sous leur forme simple. II faut remarquer que, dans ce groupe, le pyrogallol et le pyrocatechol sont des poisons mitotiques- et, qu'au moins en ce qui concerne le premier, l'activite radiopreservatrice n'apparait que pour une dose superieure a la dose minimale antimitotique. II est permis de penser que les derives cetoniques que nous avons utilises sont, a dose * Travail du Laboratoire Pasteur a I'Institut du Radium, realise avec une subvention du Commissariat k I'Energie Atomique. 64 A. LACASSAGNE, J.-F. DUPLAN ET N. P. BUU-HOI equivalente, moins toxiques pour les mitoses que les corps simples corres- pondants. Les cetones derivees dii naphtol montrent une activite indubitable en ce qui concerne le l-behenoyl-2-naphtol et plus douteuse quand il s'agit de derives du 1-naphtol, ce qui pent s'expliquer par la toxicite plus forte de ce dernier corps. Les differents derives des 2-di et 2-trihydroxyphenyl-indole sent tous inactifs, ce qui semble indiquer que la chaine aliphatique saturee doit etre 4- acylpyrogallol ^-Qcylpvrocaiechol ^-acylresorcinol 2-acyl-1-naphtol l-acj'l-2-naphtol 3-R-2-(2-4'-dihYdP0xyphenyl) indole 3-R-2-(2-3'-4'-trihydroxyphenyl);:idol8 Figure 1 fixee directement sur le cycle phenolique. II n'est, par contre, pas possible d'affirmer que la fixation de cette chaine par I'intermediare d'une fonction cetone soit necessaire. Dans le groupe des phenols, a partir de six carbones, la longueur de la chaine laterale ne semble pas influencer le pouvoir protecteur et ne parait pas non plus modifier considerablement la duree de la protection mesuree par le temps qui s'ecoule entre I'injection du compose et le moment de I'irradiation. Les resultats des experiences que nous venons de rapporter 65 F ACTION PRESERVATRICE DE DERIVES PHENOLIQUES et de notre travail anterieur^ montrent que, pour des chaines de 8 a 22 carboneSj I'activite preservatrice est maximale 45 minutes environ apres I'injection. II est possible, par contre, que les naphtols soient doues d'acti- vite meme quand ils sont lies a des chaines laterales plus courtes. On a souvent pu demontrer qu'il existait un parallelisme indiscutable entre les radioprotecteurs actifs in vitro et in vivo, tel est le cas de la cys- teine ^' ^, de la thioiiree^' ®, des dithiocarbamates'' ^ et d'un grand nombre Ta bleau I Temps entre Taux de Substances Doses mg/kg injection et survie Date des irradiation au dices 30ej. min Temoins 0/18 7e au 12e j Huile d' olive • — 20 0/12 6e - lOej Laurate de Na 300 35 0/6 8e - 12ej Pyrogallol 165 10 0/6 5e - lOej A-Behenoylpyrogallol r 750 45 4/10 lie - 21ej R=C2iH43 \ 750 150 0/6 9e - 18ej I 1.000 60 0/6 7e - 12ej Risorcinol 120 10 0/6 6e - 9ej A-Palmitoylresorcinol 400 30 0/6 7e - 12ej R=Ci5H3i Pyrocatechol 160 10 0/6 6e - lOej 4-Behenoylpyrocatichol r 600 \ 750 45 1/10 Be - 16ej R=C.,iH43 30 0/12 6e - 13ej l-Behenoyl-2-naphtol 250 35 6/10 lOe - 20ej R=C2iH43 2-Acetyl-l-naphtol 60 35 1/12 lOe - 15ej R=C2H5 2-Palmitoyl- 1-naphtol 160 30 0/6 7e - 12e.j R=Ci5H3i 2-Stearoyl- 1-naphtol 200 30 1/6 8e - 15ej R=Ci7H35 3-Docosyl-2{2'-4'-diliydroxyphenyl)indole 600 30 0/6 7e - 12ej R=C2oH4i 3-Phenyl-2{2'-3'-4'-trihydroxyplienyl)indole r 250 \ 400 30 0/6 7e - 12e.j R=C6H5 30 0/6 6e - lOej 3-n- Tetradecyl-2{2'-3'-4'-trihydroxyphenyl) - 500 30 0/6 7e - llej indole R=«-Ci4H,9 3-n-Docosyl-2- {2'-3'-4'-trihydroxyphenyl) - 750 30 0/6 6e - 12ej indole R=«-C,oH4i d'acides amines'^- ^'^. On savait aussi que les phenols etaient des accepteurs d'oxygene radioforme ^' ^^ et des protecteurs vis a vis de la degradation des polymeres'^, mais ils se montraient totalement inactifs quand ils etaient injectes a un animal avant irradiation. Les resultats que nous venons de rapporter montrent que, pour jouer dans la radioprotection biologique un role non negligeable, les phenols doivent etre introduits sous une forme moins toxique et mieux adaptee aux fonctions organiques. 66 DISCUSSION REFERENCES 1 Lacassagne, a., Duplan, J.-F. et Buu-Hoi, N. P. C.R. Acad. Sci. Paris, 1954, 238 1279. 2 Parmentier, R. C.R. Soc. Biol. Paris, 1949, 143 585. » LoisELEUR, J. Bull. Soc. Chini. biol. Paris, 1942, 24 172. « Patt, H. M., Tyree, E. B., Straube, R. L. et Smith, D. E. Science, 1949, 110 213. 5 Mole, R. H. J. Chim. phys. 1951, 48 258. « Alexander, P. et Fox, M. J. Chim. phys. 1953, 50 415. ■> Fox, M. C.R. Acad. Sci. Paris, 1954, 237 1682. 8 Bacq, Z. M., Herve, a. et Fischer, P. Bull. Acad. roy. Med. Belg. 1953, 18 226. » Bacq, Z. M. et Herve, A. J. suisse Med. 1952, 82 160. 10 Loiseleur, J. Bull. Soc. Chim. Biol. Paris, 1943, 25 22. DISCUSSION C. Burg : Apres combien de temps I'huile d'olive injectee dans le peritoine est- elle resorbee ? J. F. Duplan : Quelques jours. C. Burg : On peut se demander quel est le sort, dans ce cas, des substances injectees dans le peritoine, lorsqu'elles sont en solution dans I'huile d'olive ? J. F. Duplan : La lenteur de resorption du solvant ne permet pas de prejuger de la vitesse de diffusion, dans Forganisme, des corps qu'il sert a dissoudre. II est probable que les composes phenoliques que nous avons etudies ne diffusent qu'apres s'etre detaches de tout ou partie de leur chaine grasse. 67 INFLUENCE OF OXYGEN ON DAMAGE TO MICRO-ORGANISMS BY IONIZING IRRADIATION H. Laser Molteno Institute, University of Cambridge A NUMBER of bacteria representing strict and facultative aerobes and anaerobes, as well as some yeasts, were exposed to X-rays (190kvp, no added filtration, dose rate 6500r/inin.) at doses from 6-5 to 65xl0^r. Known cell concentrations of washed cells were irradiated in phosphate buffer -f glucose in absence of any added growth-promoting substance. During irradiation the liquid phase was either free from dissolved Og (the gas space containing N^ or Hg) or it was in equilibrium with Og of decreasing percentage (100 per cent Og ; 20 per cent or 5 per cent Og/Ng). In some experiments with 20 per cent and 5 per cent O2 the nitrogen was replaced by carbon monoxide. Immediately after irradiation a known number of cells were transferred either into fresh buffer + glucose or into a nutrient medium in manometer flasks and the rate of some metabolic processes measured for up to 10 houis. These included : Og uptake, COg production, aerobic and anaerobic fermen- tation or acid production and utilization of Hg ( Vibrio desulphuricans) . Under these conditions the increase, for example, in Og uptake or anaerobic fermen- tation with time is proportional to the increase of dry weight of bacteria, i.e. it is a true reflection of growth. The main results are as follows : (i) Og uptake, COg production and anaerobic fermentation or acid pro- duction were not significantly affected, when measured in washed non- growing cells. Aerobic fermentation of baker's yeast was slightly inhibited. [2) Growth inhibition became apparent only after a lag period which is the shorter, the higher the X-ray dose. [3) All growing cells showed an ' Og-effect ' on irradiation. The relative sensitivity for Oa-treated as compared to Ng-treated cells was approximately three-fold for some cells {Staphylococcus albus, Lactobacillus Delbriickii) . On the other hand a strictly anaerobic organism ( Vibrio desulphuricans) was unaffected by 6-5 X 10"^ r in Ng but almost completely inhibited by the same dose in air ; and the growth of vegetative forms from spores of B. subtilis was only inhibited after irradiation (2-6 X lO^r) in presence of O2 but not in Ng. [4) In order to study whether the ' Og-effect ' is dependent on a particular metabolic state of the cell or a particular equilibrium of an enzymic system involved in respiration, cells were irradiated in presence of Og while their respiration was inhibited by respiratory poisons. Thus, in the case of Sarcina lutea, it has been possible with an X-ray dose of 26,000 r to suppress almost completely the Og-effect, i.e. the cells, after removal of the poison, behaved as if they had been irradiated in Ng. The effective inhibitors were 68 DISCUSSION CO, KCN, hydroxylamine and azide. Urethane did not abolish or diminish the ' Og-effect '. The mode of the- effective poisons in their role as respiratory inhibitors is known. They all block hydrogen transfer through the respiratory enzymic system by combining with oxidized cytochrome a-j and stabilizing the remain- ing respiratory enzymic chain in the reduced form. Taking this mode of action as a guide in advancing a possible explanation for the ' Og-effect ' in irradiation, it is suggested, at least for this bacterium, that the enhancement of irradiation damage (1) involves the enzymic respiratory mechanism, (2) requires at least part of the enzymic respiratory chain to be in the oxi- dized form during irradiation. This makes it possible, that the impedance has been caused by a reducing agent. DISCUSSION F. H. SoBELS : I was interested to hear about Laser's observation that potassium cyanide exerts a protective effect against X-radiation in bacteria in view of the opposite resuhs recently obtained by myself in Drosophila. Here injection of potas- sium cyanide prior to X-radiation significantly increases the mutation rate as compared to that induced by the same dose of X-rays alone. Similarly King, Schneiderman and Sax showed that carbon monoxide pretreatment increases the frequency of X-ray induced chromosome aberrations in Tradescanlia. It seems as if the exactly opposite results of cyanide on radio-sensitivity spring from another metabolic situation in the different experimental materials used. 69 THE ACTION OF IONIZING RADIATION ON ENZYMES AND VIRUSES Ernest Pollard Biophysics Division, Sloane Physics Laboratory, Yale University The purpose of the studies which have been conducted for the past five years in the Biophysics Division at Yale University have been to develop radiation methods as a tool for the study of fundamental cellular processes. Three reviews of this work have appeared^- '^- ^. The work is, therefore, not so much directed at the understanding of radiobiological action on large organisms, as it is at developing a means of study. The following features about radiation can be exploited for the study of cells. The high energy release produced by radiation, the fact that this is localized and that the localization can be varied by choosing the conditions of irradiation, and the fact that penetration into the cell without damage of the cell wall can be achieved by ionizing radiation. The work to be described follows logically from the early work of Lea, Smith, Holmes and Markham^. These workers studied the effect of high doses of X-rays on two dry enzymes and concluded from the nature of the inactivation that a moderately reliable estimate of the molecular size of the two enzymes ribonuclease and myosin could be obtained. The principle of this work is to assume that ionizing radiation produces randomly distri- buted high energy releases which produce the great majority of their effect inside any one molecular unit. The statistics of inactivation permit the derivation of a parameter which can be called the inactivation volume in the case of irradiation where the energy release is distributed randomly in volume and a cross section in the case where energy release is confined to dense swaths of ionization. Both of these methods of irradiation were clearly understood by Lea. The techniques of modern nuclear physics permit irradiation in a very much more precise and flexible way than was available prior to the second world war. In particular the use of cyclotron-produced particles for irradiation is very attractive. A group at Yale has exploited this by modify- ing a relatively small cyclotron, with an external beam, which passes through a defocusing system on to a shutter in front of a series of samples. The shutter is operated when each sample is put in place and exposures of known beam for known times are given and in this way inactivation of quite small and relatively insensitive molecules can be obtained. The energy release by fast charged particles is well known from a com- bination of theory and experiment. These have been described in the review articles already quoted. The important feature is that the spacing between energy releases can be changed by varying the speed of the bombarding particle and that these energy releases do not spread very far from the track of the particle itself. Therefore some idea of the localization of the 70 ERNEST POLLARD energy can be obtained in terms of the energy of the particle and the number of particles per cm^ incident on the enzyme or the virus. If an enzyme, such as trypsin, is subjected to bombardment by deuterons then it is inactivated semilogarithmically according to the relation In nlfio = —SD In this formula n and rig are the activities of the enzyme after and before irradiation respectively, D is the number of deuterons per cm- incident on the trypsin, and S is the cross section, which measures the statistical probability of inactivation of a trypsin molecule. It is found that the quantity S depends on the density of ionization of a bombarding particle. This fits quite well with the idea that the slower the particle the more dense the ionization and the closer the quantity S approaches to the expected physical cross section of the molecule of trypsin. To illustrate the kinds of studies that have been carried out the inacti- vation of invertase may be considered. If this is subjected to bombardment by deuterons at varying energies and also by high speed electrons, which produce ionization effects which are substantially random in volume, then the loss of ability to digest sucrose is lost according to a semilogarithmic manner just as described for trypsin. So is the loss of ability to combine with specific antibody to invertase. The two do not, however, follow the same line. The serological activity is lost less rapidly, with more bombard- ment, than the enzymatic activity. If the temperature at which the invertase is irradiated is raised then the sensitivity of the invertase begins to increase at a temperature just below that at which enzymatic activity would be lost due to temperature alone. If the temperature is reduced to liquid air temperatures the sensitivity of the enzyme diminishes. There is a range of 50°, or so, over which the sensitivity does not vary very much, and at which the observed cross section corresponds rather closely to that expected for the physical cross section of the molecule. If in place of commercial preparations, the enzymatic activ- ity in yeast cells is studied, it is found that the same inactivation is observed. In other words the sensitivity of invertase in intact yeast cells is the same as that in commercial preparations. It is perhaps easiest to see the nature of these effects of ionizing radiation on enzyme molecules in terms of a proposed explanation. According to this explanation we consider the enzyme to be made up of protein chains, crosslinked in some way, and that a part of this whole protein molecule is essential for the enzymatic activity, and also for other specific biological activities. The same two parts need not be involved in different functions. The arrival of ionizing radiation causes the ejection of an electron from any one of the atoms in the whole molecule without there being any special tendency to select one class of molecule. This results in the ejection of an electron and leaves behind an atom which is positively charged due to the loss of an electron. The fact that the electron is missing from an atom means that it will tend to restore its lack of balance. This it may do by transfer of an electron from a neighbouring atom and such transfer may be extremely rapid. In view of this it is probable that the localization of the ionized atom changes throughout the molecule as electrons are interchanged 71 THE ACTION OF IONIZING RADIATION ON ENZYMES AND VIRUSES between one atom and another. It is felt that this process of very rapid exchange can only take place within the molecular structure where the atoms are bonded by electron exchange mechanisms. The transfer of energy therefore takes place rapidly along the chains and continually occurs untif such time as it is either lost by some method of de-excitation such as the return of an electron to an atom to restore the original condition. Until that time, however, there is a rapidly moving region of excitation, which also corresponds to a region of weak bonding since the electron exchange necessary for bonding is not present. If a molecule has an alternative configuration which can result from such a weakness then this may take place. This may correspond to the formation of an unwanted crosslinking in which case the molecule is permanently distorted and will no longer function in its biological way. Or it may be that due to thermal agitation certain weak links are present in the molecule at various places, and that the presence of an additional weak link induced by radiation near these regions may cause again the formation of an unwanted and semipermanent structure. In this way radiation induces the permanent distortion of the molecule into another form and so causes its inactivation. It is clear that such a process, while largely confined to the molecule itself, may well have energy requirements imposed on it for eflfectivc action. Thus it may not be sufficient for one bond breakage to occur : it may be necessary that three or four be produced inside the molecule. This has been found to be the case in the studies made at Yale, particularly for ionizing action on the solubility of the molecule. For this to be lost it is generafly found that three or four ionizations must occur inside the molecular structure. In view of the effects which are covered by the explanation given above, which may still not be the true explanation but which gives a means of compact description of what is Hkely to be occurring, it is possible by radia- tion action involving heavy particles of different velocities, and also fast electrons, to determine the following three things regarding a molecule. (/) Its maximum cross section, (2) its sensitivity in terms of a number of ion pairs needed for inactivation, (3) its approximate thickness. These three quantities can only be determined if irradiations under many different conditions are used. These can be illustrated by considering the irradiation of B. subtiUs cells by deuterons and electrons and subsequent assay of these cells for the two enzymes cytochromoxidase, and succinic-dehydrogenase. Studies by Pow^ell and the author'^ have shown that these enzymatic properties are lost under deuteron and electron bombardment and that the loss varies with ion density of the bombarding radiation in a manner which permits the deduction of molecular sizes and thicknesses for these two enzymes as follows. Enzyme Cytochrome oxidase Succinic dehydrogenase Molecular Weight 160,000 310,000 Length 200 A 390 A Thickness 36 A 36 A 72 ERNEST POLLARD Irradiation of" these B. subtilis cells, and of lysates of the cells, showed no difference between them and there is, therefore, for these enzymes, no property of ionizing radiation which has been shown to be dependent on the organization within the cell. One very interesting application of ionizing radiation is the study of the structm'e of viruses. This has been employed by many workers notably Lea and Salaman, and Bonet-Maury and Latarjet^. The techniques are essentially those which have just been described and have been employed, for example, by Bonet-Maury and Latarjet to deduce a structure for the internal part of vaccinia virus. The type of study which is possible is the following. A well characterized virus usually has between three and six well-known properties. These are, for example, ability to infect the host cell, the ability to attach to the host cell, the ability to interfere with other virus infections, the ability to combine with specific antiserum, the ability to kill the host cell without necessarily multiplying in it, and the ability to agglutinate red cells in the case of a certain large class of animal viruses. All of these properties can be subject to examination after irradiation by deuterons, alpha-particles and electrons. The result of doing this shows that the loss of properties is widely difTerent. Thus the loss of ability to combine serologicaly is highly insensitive. If the method of analysis de- scribed above is applied to results on this serological affinity for the case of T-1 bacteriophage, the molecular size deduced' corresponds to a molecule of molecular weight 22,000. If the ability of Newcastle disease virus to agglutinate red blood cells is studied^, the molecular size corresponds to a pair of units of molecular weight 220,000. If the infectivity is studied very much larger sizes are obtained and the manner in which this infectivity cross section varies with the ion density of the bombarding particle indicates that, in general, there must be some kind of internal structure, for most of the viruses that have been studied. This internal structure must be a rela- tively small part of the virus in the case of T-1 bacteriophage and a relatively large part of the virus in the case of southern bean mosaic virus. A detailed study of these properties leads to inferred structures for the inside of viruses which represent one way of describing the probable nature and morph- ology of viruses. It must be stressed that such pictures are a continuing matter, that as more radiation data appears the picture may change, and that in any event the inferred internal structure of viruses must be made to agree with other methods — physical, biochemical, and genetic. Neverthe- less it would appear as though virus structure might well be elucidated, at least in part by this type of radiation study. acknowledcjement The author wishes to acknowledge the great part played by his associates in the Biophysics Division at Tale University in conducting the researches on which this article is based. REFERENCES ^ Pollard, E. Advanc. biol. med. Phys. 1953, 3 153. 2 Pollard, E., Guild, W. R., Hutchinson, F. and Setlow, R. B. Prog. Biophys. 1955, 5 72. 73 THE ACTION OF IONIZING RADIATION ON ENZYMES AND VIRUSES 3 Pollard, E. Advanc. Virus Res. 1954, 2 109. * Lea, D. E., Smith, K. M., Holmes, B. and Markham, R. Parasitology, 1944, 36 110. = Powell, W. F. and Pollard, E. Radiation Res., in press. ^ Bonet-Maury, p. and Latarjet, R. This symposium. ' Pollard, E. and Setlow, J. Arch. Bioch. Biophys. 1954, 50 376. 8 Woese, C. and Pollard, E. Arch. Bioch. Biophys. 1954, 50 354. DISCUSSION M. Errera : Je voudrais signaler que Jeener et al* a montre I'existence, dans des feuilles de tabac injectees de virus de la mosaique, de deux antigenes de constantes de sedimentation et de vitesses d'electrophese differentes, qui ont des proprietes immunologiques proches de celles du virus et qui en font probablement partie. II pourrait bien s'agir de proteines identiques a celles dont on peut supprimer les proprietes immunologiques par irradiation des virus. L. Ehrenberg : Pollard determined by irradiation the same molecular weight of saccharase when it was isolated and when it was situated in the intact yeast cells. It should be noted that there are indications that the saccharase activity is located on the surface of the yeast cell.f It would be important to know whether the yeast cells were viable. * Jeener, R., Lemoine, P., Lavand'Homme, C. Bioch. Bioph. Acta, 1954, 14 321. t Thorsell, W. and Myrback, K. Arkiv Kemi, 1951 , 3 323 ; cf. Wilkes, B. G. and Palmer, E. T. J. Gen. Physiol. 1932, 16 233. 74 L'IRRADIATION DES VIRUS p. Bonet-Maury Laboratoire Curie, Institut du Radium, Paris Si longue est la liste des organismes vivants soumis, depuis la decouverte des rayons X et de la radioactivite, a Taction des radiations ionisantes, qu'il serait surprenant de ne pas y voir figurer, au moins quelques virus. Les premieres experiences, realisees en general, avec des techniques primitives et seulement avec I'idee de ' voir ce qui se passe ' ont cependant montre que I'irradiation pouvait, comme d'autres agents physiques, detruire certaines proprietes des virus, comme le pouvoir de multiplication ou les effets pathogenes pour I'homme et les animaux. Les travaux de Dessauer, Timoffeef-Ressosvky, Zimmer, Rajewsky en Allemagne, Crowther, Gray et surtout D. H. Lea en Grande-Bretagne, Lacassagne, Holweck, Mme Curie en France, Gowen, Wyckoff, Zirckle aux Etats-Unis, ont donne naissance a la radiobiologie quantique et la theorie de I'impact — treffer ou target theorie — a reussi a interpreter raisonnable- ment Tirradiation par les rayons X des organismes unicellulaires : bacteries, levures, protozoaires. Mais il faut attendre 1940, a la veille de la guerre, pour voir appliquer ces idees nouvelles aux virus et publier des travaux quantitatifs, comportant une courbe dose/effet ainsi que le calcul du volume sensible du virus, par Wollman, Holweck (Ultra-microscopie statistique), Lacassagne, Luria en France^* 2. 3^ l^^ en Grande-Bretagne^ et Gowen et Lucas aux Etats-Unis^. Durant la guerre, Lea poursuit des recherches remarquables sur la radiobiologie quantique des virus, tandis qu'en France, dans I'ignorance complete, pendant cinq ans, des travaux britanniques, mon laboratoire entreprend une etude systematique de I'irradiation des phages et des virus pathogenes^ — par les rayons a et X — pour mesurer leurs dimensions, etudier leur structure et la preparation possible de radiovaccines''. Nous avons utilise generalement des virus pathogenes non purifies, en suspension dans un milieu aqueux, renfermant des concentrations suffisantes de substances organiques protectrices, pour eliminer I'effet chimique indirect. Les virus, non purifies, sont egalement proteges, contre les eflfets chimiques de surface par la gaine de tissus cjui les enveloppe ; d'autres conditions contribuaient encore a cette protection : le titre aussi eleve que possible du virus, la determination soignee de I'origine de la courbe dose/efifet, pour les doses faibles et dans certains cas, I'irradiation dans I'air liquide [Figure 1). Les rayons alphas du radon ont ete choisis pour des raisons de commodite techniques et surtout parce que le calcul de la cible est plus simple que pour les rayons X. Pour que la rencontre d'un virus par une particule ionisante puisse etre consideree comme le 'choc actif de la theorie de I'impact, il faut deux conditions : (7) La trajectoire doit passer par le virus (probabilite d'atteinte = l). 75 L IRRADIATION DES VIRUS (2) L'energie cedee au virus durant la traversee doit etre suffisante pour produire Teffet biologique cherche (probabilite d'action = l). La densite ionique elevee le long d'une trajectoire de particule alpha, permet de considerer la deuxieme condition comme toujours remplie, meme pour les petits virus. On peut alors remplacer la notion de volume sensible par celle de section efficace pour un bombardement alpha, dont le calcul ne comporte aucune hypothese sui l'energie exacte correspondant au choc actif. La technique d'irradiation pour les rayons alphas est simple et avec une source de 30 millicuries de radium, nous avons inactive les virus les plus resistants ; les rayons X etaient produit par un tube demontable Holweck, avec une longueur d'onde moyenne de 0-9A. La partie la plus difficile de ces experiences est le titrage biologique du virus qui, facile pour les phages, necessite pour les virus pathogenes un grand nombre d'animaux et des methodes statistiques convenables. Dose p.\,/|j,3xio^ 2 i/ e 8 1000 8.000 3.000 Figure 1. Irradiation avec les rayons OL du radon du phage '^-X-174. Action de la temperature sur Vejfet indirect Toutes nos courbes d'inactivation, comme celles de Lea, et des precedents auteurs, se sont montrees lineaires en coordonnees semi-logarithmiques, c'est-a-dire des exponentielles ou courbes de Poisson a un seul choc, d'apres la theorie de I'impact. Chaque corpuscule recevant en moyenne un choc actif, pour la dose D37, reduisant de 37 pour cent le titre du virus, on calcule le diametre de la section efficace pour les rayons alphas, par la formule d^ = 6,45 X \0^/Vn37 ou drx = diametre exprimee en m[j, D37 = dose exprimee en ionisations (33 eV) par [jl3, pour permettre la comparaison des rayonnements tres divers. Le calcul du volume sensible, pour les rayons X, necessite une hypothese sur l'energie du choc actif. Nous avons choisi, comme Lea, le groupe de 3 ionisations, correspondant sensiblement a lOOeV, utilise maintenant en chimie des radiations pour calculer le rendement G. Le diametre du volume sensible se calcule par la formule rfj, = \, 76/V D37 avec les memes unites. 76 p. BONET-MAURY Lea a donne un precede general de calcul (methode du volume associe) qui utilise la densite ionique lineaire (overlapping factor F) et permet la determination du volume sensible pour tous les types de rayonnements, y compris les rayons alphas. J'ai egalement utilise, pour les rayons alphas, le graphique liant la section efficace aux dimensions moyennes du virus ; determinees par les autres methodes physiques {Figure 2). Tous nos resultats, avec ceux d'autres auteurs, ont ete groupes dans un tableau, comportant egalement les dimensions des virus obtenues par d'autres methodes physiques : microscope electronique, centrifugation, filtration. On peut dire, d'une fagon generale, que les sections efficaces alphas sont en accord raisonnable avec les dimensions du corpuscule, mesurees par les diverses methodes mecaniques. Le fait que tout le virus soit radiosensible parait surprenant pour les gros virus, auxquels on attribue generalement une Figure 2. Relation eritre la dose d'inactivation pour les rayons a et le diametre du virus {o = Bacteriophage X = Des animaux A = Des plantes 20 W 100 Dose 37 % wo 1.000 p.um,. structure ; on peut I'expliquer cependant par une migration, physique ou chimique, de I'energie produite en exces, lors de la traversee alpha. La comparaison des sections efficaces des volumes sensibles pour les rayons X, conduit a des constatations differentes. Si les diametres X et alphas sont du meme ordre pour les petits virus comme les phages S13 et (p-X-174 ou la fievre aphteuse dermotrope, il y a une difference de plus en plus significative quand la taille du virus augmente et, pour la vaccine, le diametre X n'est plus que le dixieme du diametre alpha, c'est-a-dire un rapport 1.000 pour les volumes. Get ecart peut naturellement s'expliquer par un mauvais choix des hypo- theses de calcul. II est evident que la forme spherique, utilisee en premiere approximation, ne convient pas pour les virus filiformes, comme la mosaique du tabac et le choix du choc actif n'est pas a I'abri de la critique. Si on admet cependant que, pour les rayons X employes, le volume sensible corre- spond a une realite biologique, toute difference significative entre les cibles X et alpha indiquera une structure heterogene differenciee, c'est-a-dire un certain degre d'organisation biologique. 77 l'irradiation ues virus On sera conduit a attribuer alors aux petits phages comme le SI 3 ou le cp-X-174 vuie structure homogene alors que les gros, comme le C16 montrent deja une organisation en territoires differencies. Dimensions des cinq phages par differentes methodes : Irradiation Micr. electron. Filtration Phages (Section efficace) Centrifug. X a fac. corr. 0.83 S 13 16 16 < 20 16-20 18 cp-X-174 17 18 < 20 16 18 C36 22 35 35 23 33 StK 40 50 65 60 83 C 16 36 90 90 70-85 83 II en est dememe pour les virus des animaux, I'accord de la section efficace et du volume sensible pour la fievre aphteuse dermotrope conduit a une structure homogene, tandis que nos experiences sur la vaccine, comme celles de Lea et SALAMAN^s'interpretent par une structure tout a fait heterogene, que nous con- cevons d'ailleurs de maniere un peu differente de Lea. Celuici considere un volume sensible fractionne en 110 corpuscules de 6 m[j,, repartis dans une partie inerte, tandis qu'avec Frilley et Laterjet nous admettons*^ une construc- tion en micro-briques de 22 m[jL, entierement radiosensible pour les alphas. L'analyse de la structure des virus par l'irradiation met done en evidence le passage progressif du type macromolecule homogene, attribue aux plus petits virus, au type heterogene, annon^ant la structure cellulaire des gros virus, differencies en territoires inegalement sensibles au rayonnement. Pour etendre ces conclusions, une plus large experimentation avec d'autres rayonnements est necessaire. A ce point de vue, la contribution de PoLLARD^*^ est d'un grand interet car, avec les deuterons acceleres artificiellement, il a pu faire varier la densite d'ionisation et etudier syste- matiquement I'influence sur la section efficace de la perte lineaire d'energie, c'est-a-dire de I'espacement des ionisations, pour en deduire la forme et les dimensions du volume radiosensible, par une methode differente de calcul. Si I'application aux virus des principes de la radiobiologie quantique presente un grand interet pour les virologistes, il est au moins egal pour les radiobiologistes. L'irradiation des virus, organismes vivants les plus simples, constitue, en effet, le premier chapitre de la radiobiologie et un champ d'etude particulierement favorable pour approfondir le mecanisme ele- mentaire de Taction biologique des radiations. REFERENCES 1 HoLWECK, LuRiA et WoLLMAN. C.R. Acad. Sci. Paris, 1940, 210 639. 2 Lacassagne, a. et Wollman. Ann. Inst. Pasteur, 1940, 64 5. 3 Wollman et Lacassagne, A. Ann. Inst. Pasteur, 1940, 64 5. * Lea, D. E. Action of Radiations on Living Cells. Cambridge University Press, 1946. 5 GowEN et Lucas. Science, 1939, 90 621. ^ Bonet-Maury, p. L'irradiation des Virus dans Les Ultras-virus des Maladies Animales, Maloine, 1943 ; et Les Ultras-virus des Maladies Hiimaines, Maloine, 1948. ' Bonet-Maury, P. et Olivier. C.R. Acad. Sci. Paris, 1939, 209 459. 8 Lea, D. E. et Salaman, M. H. Brit. J. exp. Path. 1942, 23 27. » Bonet-Maury, P. Ann. Inst. Pasteur, 1945, 71 46. 1° Poll.\rd, E. This symposium. 78 INACTIVATION, PAR LES RAYONS X, D'UN AGENT TRANSFORMANT DU PNEUMOCOOUE H. Ephrussi-Taylor et R. Latarjet Institut de Genetique et Institut du Radium, Paris respectivement Le TP Sr, qui confere au pneumocoque la resistance a une concentration de 2 X 10^^ pour cent de streptomycine, a ete inactive en solution aqueuse par des rayons X de 0,7-0,9 A donnes avec une intensite de 1 kr par seconde. Les faits suivants ont ete mis en evidence. [a) La courbe de survie est exponentielle, mais presente une cassure pour une survie d'autant plus basse que la solution est moins concentree [Figures 1 et 2). Cette cassure denote une heterogeneite. Celli-ci ne provient pas de la presence de particules d'ADN differant hereditairement des autres 100 Figure 1. Courbes d'inactivation du TP en solution a 10~^ pour cent dans des extraits de leviire a. 1 pour cent, 2,5 pour cent, 5 pour cent et 10 per cent e x^T Figure 2. Courbes d'inactivation du TP en extrait de levure a 1 pour cent. I TP 10~^ pour cent. II TP 10^^ pour cent. Auto-protection manifeste [en pointiller, le prolongement de la partie rectiligne de la courbe met la cassure en evidence) SxlO^r par une radioresistance plus elevee : les molecules qui survivent a une tres forte dose de rayons X n'induisent pas chez le pneumocoque la formation de TP resistant. II s'agit d'une heterogeneite dans la structure de la solu- tion, actuellement a I'etude. {h) Le TP est extremement sensible aux actions indirectes du rayonne- ment. Tandis qu'a la concentration de 1 pour cent I'extrait de levure 79 INACTIVATION, PAR LES RAYONS X, D UN AGENT TRANSFORMANT DU PNEUMOCOqUE protege completement les enzymes et les petits bacteriophages contre I'effet indirect, a la concentration de 10 pour cent cet extrait n'assure pas encore une protection complete du TP {Figure 1). L'autoprotection du TP est egalement tres important [Figure 2). Ce fait est sans doute en relation avec I'absence de membrane proteique autour de I'ADN, et aussi avec la forme filamenteuse tres dissymetrique, qui, pour un volume donne d'ADN, offre aux radicaux libres du milievi ambiant une tres grande surface acceptrice. [c) On atteint une protection a pen pres complete en operant sur des solutions en extrait de levure a 10 pour cent congelees a — 70°C. Dans ces conditions d'effet direct a peu pres pur, la courbe d'inactivation {Figure 3) 100 ^10- Figure 3. Coiirbes d'inactivation par effet direct. I du TP en extrait de levure a 10 pour cent congele. II du bacteriophage S-13 lO^T fournit une dose Z)q 37=530 krad, qui correspond, pour le poids moleculaire du TP, a la valeur P < 7,3 xlO^ Dans le calcul de ce poids, les incertitudes liees au groupement des ionisa- tions ont ete tres diminuees en irradiant conjointement le petit bacteriophage SI 3, dont le volume est approximativement connu (diametre moyen 15,4m[jL) — Figure 3. {d) La presence d'oxygene est sans influence notable sur I'inactivation du TP. Si cette inactivation est, par sa nature, voisine de ce qu'on appelle une radiomutation genique, on peut concevoir que cette derniere, resultant d'une action primaire sur I'acide nucleique, serait egalement indifferente a la presence d'oxygene. Comme les ruptures de chromosomes lui sont en revanche tres sensibles, on peut penser qu'elles resultent de I'atteinte primaire de substances differ- entes de I'ADN. L'etude de I'influence de Toxygene permettrait ainsi de dire si une altera- tion genetique consecutive a I'irradiation resulte, ou non, de I'atteinte primaire de I'acide nucleique. 80 RADIATION DEATH IN MAMMALS B. Rajewsky Max Planck - Institut fiir Biophysik, Frankfurt/Main DOSE AND SURVIVAL TIME The complete dose-mortality curves obtained on irradiating with X-rays mice, rats, and guinea-pigs show several distinct ranges of total dose over which the biological mechanism of bringing about death is different. This fact is especially true for whole body irradiation. Figure 1, for example, repre- sents the complete survival curve of white mice after a whole body irradiation by roentgen rays. The final response to irradiation is generally the pre- mature death of the animal. A special case arises when medium doses are applied over a long period of time (for about one year or more), and if a very small irradiation intensity is used. That is the field of carcinogenesis. Here cancer is the cause of the death of animals, but in this case their mean survival time has, in general, not been reduced considerably. If one looks at the entire survival curve as it is reproduced in Figure 1, one notices first that the curve covers the dose range from some lOOr to some 1 00,000 r. When small doses (under lOOr) are applied, changes take place in the irradiated body, the meaning of which as regards injury of the organism is still not known. They can be recognized mainly by changes in the blood picture. In addition there are some further observations which, however, did not lead to a well defined conception so far as the mode of action of these radiation doses is concerned. The very large doses of the order of magnitude of 200,000 r result in the practically instantaneous death of the animals. Between these extremes there are dose ranges which differ from one another in a distinctive way. These ranges are approximately as follows : (7) lOOr to l,200r, {2) l,200r to 1 5,000 r, {3) 1 5,000 r to about 30,000 r, {4) 30,000 r to about 1 00,000 r, (5) exceeding 1 00,000 r. From the results available it can be concluded that each of these dose ranges is connected with different mechanisms of injury, w'hich finally cause the death of the animals. In the first dose range numerous changes occur in different hormonal and other systems of the body. A large part of the observed symptoms is due in this case to irritation of the haematopoietic system and its connections with other functional centres of the organism. In this range an essential part is played by the time intensity factor of the radiation effects. Here the direct injury of cells is also of great importance. Especially regarding these direct cellular effects and the effects of local irradiation we generally con- sider this region as being the field of radiation therapy. This field is being studied in greatest detail at the present time. The effects of doses under lOOr, mentioned above, can probably also be classed in this range. In the second dose range final reaction to irradiation is independent of the magnitude of the applied dose. The results of the investigations we carried out show that the damage to certain organs and their functions pre- dominate if compared with direct cell-damage and other injuries of the 81 G RADIATION DEATH IN MAMMALS tissue of the body. The third dose range is characterized by a rapid reduc- tion of survival time of the irradiated animals. The mechanism producing the final effects in this dose range is apparently completely different from the mechanism prevailing in the second dose range. In the fourth dose range animals die with symptoms that seem to point to a considerable participation of the central nervous system in the mechan- ism of injury. The fifth dose range is marked by a process of damage that takes place rapidly (instantaneous death). The mechanism of these effects proceeds WOO WOOO 100000 r (O \ ! I 1 ; 1 . 1 1 1 M II. 1 T 1 I 1 ; ii - ^ A ; 13 \ - 7 \ - V ' "t^xfc- r^ - fv - W^K^Q_ — ~ *^y "^^X \ \ \ 0 \ \ - Q) \ \ 1 - .5 - < ~ ■^ 1 — §-^ \ i. _ 1 Si =0 en ' _ o J • — -2 • -3 • - \ \ - \ - \ ^ 1 - \ \ 1 1 1 1 7^ day 7day 10\ Figure 1. Dose mortality curve of white mice after whole body roentgen-irradiation. o o Curve obtained in 1943. • • Curve obtained in 1953. f\ Wm\n. /min. Lo^ doses probably from the destruction of a relatively large quantity of practically all basic substances of the organism (molecular death). We shall see subse- quently that probably each of these ranges really includes several mechanisms of injury. This can be said definitely of the first and second dose ranges. It was possible to demonstrate that the second dose range includes at least three mechanisms which are probably connected with each other. Single doses, as small as lOOr to 200 r, already lead to a premature death of the animals. Especially remarkable is the occurrence of a dose range, varying slightly with the species and resistance of the animals, at least from l,200r to 1 5,000 r. The mean survival time for white mice amounts to 3 • 5 days and is independent of the applied dose. This horizontal part of the 82 B. RAJEWSKY curve is followed with increasing doses by a steep fall in the survival time till instantaneous death of the animals occurs (few seconds). A new symptom of injury appears when doses of approximately 30,000 r to 1 00,000 r are used. Then the irradiated animals suffer from tetanic convulsions. The lower curve of Figure 3B representing the dependence of the appearance of convulsions on the dose is similar to the general curve of injury. The injury in the 'dose-independent' range is to a great extent inde- pendent of the intensity of radiation. The range studied was for single doses lasting from 2-5 seconds to 10 hours {i.e. radiation intensities varied by a factor of 14,400). Similar effects also appear in the cases of other animals. The dose range for constant survival time differs for different species both in their extent Doses X 125 250 500 WOO 2000 WOO 6000 1S000 3Z000 6WO0 128000 3-U 36 3-8 LoQ doses u-8 SO , guinea pigs {-\ [-) and rats Figure 2. Dose mortality curves of white mice ( (• •) after whole body irradiation and in the length of the mean survival time. The differences, however, are not big. Figure 2 represents the results of corresponding irradiations of white mice, guinea-pigs, and rats. In the following we will refer for the sake of brevity to a 3-5 day-effect also with rats. PHYSIOLOGICAL PROCESSES While instantaneous death {i.e. within a few seconds) after application of large doses may probably be regarded as 'molecular' death caused by destruction of the organism's basic substances, the 3-5-day death is un- doubtedly connected with more or less complicated physiological processes in the irradiated body. Therefore this effect (first described by the author in 1943) was of special interest. Further experiments in this direction were interrupted unfortunately by the war. The studies were resumed in 1948 with my co-workers ^ (Heuse, Aurand, Wilhelm, Pauly, Gerber, Parchwitz and Winkler) using an X-ray set (Heuse and Rajewski^) 83 RADIATION DEATH IN MAMMALS of very high power, the construction of which had ah^eady been begun in 1940. The plant operates at a maximum of 50 kV, 2 A tube current (permanently) and supplies up to 1,000, 000 r/min. Irradiation of animals is carried out in rotating plastic containers so as to obtain a homogeneous irradiation. The results of these experiments^ led to the recognition of a second effect {i.e. a 7-day effect) which is observed when certain parts of the body are shielded. In the first series of experiments either the head was covered with lead and the trimk alone was irradiated or the trunk was covered with lead and the head alone was exposed. Irradiation of the trunk again gave the 3-5-day effect. With larger doses, however, the survival time decreased more slowly than with whole body irradiation. When only the head is moo 10000 100000 r ^ ill:' : •-o_o_^_ \ - ^ '"•^E I n \ \ 7CC0 10000 100000 r >-, lOday May 70 h 1\ lOmin. 1m\n. -2 -3 3 y \ \ I.I ' \ \ ^S\^ -- - 1 III! 1 1 1 1 1 ' -4. % \ 7(9day 7 day 7^K iy\. 7£'min. 7min. i- 5 3 V 5 Lo^ doses V Log doses r A B Figure 3. Survival time of white mice after irradiating the whole body ( ) , and the head only (o o) and beginning of convulsions after head irradiation (• •) irradiated the entire course of the survival curve was essentially the same as for whole body irradiation, except that the survival time for the dose- independent range was seven days and the range is shortened (approximately from 2,500 r to 1 2,000 r). We were led to conclude that two distinct mechanisms were operating in whole body irradiation in the dose-indepen- dent range of the survival curve. With whole body irradiation a 3-5-day efTect, showing itself more rapidly, naturally hides the 7-day effect, which can only appear if the mechanism leading to the 3 • 5-day effect does not act. If large doses are applied the injuries connected with the 7-day efTect probably come to the fore with a shorter latent period. Furthermore, it would appear that the 3 • 5-day efTect is essentially connected with irradiation of the trunk. Figure 3 represents the results of the tests with white mice described above. 84 B. RAJEWSKY Three interrelations for the 3 • 5-day effect seem possible : (7) An important organ of the body is critically injured after application of a certain dose. The injury requires a latent period of 3-5 days until death takes place. Further injuries, which appear when the dose is increased, take a longer time to be lethal and are therefore without any significance as regards the 3 • 5 day effect. (2) After reaching the minimum dose in the independent range a process is started in the body of the irradiated animal by various irradiation injuries, which finally causes the death of the animal within 3-5 days. (3) The changes produced in the body in the dose-independent range are of such kind that continued injuries are compensated by the function of one or several organs. This system of compensation breaks down if a much too large dose is applied. Based on the studies of Miller, Hammond, Tompkins and Shorter^, injury of the intestine, leading to an increased permeability to bacteria and consequently to bacteraemia, appears as a possible mechanism, and Quastler (verbal communication), after confirming the 3-5-day effect, Table I. — Effect of antibiotics on survival time No. of Dose Antibiotic Mean sur- Species animals r used vival time c Mice 62 1,500 penicillin and streptomycin 3-5d+3h •±13-9h Mice 25 1,500 untreated 3-5d-8h ±10-8h Rats 15 1,500 penicillin and streptomycin 3-5d+2h ±7-5h Rats 15 U500 untreated 3-5d-7h ±7-2h believed that it is mainly due to damage of the intestinal epithelia. It was necessary, therefore, to examine this question. For this purpose white rats and white mice were treated with penicillin and streptomycin before irradiation and then were irradiated with different doses. The 3-5-day effect was maintained and the treated animals showed no significant differ- ence from the untreated animals [see Table I). Blood cultures from the control and antibiotic-treated animals were all negative. Consequently bacteraemia cannot be the cause of the 3-5-day effect. The two other possibilities mentioned above were therefore examined using a different technique of lead shielding. The experiments were carried out in such a way that in one series the amount of shielding of the animal's body was increased progressively from the cranial end. In the other series shielding was started at the caudal end portion and increased towards the head. As can be seen from Figure 4A the two injury curves overlap at a point which corresponds to a life span of 3-5 days ; (/) shows both the 3-5-day and the 7-day effect, while from the other curve (//) only the 3 • 5-day effect can be observed, which hides the 7-day effect in whole body irradiation. 85 RADIATION DEATH IN MAMMALS The anatomical location of the cross over 'point' for the 3-5-day effect can be seen from the radiograph of the mouse used as abscissa in Figure 4 A and B. Figure 4. Survival time of white mice after partial body irradiation with 8,000 r. Diagram A shows the survival time as a function of the amount of the body shielded with lead. I Caudal region of the body shielded up to point •. // Cranial region of the body shielded up to point o. Diagram B shows the survival time after slit irradiation in relation to the position of the 5 mm slit in regard to the body 2 3 Posiiion of fhe s/if relafi\/e to the body (taken as abscissa) It corresponds to the region where important organs of the body (liver, spleen, kidney, and a part of the intestine) are located. 86 B. RAJEWSKY Curve (/) in Figure 4 also presents a further dose-independent range of about 5-dav mean survival time between the two constancy ranges of the 3-5-day and 7-day effects. The meaning and more precise localization of this new effect has not yet been clearly recognized. Anatomically considered, however, the effect is not connected with irradiation of the adrenal, or the pituitary gland region. Further investigations on this point are in progress. ' Slit irradiation ' was attempted to locate more exactly the critical organs. With these irradiations the whole animal's body is shielded by lead with the exception of a 5-mm wide slit. In this way only the part of the body actually placed under the slit was irradiated. The slit was brought above different regions of the animal's body in a series of irradiations. The results of these experiments are summarized in the diagram B in Figure 4. It can be seen that the irradiation of only a narrow strip of the animal's body is able to produce both the 3 • 5-day and the 7-day effects. The 3-5-day effect can be obtained by irradiating only the region of the abdominal organs mentioned above, especially by irradiating a zone containing the adrenal and the kidney, while the 7-day effect can be obtained by irradiating the region of the pituitary gland. Slit irradiation of other parts of the body decreased the mean survival time, although to a very much smaller degree than irradiation of the critical regions just mentioned. These results indicate that the two dose independent survival times were essentially due to local injuries and the next step was to determine the relative importance of the different organs located in the sensitive regions. The following experiments were carried out with this end in view : (i) Animals were irradiated, which were splenectomized, adrenalecto- mized or hypophysectomized before irradiation. {2) Individual organs of the animals were irradiated in situ : adrenal, kidney, liver, spleen, intestine, and pituitary gland. (3) Different combinations of these organs were irradiated in situ. (4) An attempt was made to influence the reactions by the administration of certain substances. Details of these experiments will be given elsewhere^ and the most important results only will be summarized. Adrenal — White rats were exposed to radiation over the whole dose- independent range from 800 to 1 5,000 r, but no constant survival time was observed in adrenalectomized animals, and a significant dependency of survival time on the applied dose {i.e. a rapid fall of the medium survival time with increasing doses ; curve -| \- in Figure 5) was found. Irradiation of the adrenal in situ, while all other parts of the animal's body were shielded with lead, did not produce a direct effect ; during the time of observation which lasted for 6 weeks, the animals stayed alive. In another series of irradiations the adrenalectomized animals were treated with 5 mg of cortisone twice daily after irradiation. These cortisone- treated animals showed again the full dose-independent range of the 3-5-day effect. It is, however, remarkable that the dose range over which the 3-5-day efTect occurs was not extended by treatment with cortisone — after further increase of the dose up to approximately 1 5,000 r a rapid fall of the animals' mean survival time occurred according to the general injury curve. 87 RADIATION DEATH IN MAMMALS Pituitary gland — Animals that had been hypophysectomized and irradi- ated with different doses showed a reaction similar to that of the adrenalecto- mized animals. Here also the 3 • 5-day effect disappears and a distinct dependence (Curve o o in Figure 5) of the effect on dose appears. The course of the survival curve is slightly different from that of the adrenal- ectomized animals. Again irradiation of the pituitary gland alone {in situ) produced no effect in the six-week period of observation. Spleen — Splenectomized animals given whole body irradiation with different doses in the independent range (3-5-day effect) behaved like animals that had not been operated; here the complete 3-5-day effect was observed. The mean survival time amounted to somewhat above 3-5 days, the difference, however, is only small. Results of these tests are shown in Figure 5. 700 70 'r >\ N 7L ^ N ^ \ •^ X- \ \ \ \ s \ \ \ Figure 5. Survival time of white rats after irradiation in the dose range l,000r-15,000r • • irra- diated controls [3 • 5-day effect) , -\ \- irradi- ated splenectomized animals, o — o irradiated adrenalectomized animals, X- X irradiated hypophysectomized animals. 2000 6000 70000 Doses — ► 19000 78000 V Intestine — Irradiation of the intestine in situ did not have a significant effect. The survival time was long. The applied doses were within the 3-5-day dosage range. Irradiations of more than one organ in situ — The applied doses were within the range of the 3 - 5-day effect. When liver, spleen, kidney, and intestine were irradiated simultaneously an effect was apparent and the survival time fell to approximately 4 days ; but a constant range could not be definitely determined, because the scatter of the results was relatively great. We hope to answer this question soon when conditions of irradiation are modified. In addition, the following combinations of organs were chosen : {a) liver, spleen, and kidney, (b) liver, spleen, and intestine, {c) spleen, kidney, and intestine, [d) liver and kidney. Liver and spleen as well as liver and intestine were irradiated with 1 5,000 r in situ. In all cases the survival time was reduced gradually and differently. The effect was most obvious with all those combinations in which the liver participated. The experiments are still in progress and the interpretation has to wait until all 88 B. RAJEWSKY doses in the independent range have been tried out. At present we can only say that simiUtaneous irradiation of two or more organs shows a clear effect on the survival time, and is therefore in contrast to irradiation of one isolated organ only. Participation of the liver intensifies the effects. 10 ^gm 100 T -g 2eo ^290 ^220 i 1 1 %'200 \180 — R6- % 160 >, no . \ 120 ^ 100 so ' 1 1 ! ' 1 1 1 I 1 350v ^ ^ 1 1 1 1 1 ' 1 M j l| 1 1 800 V 1 \ 1 1 i -\ -Ro- , y\\ y ' M 1 1 ! ! i lOOOv ! 1 t Ro ' 1 I ;\ \ ; \ i 1 1 1200r Ro 1 1 ■ 1 2500 V 1 Ro' - - 1 i • 1 1 \ \ ' Vv ^ 200 00 T - Ro' — - i \^ -1- \~A — ' 1 12 3 9 12 3 9 12 3 9 12 3 12 3 12 3 Figure 6a. Urinary excretion of corticoids of mice after whole body irradiation. 12 3 days III; fn ^n-m 100 T ^ %.^300 ^ 1200 53 %.1100 ^ 1000 ■Si 900 Ht " 1 § :i 800 1 ^ 700 -2? 600 ^ 500 WO ' i 1 i 1 800 T 1 j / - / 1 r \ - o' I H r\ 11 I ■ 1 1 i 3 ^ 1 li_ 1000 r R y - - T 1 _i 1200 T 7^ 1 -y- k ; J \ 'i \ \ \ -H 1- 1 i i 2500 T R o' 1 1 I \ j \ y 1/ ^ ' 1 1 20000 r [ '8 "111 i \ ( I , aA 1 \ 1 2 3 14 123 123 12 3 12 3 days Figure 6b. Urinary excretion of 17-kelosteroids after whole body irradiation The influence of irradiation on some hormonal systems within the 3-5-day dose range was examined by analysing the urinary excretion of the corticoids and the excretion of 17-ketosteroids of the irradiated animals''. With doses less than those necessary for the 3 • 5-day effect an increase of excretion appears in both cases. At the beginning of the independent range this increase stops relatively abruptly and is followed by a decrease of the excretion. 89 RADIATION DEATH IN MAMMALS In the case of the excretion of 1 7-ketosteroids the dose of 1,000 r has no effect. Decrease of excretion starts at 1 ,200 r and takes place one day after irradia- tion. The excretion of corticoids decreases at l,000r, beginning immedi- ately after irradiation. The results of these tests are illustrated in Figure 6. Blood — Some changes in the blood after irradiation with large doses were examined. For clinical purposes after irradiation the changes in the 'pro- thrombin-time ' was studied in detail. These examinations carried out with RoMER and Beller', showed a clear effect on the blood of m vitro irradiation {see Figure 7). Investigations of metabolism — The metabolism of tissues irradiated in vitro was measured by the method of Warburg^"- ^^ These investigations are still 700 % 90 80 70 60 50 w- 30- 20- 10- Figure 7. Decrease of ''prothrombin time ' after irradiation of blood in vitro ^0, r\ 200 V-00 600 800 WOO Doses kr J I I I I 200 900 600 800 10^ Doses kr Figure 8. Tissue respiration after irradiation in vitro of {A) liver and {B) spleen of white mice ¥00 eoo Doses in progress. Figure 8 shows two examples of the results obtained, i.e. respira- tion of liver and spleen tissue slides after irradiation with high doses. Especially the 'residual' respiration which is preserved after doses of 1,000,000 r had been applied, should be noted. The curves are analogous to those curves obtained earlier by Rajewsky and Inouye^ by tissue irradiation with alpha-particles. FRACTIONATION OF DOSE To clarify the 3-5-day effect we investigated the time intensity factor in this range. As already mentioned, change of irradiation time from 2-5 seconds to 10 hours gave no changes of the 3-5-day effect. We therefore studied, in co-operation with Aurand^, the effect of dose fractionation. I shall illustrate our results with only one example. In Figure 9 the curves for the dose effect are represented for a single and for fractionated irradiation, 90 B. RAJEWSKY respectively with a total dose of l,200r and a time interval of 24 hours between the two part-doses of 600 r each. 1 ' ' . 7200 T A (65 animals) 1 t 600 r BOOrB (66 animals) Figure 9. Percentage of animals dead at different limes after single and fractionated whole body irradiation {total dose l,200r). A single irradiation, B fractionated irradiation with two 600 r doses at 24-hours interval It is not the mean survival time that is shown in this diagram, but the percentage of animals which died at different days after irradiation. A clear effect of fractionating of the dose is apparent. SUMMARY The dose-mortality curve for whole body irradiation of white mice has been determined over the range from 250 r to 200,000 r. The dose mortality curve obtained can be divided into several dose ranges and may be connected with difTerent mechanisms of damage. Irradiation of rats and guinea-pigs showed that the dose efTect curves were similar to that found for mice. With very high doses the survival time is decreased to times which correspond practically to instant death. The mortality curve in the dose range of approximately l,000r to 1 5,000 r shows a plateau where life span of the irradiated animals remains inde- pendent of the dose. It could be shown by irradiation with lead-shielding of part of the body only that this dose range contains three different constant survival times, corresponding perhaps to three different mechanisms of action : (a) 3-5-day efTect (whole body irradiation and trunk irradiation), (b) 5-day effect (irradiation of the central trunk region), (r) 7-day efTect (irradiation of the head). By irradiation of difTerent parts of the body through a slit v/e could localize the efTects mentioned above, in certain regions of the body. Since irradia- tion of certain localized strips of 5 mm width were sufficient to cause these efTects without irradiation of the other parts of the body. The irradiation of animals from which single organs had been removed before irradiation and irradiation of some organs in situ have shown that the adrenal and the pituitary glands are of decisive importance in regard to the development of the 3-5-day and the 7-day efTect respectively. In the cases of the adrenalectomized and hypophysectomized animals no dose-inde- pendent ranges were found and survival time was strongly dose dependent. Irradiations of these organs alone (exteriorized or in situ) did not decrease survival time significantly. Apparently not only injury of the organ itself but also disturbances of the functional systems in which it participates are necessary for the lethal efTects to occur. Treatment with cortisone of the 91 RADIATION DEATH IN MAMMALS adrenalectomized animal restored the 3 • 5-day effect. The irradiation of other organs (single and combined : liver, spleen, kidney, intestine) showed a special role for the liver. The irradiation of the splenectomized animals did not result in a change in the 3-5-day effect. Treatment with penicillin and streptomycin also did not influence the 3 • 5-day effect. After in vitro irradiation with high doses, examinations of blood ('prothrombin time') and measurements of tissue respiration were carried out. To clarify the 3-5-day effect the irradiation intensity was changed or the dose was fractionated. While change in dose rate showed no effect, a significant difference was found if the same dose was divided in two parts. REFERENCES 1 Rajewsky, B. ' Naturforschung und Medizin in Deutschland 1939-1946 (Fiat- Review of German Science 1939-1946) ' Bd. 21 Biophjsik Teil I. .Dieterich'sche Verlagsbuchhandlung, Wiesbaden, 1948. 2 Heuse, O. Z- angew. Physik, 1953, 5 361. 3 Ph. Miller, C, Hammond, C. W., Tompkins, M. and Shorter, G. J. Lab. din. Med. 1951, 38 331, and 1952,39 462. * Rajewsky, B., Heuse, O. and Aurand, K. Z- Naturf. 1953, 8b 157. ^ Rajewsky, B. and co-workers. Z- Natiirf., in press ; Strahlentherapie, in press. * WiLHELM, G. Strahlentherapie, in press. ■^ ROMER, H. and Beller, F. K. 7. International Congress of Radiology, Copen- hagen 1953. * Rajewsky, B. and Inouye, K. Naturwissemchaften, 1937, 25 540. ^ Aurand, K. Strahlentherapie, in press. lOAuRAND, K. and Panly, H. Z- Naturf. 1954, 9b 506. i^Panly, H. Strahlentherapie, in press. DISCUSSION M. V. Haigh : In work with Dr. Edith Paterson at the Christie Hospital, Man- chester, on whole body irradiation of Rhesus monkeys, we also find evidence for a so-called ' protective effect ' of an initial dose of X-ray followed by a second dose at varying intervals later. In this time factor work, our L.D. 50 (30 days) is determined by the probit method. For single session irradiation a series of animals were irradi- ated at 450 r, 500 r, 550 r, 600 r, and 650 r respectively and the L.D. 50 was found to lie between 500 r and 550 r, 20 animals being used in these latter two groups. In the 'time factor' experiments the initial dose of X-ray was fixed at 260 r (approximately \ L.D. 50 for the single session). One group of monkeys then received a second dose of X-rays seven days later and the total dose of X-rays was then found to lie between 700 r and 750 r at L.D. 50 levels. Thus the comple- mentary dose to the initial 260 r at 7-day interval is lower than the L.D. 50 dose for single session. This shows that at 7 days the animals had not recovered from the initial 260 r, and it has not shown a ' protective ' effect. A second group of monkeys given an initial dose of 260 r were irradiated to L.D. 50 levels after an interval of 20 days. The L.D. 50 was found to lie between 850 r and 900 r (on the probit graph at 880 r) and here the complementary dose of X-rays is 880 r — 260 r = 620 r which exceeds the L.D. 50 level for single session. Here the animals have recovered from their inidal 260 r and are apparently protected from a higher dose than they would have survived in single session. This has also been shown to be true in mice* The work has not been extended beyond the 20-day interval so that we have no evidence as to whether after 30 days the protective effect of the initial dose is not any longer apparent. * Paterson, Gilbert, Matthews. Brit. J. Rad. XXV No. 296, Aug. 1952. 92 ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS Maurice Errera Laboratoire de Morphologic animale, Universite libre de Bruxelles In order to understand the mechanisms of the physical effects of radia- tions, it is important to distinguish between primary and secondary effects. From a l:)iochemical or biological standpoint, these must not be confused with initial (or immediate) and late effects which occur through a chain of yet unknown events. Effects observable in a biological system after very short delays may be tentatively classified as initial. Amongst them, retardation and inhibition of mitosis, chromosome breakages, mutations, have been observed very shortly after irradiation, but often these effects become more conspicuous if some time or several cell generations elapse before observa- tion ; on the other hand unless very high dosages are used killing effects are usually observable only after several cell divisions. Whether these fundamental effects are related to one another is still an open question and the answer will depend on whether it is possible to ascribe them to some com- mon initial biochemical step. The search for cytological damage will help to identify the cellular components liable to be altered initially : both damage to the nucleus (chromosome breakage or stickiness, altered morphology of the nucleolus) or to the cytoplasm (swelling, vacuolization, change in staining of mitochondria) have been observed. Attacking the problem from the other end, physical chemists and bio- chemists have usually looked for radiation effects on purified cellular com- ponents treated in vitro (effects on lipids, proteins, polysaccharides or even more simple molecules) . Unfortunately nothing proves that cell components behave in a similar way outside and inside the cell, where they often belong to complex structmes and where they are in close contact with numerous protecting agents. In our opinion, it is of fundamental importance to look for effects on cel- lular components which can be demonstrated immediately after irradiation : the longer one waits, even if smaller dosages are used, the more numerous will be the compounds liable to be altered by secondary biochemical re- actions— small initial biochemical alterations (which as we have seen may be direct or indirect for the physicist) may lead to important secondary effects and it will become more and more difficult to trace the chain of reactions induced by the radiations. We do not want to minimize the importance of these secondary effects, which may have considerable biological conse- quences, but for the biophysicist or the biochemist they will hardly help to solve the fundamental mechanisms one is looking for. It is a priori difficult to predict which cellular compounds will be altered when irradiated in vivo, and which will be the biological consequence of such alterations : our knowledge of ' key ' constituents is still very limited. So 93 ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS far radiobiological work has been of great importance to analyse the possible mechanisms of action of radiations and one is just beginning to uncover the possible mechanisms of primary steps. Unfortunately this has led to very little information concerning the cellular constituents whose alterations produce biological effects. (7) Effects on proteins irradiated in vivo Physical chemists may study the action of radiations on various chemical or physical properties of proteins, if these are treated pure or in solution. This is not possible when the same constituents are irradiated in vivo : methods of purification are not yet adequate and biochemical properties of proteins must be relied on : enzymes may fortunately be studied a very short time after irradiation of whole organisms in rapidly prepared extracts or homogenates : we believe that effects on enzymes found immediately after the irradiation of a variety of diflferent cells or organisms would be of fundamental importance. Barron and his co-workers studied the effects of X-rays on several enzymes in solution and were able to distinguish between two classes of differently susceptible enzymes : those whose activity depends on reduced — SH groups and which are inactivated with high ionic yields (adenosine triphosphatase, hexokinase, succinic dehydrogenase, triose phosphate dehydrogenase) and those whose inactivation needs a higher expenditure of energy (such as ^-amino-acid oxidase, carboxypeptidase, trypsin) and whose activity is not dependent on — SH groups". This scheme is certainly not a definite one, for ribonviclease, classified as radio-resistant by Barron, has recently been shown to be dependent on — SH groups^^^; and the relative difficulties to oxidize these groups by ionizing radiations remains to be explained. But — SH enzymes are within the cell in close contact with reducing agents like glutathione, ascorbic acid or other types of protecting agents and their oxidation in vivo by ionizing radiations, although highly probable, has not been proved to be of direct importance for the cell. The importance of Barron's contribution is to have found specific chemical changes respon- sible for biochemical effects and to have shown that these eflfects may be of great biological importances^. Enzymes have, however, other chemical groups indispensable for their biochemical effectiveness and these may also be susceptible to radiations : this chapter of enzymology is still in its infancy and only a few active groups of a limited number of enzymes are known so far. Free carboxyl groups are needed for the activity of insulin and lyzozyme, some free phenol or indole groups as well as amide or guanidyl groups are needed for both these enzymes and also for trypsin ; S — S groups are required for insulin whilst free amino groups are needed for lyzozyme ^2. Most of these groups are known to be susceptible to chemical effects of ionizing radiations and it is probable that if radiation eflfects on specified protein side groups were better understood one might be able to deduce, as Barron did for — SH groups, which ones of these other groups are most important. Research of this type might lead to the discovery of other protective mechanisms than those already found. We have summarized in Table I what is known of the effect of ionizing radiations on different individual enzymes. On examination of these data, 94 MAURICE ERRERA Table I. — Effects of ionizing radiation on individual enzymes Enzyme Dosage r Tissue Effect observed Author-reference i Adenosine tri- 1 1 640 spleen increase Ashwell & Hickman^ phosphatase 100-800 rat thymus negligible Thomson et al'- — kidney, spleen, liver none Fisher et al^^ Arginase 500 rat, liver none Ludewig & Chanutin^* embryonic tis- slight inhibition Holmes" sue Carboxylase 72.000 yeasts none Aldous & Steward 2 Catalase 500 rat liver none Ludewig & Chanutin*^ 800 mouse liver inhibition of acti- vity begins 2 mins. after irradiation Feinstein et al'-^ 100-500 mouse liver inhibition possibly due to serum cata- lase inhibitor Kazuo Mori et al*^ 72,000 yeasts none Aldous & Steward 2 48,000 growing bar- ley coleoptyle slight rise irradia- Forssberg^i 55,000 Brown Pierce carcinoma slight rise ted in vitro 83,000 rat liver, Jen- sen sarcoma slight rise Cathepsin L50 rat, mouse liver increase (perhaps due to destruction of inhibitor) Ballin & Feinstein' Choline oxidase 200-800 rat liver none Kunkel & Phillips" 600 none Dinning et al^^ Roth et a/" Dehydrogenase : alcoholic 72,000 yeast none Aldous & Steward^ lactic — rat kidney liver-spleen none none Fisher et al-^ malic 800 rat thymus negligible Thomson et a!''- succinic 800 negligible 400-800 rat liver none Le May^2 20,000 sea urchin sperm inhibition Barron el al^" Succinoxidase — rat liver, kidney none Fisher et al'^^ — rat spleen inhibition reversed by SH Triosephosphate 640 spleen homo- genate none Hickman & Ashwell" Deoxyribonu- 500 rat liver slight inhibition Douglas et a/'* clease spleen increase from 4 to 24 hours 500,000 tetrah^-mena none (in homogen. immediately after irradiation) Eichel & Roth2* Esterase 500 rat liver none Ludewig & Chanutin^" Hexokinase 72.000 yeast slight inhibition Aldous & Steward^ 500 spleen, liver slight increase kidnev 95 ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS Table I. Effects of ionizing radiation on individual enzymes — continued Enzyme Dosage r Tissue Effect observed Author-reference Phosphatase 500 rat, thymus, increase during first 1 Ludewig & Chanutin*" (alkaline) liver day rat liver none Ludewig & Chanutin^" 600 rat liver temporary decrease 6 first hours Petrakis et al'>^ 600 rat intestine increase in nuclei and nucleoli of Lieberkuhn crypts Ross & Ely62 bone marrow permanent inhibi- tion for dosages >1500r Woodard & Spiers'^ Ribonuclease 500,000 tetrahymena none Eichel & Roth24 600 rat liver initial rise followed by drop Roth et al^^^ Rhodanese 500 rat liver none Ludewig & Chanutin'^* Transaminase 1,500 rat heart none Brini« rat duodenum inhibition after a few days Brin>« rat liver rise after 2-5 days Brini'i rat spleen inhibition Brinis Tryptophane 1,000 rat liver rise after a few Thomson & Mikuta"* peroxidase hours (not if ad- renalectomized) Xanthine oxidase 600 rat liver no initial action followed by rise Roth et al^^^ Zymase 72,000 yeast slight drop Aldous & Steward 2 one immediately sees that, when irradiated in vivo, most proteins studied so far are very radio-resistant. A same enzyme, in different tissues, may show a variety of responses to irradiation according to the experiment and even — SH enzymes Hke adenosine triphosphatase, hexokinase and ribo- nuclease do not appear to be inactivated by dosages which initiate profound biological damage. (2) Energy sources If instead of individual enzymes, complete chain reactions like glycolysis, respiration or oxidative phosphorylations are investigated, more promising results are obtained. Classical experiments of Crabtree and Gray^'^ have shown that irradiation of rat retinal tissue with l,000r inhibits the formation of lactic acid without altering respiration. Holmes*^ discovered that lactic dehydrogenase was more radio-sensitive in tissues of young embryos than in more mature organisms. Hickman and Ashwell obtained similar inhibitions in mouse spleen homogenates after total body irradiation**^ ; but triose phosphate dehydrogenase (an — SH enzyme) was not affected. The block seems to result from the alteration of an adenine nucleotide phosphorous acceptor and such inhibitions of glycolysis may be related to the fact that liver glycogen increases after in^adiation, even in fasting animals (McKee^s, Ross and Ely^^). 96 MAURICE ERRERA Respiration also responds in some instances to irradiation. The most systematic studies were performed b)' Barron who showed a decrease in the respiration of irradiated tissue sHces and grasshopper eggs. This effect was attributed to the radio-oxidation of the — SH groups of succinic dehydro- genase (Barron, Gasvoda and Flood ^".) Similar results were obtained on liver respiration by Marsili and Paleotti^'. However Le May^- did not find any inhibition of succinoxidase, succinic-dehydrogenase or cytochrome oxidase, and Dubois also found no inhibition-^. In some cases, as reported by BiLLEN, Stapleton and Hollaender, respiration of E. coli is even increased after irradiation^*. An effect of the Krebs cycle seems nevertheless apparent after in vivo irradiation of mice injected with radioactive glucose : there is a marked decrease in the radioactive CO2 produced (Hevesy and Forssberg^^). However all tissues do not respond similarly : C^^Og production is increased immediately after irradiation in the bone marrow (Altman, Richmond and Salomon^). Dubois and Kochran^" have, on the other hand, shown an inhibition of citric acid synthesis in bone marrow. These results indicate that, at least in some tissues, the large cytoplasmic granules (mitochondria) have been affected ; this has also been shown cytochemically by Janus green staining*'*. This is also supported by the numerous instances of inhibi- tion of phosphorylation. Ashwell and Hickman^ showed that spleen homogenates of irradiated animals synthesize less energy rich P bonds, althouarh their succinoxidase is not affected. This inhibition increases during the first 3 days after irradiation, but may in some instances be demon- strated as early as one hour after irradiation (Potter and Bethell^"). Van Bekkum obtained similar results in spleen'*, but in E. coli no inhibition of phosphorylations seems to occur, although one does find a leakage of ATP into the medium^* and Florsheim found no inhibition of phosphorylations in mouse brain^". These inhibitions may be the result of the inactivation of a single enzyme of the chain of reactions if it exists in limiting amounts and if no protecting agent is available at the time of irradiation, as has already been pointed out by BoELL^'\ Thus most of these complex systems may be, but are not always (it may depend on the tissue or experimental conditions), inhibited by irradiation. Very st-imulating experiments by Sherman^^' ^^ may throw some light on these discrepancies. If yeasts are grown on a medium rich in nitrogen, one finds no eflfects of X-rays on glycolysis ; but if these organisms are grown on a synthetic medium poor in nitrogen, dosages of about 30. lO^r will be inhibitory. This may mean that competitive constituents have been eliminated by the low nitrogen diet and the enzymes of glycolysis become more exposed. (5) Synthesis of lipids, proteins and nucleic acids As we have just seen, radiation eflfects on isolated proteins or on energy yielding systems do not appear to be of constant importance : in some cells the activity of these systems may even be increased after irradiation. It was therefore necessary to look for eflfects on systems of a greater complexity still. 97 H ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS Inhibition of protein synthesis or of incorporation of amino acids into proteins does not appear to be of crucial importance either : many instances where these are not immediately affected have been described as can be Table II. Effects of ionizing radiations on lipid and protein metabolism Dosage V Effect observed Author-reference I Protein metabolism Nitrogen fixation in Azo- inhibition Whelden et aP^ tobacter Incorporation of glycine- 500-800 no effect in spleen and Abrams ^ i^C (rat, rabbit) intestine Incorporation of alanine- inhibition after 2 hours Hempelman et aP^ "C increase after 4 days Synthesis of haemoglobin 800 increased in spleen in Richmond, Altman & (rabbit) bone marrow, initial rise in hemin followed by drop ; no altera- tion of globin meta- bolism Salomon*! — increased in reticulo- cytes Bacq et aP Incorporation of ^*C- 880 increased in brain, Hevesy & Dreyfus^* acetate (mice) liver, plasma Antigen formation — anti red cell antibody 700 inliibition from first Taliaferro et al'" (rabbits) day incorporation of ^^S in — inhibition Stevens et aP^ antibodies antibody formation — inhibition, not as Jacobson*^ (rabbit) • marked if spleen and appendix are shielded Adaptive enzymes — lactase of E. coli 20,000 no effect Yanovsky'' maltozymase and galac- 365,000 no inhibition but slight Baron et aP tozymase (yeast) increase in the lag period hydrogenlyase (resting 60,000 complete inhibition Billen & Lichsteini3 E. coli) (no effect on pre- formed enzyme) galactozymase 48,500 no inhibition Brandt, Freeman & Swenson^'^* II Lipid metabolism Fatty acid synthesis bone 800 immediate 2 - 3-fold Altman, Richmond & marrow (rabbit) increase Salomon^ Acetate incorporation 880 increased Hevesy & Dreyfus^' (mice) '*P incorporation in liver 1,000 increased Entenman & Wein- slices (rat) man^* seen from Table II. Synthesis of adaptive enzymes has been inhibited only in a few cases by X-rays, although such an affect is usual with ultraviolet rays. Synthesis of adaptive enzymes is probably related to the presence of 98 MAURICE ERRERA cytoplasmic cell particles. If the cells are made to divide in the absence of the adaptive substrate Spiegelman^^ has calculated that these particles would be diluted between the daughter cells and eventually lost. It would be of interest to investigate if the synthesis of adaptive enzymes is more sensitive to irradiation after a culture of micro-organisms has gone through rapid cell divisions : it would furthermore give an experimental check to Spiegelman's attractive hypothesis. Concerning fatty acids synthesis, one has not found any inhibition by radiation. As this biochemical activity is performed by mitochondria, one must conclude that their sites, responsible for fatty acid synthesis, are not radio-sensitive. It would be of interest, as we have seen that oxidative phosphorylation which is also a mitochondrial activity, may be inhibited, to study different enzymes bound to mitochondria and see if the ones pre- sumed to be bound to the same class of particles undergo inactivation simultaneously or not. Experiments on the synthesis of nucleic acids have given more encouraging results and an inhibition of the synthesis of deoxyribonucleic acid (DNA) has constantly been found in irradiated cells, whereas inhibition of ribo- nucleic acid synthesis or turnover is somewhat less marked or synthesis may even be increased. This is apparent from the work of Hevesy^', Holmes*^, Abrams^, Forssberg^^% Kelly*'' *^, BAcq^ and others and from some of the work performed on bacterial viruses (Latarjet^*') . As we have pointed out earlier, the inhibition of DNA synthesis may be the result of one of the following causes : (a) Inhibition of the synthesis of DNA precursors : this mechanism seems ■ improbable because the synthesis of mononucleotides in yeast or liver cells is not inhibited (Sherman and Forssberg^'^) . Similar results obtained with E. coli with ultra-violet light point to the same conclusion : various low molecular weight derivatives of DNA begin to accumulate immediately after irradiation when the synthesis of DNA is blocked (Kanazir*'*). These data indicate that the block in DNA synthesis probably operates at later stages of this chain reaction. {b) DNA metabolism could become abnormal as a consequence of the incorporation into the normal chain of reactions of an unusual precursor which could be formed by the effects of irradiation on some building block. Many instances of the incorporation of chemical analogues of nitrogen bases have been described in the case of RNA metabolism in mice, bacteria or viruses, and one knows also that abnormal ribosides may be formed by exchange enzymatic reactions (Friedkin^^). {c) Several authors have looked for immediate physical or chemical effects of radiations on DNA irradiated in vivo or in situ. The earliest of these attempts was made on nucleated erythrocytes : the gels obtained with the nuclei of irradiated cells are less rigid than those of controls and one knows that the rigidity of these gels depends upon the presence of ' intact ' DNA'-^i' 22. More recently Limperos and Mosher^^ found that the DNA extracted from thymus of irradiated mice, immediately after irradiation, contained a decreased purine to pyrimidine ratio, ' depolymerization ' being observable only 24 hours later. Harrington and Koza^* found that the affinity for methyl green of the nuclei of irradiated embryos was diminished 99 ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS after 24 hours. These experiments indicate a possible effect on DNA or on the nucleoproteins. However Euler and Hahn^^ found no akeration of physical-chemical properties of deoxyribonucleoproteins extracted from irradiated thymus nuclei ; but unfortunately no quantitative recovery was attempted ; also at the time of the experiment the methods of extraction were perhaps not so gentle as they are now. Anderson^ has recently shown that the high radio-sensitivity of DNA was a test for its degree of depolymerization, which decreases very rapidly in homogenates. More recently, Kaufman^^ found no decrease in the affinity for methyl green of the nuclei of irradiated onion root tips but there is an increase in affinity for fast green and the author thinks this is probably the reason for the smaller degree of swelling of irradi- ated cell nviclei when they are placed in trypsin solution. It must be emphasized that in all these data, tending to show that DNA or nucleoproteins may be affected a very short time after irradiation, most of the evidence so far obtained is very indirect. One method which might give interesting results would be to compare the radio-sensitivity (based on chemical or physical tests) of DNA irradiated dry and at low temperature where indirect effects are reduced to a minimum, in vitro and in organisms like bacteria. One must not forget that at least for DNA, biological tests are much more sensitive to minute alterations of the molecule than physical- chemical ones. This appears clearly from the work of Zamenhof^^ on the stability of the transforming factor ; the study of the transforming activity of DNA extracted immediately after irradiation of the bacteria would indi- cate perhaps if an immediate alteration of DNA is to be found. Actually the results presented in this symposium by Ephrussi-Taylor and Latarjet indicate that such an immediate alteration does occur. Some more direct evidence can be extrapolated from work done with bacteriophage. The inhibition of phage multiplication when infected bacteria are irradiated does seem to indicate that the phage itself is altered because normal phage is capable of multiplying in heavily irradiated bacteria. That unmodified DNA is necessary for phage multiplication is proved by an experiment of Hershey where phage is inactivated by the transmutation of ^^P atoms of its nucleotides^^. Biological effects become apparent only where some cellular constituents are ' limiting ' : biochemical mutations in micro-organisms can be observed only when some important metabolic pathway is interrupted ; this occurs if some building block, normally synthesized by the enzyme lost as a con- sequence of the mutation, is not available for the cell. In more complex organisms it is possible that a chain of reactions may be blocked by radiations in one area but that the products continue to be supplied from another site which is less radio-sensitive. In this case no radiation effect may be observed. To produce observably biological, biochemical, or biophysical lesions the conditions must be such that the factor studied is ' limiting '. The experiments on glycolysis of Sherman, mentioned previously, support this view. The influence of the ' ploidy ' of a cell on its radio-sensitivity is another illustration of this concept. Latarjet and Ephrussi^^ have observed that haploid yeasts are killed exponentially whereas if they are diploid the killing curve becomes sigmoid. A similar effect was found by Zirkle and Tobias''^ : 100 MAURICE ERRERA In the case of the haploid strain, the alteration of a single genetically indispensable ' site ' is sufficient to obtain a killing effect ; two such ' sites ' must be destroyed in the case of a diploid strain. The same explanation might be given to the radio-resistance of the B/r strain of E. coli as compared to the wild B type. CONCLUSION As a conclusion to this discussion, the only clearly apparent immediate effect of ionizing radiation appears to be on DNA metabolism, perhaps through some alterations of the structure of DXA or nucleoprotein itself. This does not necessarily mean that the DNA-containing cell constituents are more sensitive to ionizing radiations than others, but effects on DXA constituents may perhaps be more conspicuous because of their controlling role on cellular processes and perhaps their greater specificity. Conditions on which other cell constituents can be made limiting are not yet well understood. We mentioned the hypothesis of Spiegelman that micro-organisms grown in the absence of adaptive substrate lose their capacity of forming adaptive enzymes, perhaps through the loss of some specialized cell particle. Similarly, Lwoff observed that rapidly dividing Euglena finally lose their chloroplasts. It becomes conceivable that under certain conditions (starvation, rapid division, etc.) limiting conditions might be obtained and could help to explain some of the conflicting results discussed in this paper f effects on protein synthesis or respiration). REFERENCES 1 Abrams, R. Arch. Biochem. Biophys. 1951, 30 90. 2 Aldous, J. G. and Steward K. B. Rev. canad. Biol. 1952. 11 49. ^ Altman, K. I., Richmond, J. E. and Salomon, K. Biochim. biophys. Acta, 1951, 7 460. * Anderson, N. G. Fed. Proc. 1954, 13 3. 5 AsHWELL, C. and Hickman. J. J. biol. Chem. 1953, 201 651. ^ BAcq, Z., personal communication. 7 Ballin, J. C. and Feinstein, R. N. Fed. Proc. 1952, 11 184. * Baron, L. S., Spiegelman, S. and Ql-astler, H. J. gen. Physiol. 1953, 36 631. * Barron, E. S. G.. Dickman, S., Munk, J. A. and Singer, T. S. J. gen. Physiol., 1949, 32 537. 10 Barron, E. S. G., Gasvoda, B. and Flood, V. Biol. Bull.. Wood's Hole, 1949, 97 144. 11 Barron, E. S. G. and Seki, S. L. J. gen. Physiol. 1952, 35 865. 12 Barron, E. S. G. and Johnson, Ph. Arch. Biochem. Biophys. 1954, 48 149. 13 Billen, D. and Lichstein, H. C. J. Bact. 1952, 63 533. 1^ Billen, D., Stapleton, G. E. and Hollaender, A. J. Bact., 1953. 65 131. 15 Boell, E. J. J. cell. comp. Physiol. 1952, Suppl. 2 19. 15^ Brandt, C. G., Freeman, P.J. and Swenson, P. A. Scietice, 1951, 113 383. 16 Brin, M., Fed. Proc. 1952, 11 190. 1- Crabtree. H. G. and Gray, L. H. Brit. J. Radiol. 1939, .V.^. 12 39. !■* Crabtree, H. G. Biochem. J. 1951, 29 2334. 1* Dinning, J. S., Meschan, I., Keith, C. K. and Day, P. L. Proc. Sac. exp. Biol. 1950, 74 776. 19 Douglass, C. D., Fellas. \'. M., Mesch.^n, I. and Day, P. L. Fed. Proc. 1954, 13 201. 20 Dubois, K. P. and Cochran, K. W. Proc. Sac. exp. Biol. 1951, 76 422. 101 ACTION OF IONIZING RADIATIONS ON CELL CONSTITUENTS 21 Errera, M. ColdSpr. Harb. Symp. quant. Biol. 1947, 12 60. 22 Errera, M., Ann. Soc. Sc. med. nat. Brux. 1951, 5 65. 23 Errera, M. et Herve, A. Mecanisme de V Action biologiqne des Radiations. 1951, Liege, Desoer ; Paris, Masson. 2* EiCHEL, H. J. and Roth, J. S. Biol. Bull., Wood's Hole, 1953, 104 351. 25 Entenman, C. and Weinman, E. O. Fed. Proc. 1952, 11 44. 2« EuLER, H. V. and Hevesy, G. Kgl. dansk. Vid. Selsk. Biol. Med. 1942, 17 3. 2' EuLER, H. V. and Hahn, L. Acta radiol. Stockh. 1946, 27 269 28 Feinstein, R. N., Butler, C. L. and Hendley, D. D. Science, 1950, 111 149. 2* Fisher, M. A., Purvis Coulter, E. and Costello, M. J. Proc. Soc. exp. Biol. 1953, 83 266. 3" Florsheim, W. H., Doerbach, C. and Morton, M. E. Proc. Soc. exp. Biol. 1952, 81 121. 31 Forssberg, a. Ark. Kemi, 1945, 21 A no. 7. 32 Fraenkel Conrat, J. and H. Biochim. biophys. Acta, 1950, 5 89. 32"' Fraenkel Conrat, H. Arch. Biochem. Biophys. 1950, 27 109. 33 Friedkin, M. Fed. Proc. 1952, 11 216. 3* Harrington, N.J. and Koza, R. W. Biol. Bull., Wood's Hole, 1951, 101 138. 35 Hempelman, L. H., Carr, S., Franz, I. D., Masters, R. and Lamdin, E. Fed. Proc. 1950, 9 183. 38 Hershey, a. D., Kamen, M. D., Kennedy, J. W. and Gest, H. J. gen. Physiol. 1951, 34 305. 37 Hevesy, G. Advanc. Enzymol. 1947, 7 111. 38 Hevesy, G. and Forssberg, A. Nature, Lond. 1951, 168 692. 39 Hevesy, G. and Dreyfus, D. Ark. Kemi, 1952, 4 337. 40 Hickman, J. and Ashwell, G. J. biol. Chem. 1953, 205 651. "1 Holmes, B. E. Biochem. J. 1935, 29 2285. 42 Holmes, B. E. Brit. J. Radiol. N.S. 1949, 22 487. *3 Jacobson, L. O., Robson, M. J., Marks, E. K. Proc. Soc. exp. Biol. 1950, 75 145. 4* Kanazir, D. and Errera, M. Biochim. biophys. Acta. 1954, 14 62. 45 Kaufman, B. P., McDonald, M. R., Gay, H., Rowan, M. E. and Moore, E. C. Tearb. Carnegie Instn. 1951, 50 203. 48 Kazuo Mori, Seiko Momoki and Hisashi Ito. Igaku to Seibutzaku, 1951, 18 303 ; Chem. Abstr. 1951, 45 9580. 4' Kelly, L. and Jones, H. B. Proc. Soc. exp. Biol. 1950, 74 493. 48 Kelly, L. and Payne, A. Fed. Proc. 1952, 11 256. 48* Klein, G. and Forssberg, A. Exp. Cell. Res. 1954, 6 211. 49 Kunkel, H. O. and Phillips, P. H. Chem. Abstr. 1951, 45 9580 ; Arch. Biochem. Biophys. 1952, 37 366. 50 Latarjet, R. J. gen. Physiol. 1948, 31 529. 51 Latarjet, R. and Ephrussi, B. C.R. Acad. Sci., Paris, 1949, 229 306. 51* Ledoux, L. Biochim. biophys. Acta, 1953, 11 517. 52 Le May, M. Proc. Soc. exp. Biol. 1951, 77 337. 5 3 LiMPEROs, G. and Mosher, W. A. Amer. J. Roentgenol. 1950, 63 681. 54 Ludewig, S. and Chanutin, A. Arch. Biochem. Biophys. 1950, 29 441. 55 Ludewig, S., Chanutin, A. and Leutz, E. A. Amer. J. Physiol. 1950. 163 648. 5* Lwoff, a. C.N.R.S., colloques internal., Unites biologiques donees de continuiti genetique. 1948, Hermann, Paris. 5' Marsili, G. and Paleotti, M. Bol. Soc. ital. biol. sperim. 1952, 28 1269. 58 McKee, R. W. Fed. Proc. 1952, 11 256. 59 Petrakis, N. L., Ashler, F. M., Ferkel, R. L., Naval Radiol. Dep. Lab. Rep. AD 126 B ; Chem. Abstr. 1950, 44 2581. 60 Potter, R. L. and Bethel, F. H. Fed. Proc. 1952, 11 270. «i Richmond, J. E., Altman, K. L and Salomon, K. J. biol. Chem. 1951, 190 817. 102 MAURICE ERRERA «2 Ross, M. H. and Ely, J. O. Amer. J. Roentgenol. 1949, 62 723. «» Ross, M. H. and Ely, J. O. J. cell. comp. Physiol. 1951, 37 163. 63a Roth. J. S., Eichel, H. J., Wase, A. W., Alper, C. and Boyd, B. J. Arch. Biochem. Biophys. 1953, 44 95. 6* ScHERER, E. and Ringelb, D. Strahlentherapie, 1953, 90 34. «5 Sherman, F. G. Fed. Proc. 1951, 10 125. «s Sherman, F. G. Experientia, 1952, 8 429. «' Sherman, F. G. and Forssberg, A. Arch. Biochem. Biophys. 1954, 48 293. 68 Spiegelman, S., De Lorenzo, W. F., Campbell, A. M. Proc. nat. Acad. Set. Wash. 1951, 37 513. 69 Stevens, K. M., Gray, I., Schwartz, M. S., Lighter, R. J., Highland, G. P. and Dubois, K. G. Amer. J. Physiol. 1953, 175 141. ^o Taliaferro, W. H., Taliaferro, L. G. and Janssen, E. F. Science, 1952, 115 487. '1 Thompson, C. B. Amer. J. Physiol. 1952, 170 731. ^2 Thomson, J. F., Tourtelotte, W. W. and Cattar, M. S. Proc. Soc. e.xp. Biol. 1952, 80 268. ■3 Thompson. J. F. and Mikuta, E. T. Proc. Soc. exp. Biol. 1954, 85 29. "* \'an Bekkum, D. VV., Jongepier, H. J., Nieuwekerk, H. T. M. and Cohen, J. A. Brit. J. Radiol. 1954, N.S. 27 127. '» VVhelden, R. M., Enzman, E. V. and Haskins, C. P. J. gen. Physiol. 1941, 24 789. "6 VVooDARD, H. Q. and Spiers. F. W. Brit. J. Radiol. 1953, N.S. 26 38. '7 Yanofsky, Ch. J. Bad. 1953, 65 383. ■^8 Zamenhof, S., Griboff, G. and Marullo, N. Biochim. biophys. Acta, 1954, 13 459. '9 Zirkle, R. E. and Tobias, C. A. Arch. Biochem. Biophys. 1953, 47 282. 103 RELATIVE BIOLOGICAL EFFICIENCY OF FAST NEUTRONS AND GAMMA RADIATION FOR CHRONIC IRRADIATION OF MICE R. H. Mole, R. J. Munson and G. J. Neary Radiobiological Research Unit, Medical Research Council, Harwell A GRAPHITE pile at A.E.R.E. Harwell has been used for dally irradiation of mice with fast neutrons substantially free from gamma radiation. Sterilization of male and of female mice and a reducdon in the weight of the testis have provided sensitive indicators of radiation damage. Dose- response curves for fast neutrons and cobalt gamma radiation have been found to be very similar : there has been no evidence so far of any qualitative difference. Relative biological efficiency factors lay between 3 and 8 for exposures ranging from 24 hours to 5 months : there are definite differences between different strains of mouse. An experiment to determine the relative biological efficiency for lethal effects of low daily irradiation has been started but no results are yet available. DISCUSSION L. Ehrenberg : The authors found the relative biological efficiency of neutrons to vary between different mouse races, and the reason for this has been attributed to different sensitivities to gamma rays. I want to point out that this result is similar to those obtained in plant material : several physiological factors have an influence on the sensitivity to sparsely ionizing radiations, but not on the sensitivity to fast neutrons. This difference between the two types of radiations is probably due to a different mechanism of action. 104 REMARKS CONCERNING SULPHYDRYL PROTECTION AGAINST MAMMALIAN RADIATION INJURY Harvey M. Patt Division of Biological and Medical Research, Argonne National Laboratory, Lemont, Illinois The modification of radiation effects by chemical agents has been and continues to be an important focus in radiobiological investigation. We have learned that a number of substances have the capacity for modifying a variety of responses to ionizing radiations. Viewed in an historical perspective, a number of landmarks appear along the way, but it is perhaps sufficient for our purpose simply to recall the early interest and accomplish- ment in this area. Armed with some understanding of the radiolysis of water and cognisant of its potential contribution to the development of radiation injury in plants and animals, present investigators have greatly extended the possibilities for modification of radiation effects. The list of chemicals that protect against one or another radiation effect ranges from sugar to alcohol, from the familiar epinephrine to the esoteric para-amino- propiophenone. It includes sodium nitrite, cyanide, and pitressin as well as cysteine, glutathione, cysteinamine and other amines. These substances are by no means equally effective and, no doubt, differ somewhat in their mode of action. The more recent developments have been reviewed else- where^. We may note in passing, however, the first definitive experiments on the oxygen effect by Thoday and Read^-^ and the chemical protective effects in animals described by our own group^- ^ and by Chapman^- ' BAcq^' ^ and their collaborators. While certain of the facts of chemical modification are reasonably well established, the mechanisms underlying the facts are frequently obscure or at best only circumstantial. We may define chemical protection for our purpose as the reduction of a given response by administration of a suitable agent prior to irradiation. One is inclined generally to interpret protective phenomena of this sort in radiochemical rather than in biological terms. This implies, as in the case of anoxia, that the protection is due, for the most part, to a decrease in the biological effectiveness of the radiation rather than to a basic change in the sensitivity of the responding system. We will be concerned here mainly with the effects of cysteine which, in certain respects at least, may be thought of as a characteristic example of this type of pro- tection. Other pharmacological agents that are related to oxygen avail- ability in one form or another probably also fall into this classification. This may obtain even for the paradoxical cyanide, which protects some species but not others, potentiates effects on isolated tissues and is evidently neutral to suspensions of isolated cells^. 105 SULPHYDRYL PROTECTION AGAINST MAMMALIAN RADIATION INJURY For the sake of completeness it should be noted that there is another category of modifying factor, one that is concerned apparently with the events responsible for injury to specific physiological systems or to recovery from such injury. Tissue shielding and transplants, pretreatments with phenylhydrazine, foreign protein, oestrogens and other hormones are examples of these rather specific procedures. There is reason to believe that protection by suitable sulphydryl sub- stances, e.g. cysteine, is a fairly general phenomenon and that both de- structive and regenerative processes may be modified. This is apparent particularly when protective effects can be quantitated in terms of radiation dose-biological response parameters. Although the desirability of such evaluation is perhaps obvious, it is worthy of emphasis in view of inferences that are sometimes drawn from single dose determinations or semi-quanti- tative measurements. Under appropriate experimental conditions, cysteine and other sulphydryl substances have been shown to diminish many of the effects of gamma and X-irradiation, e.g. lethality of bacteria, isolated cells and animals, chromosome breakage in plant cells, mitotic block in corneal epithelium, decreased nucleic acid turnover, enzymic changes in spleen and thymus, lenticular opacities, epilation, splenic atrophy, leucopenia and anaemia^' ^^. Cysteine affords a rather uniform protective action against lethality, splenic involution, lymphopenia and granulocytopenia in the intact animaP and the killing of thymocytes and ascites tumour cells in vitro^^. It is worth noting that d and 1 cysteine are equally effective in mice but that all sulphydryl substances are not protective to animals, perhaps because of differences in their biological fate. The latter is a complicating factor, not infrequently overlooked, which must enter into interpretation of in vivo findings. The action of cysteine and p-mercaptoethylamine in mice appears to be similar^-. The latter is more efficient but is also more toxic than the former. It is of interest that maximally tolerated doses of each give equiva- lent protection against acute radiation lethality ; suboptimal amounts seem to be completely additive. Further indication of their similarity of action is apparent also from enzyme protection studies. As shown by Petersen and DuBois^^ prior administration of cysteine or [i-mercapto- ethylamine in roughly equimolar amounts has a comparable effect on the radiation-induced increase in adenosine triphosphatase and 5-nucleotidase activities of spleen and thymus. The degree of protection is a function of cysteine dose and is independent of radiation dose, at least within certain limits. The effect is manifest, therefore, by a change in slope of the radiation dose-response curve. Results obtained in mice with a split-exposure procedure, in which a standard dosage of cysteine was given between two radiation fractions or a propor- tional amount preceded each fraction, suggest that (7) irradiation just prior to cysteine administration does not influence the protection against subse- quent exposure ; (2) the lethal potential of small dosages of radiation cannot be reversed by cysteine ; and (5) the protective action of two suitably spaced cysteine injections is additive^. Essentially similar relationships have been found for the oxygen effect in plants i*. Unlike the picture with animals, there is a definite, although lesser, protective effect when cysteine 106 HARVEY M. PATT is added to a thymus lymphocyte suspension immediately after X-irradia- tion^^. This can be attributed only in part to the persistence of toxic substances in the medium. Differences in the time course of protection of various systems may be related perhaps to differences in the kinetics of reactions with cysteine and in the time constants for development of irre- versible injury. Thymus cells suspended in the test tube represent a fairly sluggish system in which oxygen is rate limiting for the disappearance of cysteine. On the other hand, the systems most responsible for acute lethality of the living animal are those with a rapid turnover in which the chances for intersection of a chain of events are limited accordingly. The possibility exists that some in vivo responses may be amenable to prompt post-irradiation modification by chemicals of this type. Of considerable interest is the fact that cysteine protection in mice is an inverse function of ionization density, being less for fast neutrons than for gamma rays or X-rays^*'. This finding parallels the oxygen effect in irradi- ated systems and is supported by other data^^ It may be stated that cysteine does not protect thymus lymphocytes in the absence of oxygen i^. The resistance of packed thymocytes and the failure of cysteine to protect them may be attributed to their hypoxic state. Cells equilibrated with oxygen before packing by centrifugation appear to be as sensitive as cells in suspension and are readily protected. This is interpreted as evidence for a cellular site of action which is implicated also from studies with tumour fragments. It should be noted that there is a rapid uptake of oxygen and an increase in lactic acid when cysteine is added to the thymic cell suspen- sion. These data suggest perhaps that its action is related to, although not necessarily the direct consequence of, the availability of intracellular oxygen. Potentiation of the protection of the intact animal by suitable dosages of dinitrophenol or sodium nitrite or by 10 per cent oxygen^^ its inhibition by high oxygen tensions i^, and the finding of an increase in the arteriovenous oxygen difference after cysteine injection i'* may also be thought of as sup- portive evidence for this interpretation. It should be remarked that the oxygen content of mixed venous blood does not necessarily reflect the situa- tion in various loci ; hence the failure to detect a change with [i-mercapto- ethylamine^o does not exclude a possible role of oxygen in the protective mechanism. It may be noted also that the dosages used were considerably below those required for protective effects in other species. Certain parallels have been drawn between oxygen poisoning and X- irradiation and may signify a common primary mechanism of action through oxidizing free radical formation as suggested by Gerschman et aP^. It may be noted in particular that some substances that are protective against X-ray injury are also effective against oxygen poisoning. It is well to recall that the time course of the two syndromes differs widely unless many thous- ands of roentgens are used. This may reflect differences in concentration and distribution of the toxic intermediates produced in the two situations, although other interpretations are possible and perhaps more likely. It appears that mechanisms other than the immediate oxidative reactions induced by activated water may also be involved in this form of protection against X-radiation. It has been observed, for example, that protection of thymocytes in suspension by cysteine is dependent upon temperature during 107 SULPHYDRYL PROTECTION AGAINST MAMMALIAN RADIATION INJURY the first 30 to 60 minutes after irradiationii. A brief period of chilling at 2°C immediately after X-ray exposure completely reverses the effect. Temperature dependence of cysteine action before irradiation has also been shown. The latter may be thought of perhaps in terms of a decreased rate of reaction with oxygen in the cold. An explanation of the post-irradiation temperature dependence for cysteine protection of thymus lymphocytes is not readily apparent, but it is clear that we must also inquire about the role of delayed and of chain reactions in the interpretation of these phenomena. The pathways for reaction or degradation of organic radicals formed during irradiation may depend upon oxygen availability in addition to other factors such as pH, temperature and the redox potential". It is possible that decreased oxidation of cysteine during a critical post-irradiation period frees oxygen for reaction with chemical intermediates which might other- wise be directed into other channels. The results with thymocytes may be contrasted with chemical protective effects in bacteria. Several modes of action apparently prevail in the protection of the various systems, owing perhaps to differences in the nature of the chemical intermediates and their reactions, the time required for development of irreversible injury, and the relative contribution of the intra- and extra-cellular phases to the over- all effect. Uniformity of the protection (dose reduction) by cysteine against a number of radiation sequelae in the intact animal suggests that the decisive action occurs at an early stage in the chain of events. It does not necessarily imply protection against all radiation changes since different primary mechanisms may be involved in their development. It should be emphasized that there is scant information concerning protection against the more chronic sequelae of irradiation, although one might suppose a priori that some effectiveness would also be manifest here. For the present, these modifying effects may be interpreted best in terms of the more immediate physicochemical ramifi- cations of energy transfer. Protective chemicals of this sort may alter the effective radical concentration {e.g. of OH or HOg) either directly or indirectly by relative depletion of oxygen. Effects on chain reactions initiated by the primary interactions may also be involved. The experi- mental findings in general are consistent with such assumptions and there is little basis at the moment for postulating a more selective in vivo protection of one or another biochemical entity or physiological mechanism by this class of chemicals. Reference has been made from time to time to the possibility of a primary attack of ionizing radiation on sulphydryl groups generally 2^. More recently, it has been suggested that inactivation of Coenzyme A may be of importance in the initiation of cellular damage by irradiation 2^. There is now ample evidence that sulphydryl enzyme inactivation is neither a selec- tive nor a uniform process in vivo^- 1". The possibility that effects of this sort may occur in areas where — SH groups are few and their inactivation is critical cannot be ruled out, however. It should be noted that there is no evidence for an over-all destruction of Coenzyme A by whole body X- irradiation. As shown by Thomson and Mikuta^s, rats given^ lOOOr retain their capacity to acetylate p-aminobenzoic acid and sulphanilamide for at least 3 days after exposure. While it is true that CoA levels in the 108 HARVEY M. PATT lens are decreased as cataract develops, this is a delayed and not an immedi- ate effect of X-irradiation^*. Evidence for a post-irradiation effect of p-mercaptoethylamine in the liver shielded animal, perhaps suggestive of a biochemical locus, is, at best, conflicting^^' 2^. No doubt, various bio- chemical and physiological states can profoundly affect radio-sensitivity. Protection of mammalian systems with chemicals such as cysteine and P-mercaptoethylamine appears, however, from the preponderance of circum- stantial evidence, to be related mainly to an effect on energy transfer mechanisms rather than to a specific effect on one or another biological determinant of injury or recovery. REFERENCES 1 Patt, H. M. Physiol. Rev. 1953, 33 35. 2 Thoday, J. M. and Read, J. Nature, Land. 1947, 160 608. 3 Thoday, J. M. and Read, J. ibid, 1949, 163 133. * Patt, H. M.. Tyree, E. B., Straube, R. L. and Smith, D. E. Science, 1949, 110 213. ^ Patt, H. M., Mayer, S. H., Straube, R. L. and Jackson, E. M. J. cell. comp. Physiol. 1953, 42 327. ® Chapman, W. H., Sipe, C. R., Eltzholtz, D. C, Cronkite, E. P. and Chambers, F. W., Jr. Radiology, 1950, 55 865. ' Cronkite, E. P., Brecher, G. and Chapman, W. H. Milit. Surg. 1951, 109 294. * BAcq, Z. M., Herve, a., Lecomte, J. and P'ischer, P. Science, 1950, 111 356. ^ BAca, Z. M. and Herve, A. Bull. Acad. ray. Med. Belg. 1952, 17 13. i» Patt, H. M. Anmi. Rev. Physiol. 1954, 16 51. 11 Patt, H. M. Ann. TV. T. Acad. Sci. 1954, in press. 12 Straube, R. L. and Patt, H. M. Proc. Soc. exp. Biol. Med. 1953, 84 702. 13 Petersen, D. F. and DuBois, K. P. Fed. Proc. 1954, 13 394. i* Read, J. Brit. J. Radiol. 1952, 25 336. 15 Paxt, H. M., Blackford, M. E. and Straube, R. L. Proc. Soc. exp. Biol. Med. 1952, 80 92. i« Patt, H. M., Clark, J. W. and Vogel, H. H., Jr. ibid, 1953, 84 189. 17 Mayer, S. H. and Patt, H. M. Fed. Proc. 1953, 12 94. 18 Salerno, P. R. and Friedell. H. L. ibid, 1953, 12 364. 1^ Salerno, P. R. and Friedell, H. L. Radiation Res. 1954, in press. 20 Charlier, R. Proc. Soc. exp. Biol. Med. 1954, 86 290. 21 Gerschman, R., Gilbert, D. L., Nye, S. W., Dwyer, P. and Fenn, W. O. Science, 1954, 119 623. 22 Selected papers : 'The Chemistry of Biological After-EfFects of Ultraviolet and Ionizing Radiations.' Brit. J. Radiol. 1954, 27 36, 117. 23 Barron, E. S. G. Symposium on Radiobiology, p. 216 Ed. by J. J. Nickson. Wiley, New York, 1952. 2* Bacq^, Z. M., Dechamps, G., Fischer, P., Herve, A., Le Bihan, H., LeComte, J., PiROTTE, M. and Rayet, P. Science, 1953, 117 633. 25 Thomson, J. F. and Mikuta, E. T. Proc. Soc. exp. Biol. Med. 1954, 86 487. 2^ Maisin, J. H., Lambert, G., Mandart, M. and Maisin, H. Nature, Lond. 1953, 171971. 109 LE METABOLISME DE LA CYSTEAMINE W. G. Verly*, G. Koch et S. Gregoire Laboratoires de Pathologic Generale et de Biochimie ; Universite de Liege La [i-mercaptoethylamine ou cysteamine est une des molecules qui forment le coenzyme A, le catalyseur des acylations biologiques et de la reaction de condensation du groupe acetyle active avec Toxaloacetate pour former du citrate, premiere etape du cycle de Krebs. La cysteamine ne semble pas etre utilisee pour la biosynthese de la pantetheine ou du coenzyme A ; elle apparait, a Vetat combine, au cours de la biosynthese de ces substances par decarboxylation d'un residu cysteine. De la cysteamine libre pourrait se former dans les tissus par hydrolyse du coenzyme A ou de la pantetheine ; Gregory et Lipmann ont trouve un enzyme qui catalyse cette reaction d'hydrolyse dans le foie de certains oiseaux. Bac'q a montre que 3 mg de cysteamine, injectes dans le peritoine, protegent des souris de 20 g contie 700 r de rayons X, dose mortelle pour 100 pour cent des animaux non proteges. De la cysteamine marquee avec du ^^S a ete preparee par Rayet et Urbain. 3mg de cette cysteamine ont ete injectes dans le peritoine de souris de 20 g et I'on a etudie la persistance du soufre radioactif dans le corps de la souris en fonction du temps apres I'injection, et la distribution du ^^S entre les organes. Le ^^S, par gramme de tissu, est plus eleve dans le foie, les reins et I'intestin (+ pancreas) que dans le reste du corps ^. 24 heures apres I'injection, 34 pour cent du ^^S se trouvent encore dans le corps de la souris^. La protection contre le rayonnement X observee apres I'administration de la mime quantite de cysteamine a une souris de mime poids est de tres courte duree ; la plus grande partie de la radioactivite presente dans I'organisme 24 heures apres I'injection ne se trouve vraisem- blablement plus dans des molecules de cysteamine. On a dose le ^^S present dans des molecules de cysteamine et de cystamine (P : p'-diaminodiethyl-disulfure) dans les tissus de la souris apres injection de la meme dose de ^^S-cysteamine^. 24 heures apres I'injection, 2 pour cent seulement de la cysteamine se trouvent intacts — ou sous forme de cysta- mine— dans le corps de I'animal. La moitie de la cysteamine marquee disparait des tissus en 40 minutes environ. La presque totalite du ^^S present dans I'organisme 15 minutes apres I'injection de 3mg de cysteamine marquee a une souris de 20 g, se trouve dans des molecules de cysteamine ou de cystamine ; 24 heures apres I'injec- tion, on trouve encore 34 pour cent du soufre radioactif injecte, mais 2 pour cent seulement se trouvent dans des molecules de cysteamine et de cystamine. II semble que, pendant les premieres minutes, la disparition de la ^^S- cysteamine soit due principalement a I'excretion urinaire de ^^S-cysteamine- * Associe du Fonds National Beige de la Recherche Scientifique. 110 W. G. VERLY, G. KOCH ET S. GREGOIRE cystamine ; dans la suite, la perte de cysteamine marquee est due davantage a un catabolisme dans les tissus ; le rein elimine a ce moment lui melange de ^^S-cysteamine-cystamine et de catabolites marques. Dans le but d'identifier les produits du catabolisme de la cysteamine, on a injecte dans la veine femorale d'un chien de 7 kg, 104mg de cysteamine marquee avec du soufre radioactiP. En 8 heures, 16 pour cent du ^^S injecte ont ete excretes dans les urines ; 4 pour cent se trouvent dans des molecules de cysteamine ou de cystamine. La cysteamine-cystamine radio- active est surtout excretee pendant les deux premieres heures ; le quatrieme echantillon d'urine analyse (6-8 heures apres I'injection) n'en contient pratiquement plus. On pent conclure que, des 104mg de cysteamine injectes a ce chien de 7 kg, 4 a 5 poiu' cent seulement ont ete excretes sous cette meme forme dans I'urine. Une fraction importante du ^''S urinaire se trouve dans des molecules de sulfates ; on observe egalement une excretion de taurine marquee. Eldjarn a aussi observe la transformation en taurine et sulfates de la cystamine donnee au rat, au lapin et a I'homme. Cysteamine-cystamine, taurine et sulfates ne suffisent pas pour rendre compte de la totalite du ^^S excrete dans I'urine ; il existe d'autres meta- bolites marques qui n'ont pas encore ete identifies. REFERENCES 1 Verly, W. G., BAcq, Z. M., Rayet, P. et Urbain, M. F. Biochem. biophys. Acta, 1954, 13 233. - Verly, W. G., Gregoire, S., Rayet, P. et Urbain, M. F. Biochem. J., 1954, 58 660! 3 Verly, W. G. et Koch, G. ibid, 1954, 58 663. Ill STUDIES ON THE MECHANISM OF RADIATION PROTECTION AND RECOVERY WITH CYSTEAMINE AND P-MERCAPTOETHANOL * Alexander Hollaender and C. O. Doudney Biology Division, Oak Ridge National Laboratory, Tennessee I N an effort to elucidate the mechanism involved in the protection of living material from X- and gamma radiation, a comparison was made of the protective efficiency of cysteamine (p-mercaptoethylamine) and ^-mercapto- ethanol on Escherichia coli B/r. The latter compound was chosen for study 1-0 Molar concn. of cysteamine 0-002 0006 001 0-02 om "I — I — r Cysfeamtne p-mercapfoef Hanoi Figure 1. The effect of cysteamine and fj-mercaptoethanol on the survival of E. coli Bjr during exposure to 60 kr of 250-kVp X-rays 0-025 0-05 0-095 0-1 Molar concn. of ji-mercaptoethanol because it resembles cysteamine very closely in structure and in chemical properties. The effect of concentration on protection against X-ray damage to E. coli B/r at 60 kr for these two compounds is depicted in Figure 1 (upper scale, cysteamine ; lower scale, mercaptoethanol). Cysteamine reaches its maximum protective level at 0 • 006 M, and the plateau for mercaptoethanol is about 0 • 1 M. * Work performed under Contract No. W-7405-eng-26 for the U.S. Atomic Energy Commission. 112 ALEXANDER HOLLAENDER AND C. O. DOUDNEY Figure 2 shows a comparison of the protective ability of the two compounds on E. coli irradiated in a balanced salt solution at 2°C. More than 99-9 per cent of the irradiated bacteria are killed at 60 kr, but in cysteamine at the same dose, only about half of the bacteria are killed. With increasing energy in excess of 60 kr the protective ability of cysteamine decreases markedly and reaches another level of protection at about 90 kr. At submaximally protective concentrations of cysteamine the decline in protective ability comes at correspondingly lower energy levels. However, Figure 2. The effect of cysteamine and ^-mercaptoethanol on survival of X-irradiated E. coli 5/r TO-^ 10' 10' Cysfsaminc fo OS M ) \ p-Mercapfoethanol \ (o-m) \ \ \ .§ , \ R* + H2O ; and reaction with another radical has to occur before the radical is abolished, e.g. R® + OH® > ROH. It would appear that effective protective compounds should be those which facilitate a double reaction involving two radicals. Chelating compounds which, as Bacq, Alexander and Fox pointed out, are very effective are those with two functional groups in close proximity and these are favourably constituted for such a double reaction to occur, e.g. NH2 /NH® /NH® H2O /NH2 \SH SH2 SH® SOH The advantage of a mixed amino-sulphydryl compound may be in the greater size of the S atom, which permits a closer contact of the two functional groups. M. Magat : Je voudrais donner le point de vue d'un physico-chimiste sur le mode d'action des agents protecteurs du type de la cysteamine dans le cas d'action indirecte. Je pense que, si mon analyse est correcte, elle permettra de prevoir la nature des composes chimiques que Ton pourrait envisager comme des protecteurs possibles. La plupart des reactions d'oxydation, de cracking etc, debutent de la maniere suivante : un radical primaire R'j forme par un processus quelconque (dans notre cas par le rayonnement ionisant) arrache un atome, en general un atome d'hydro- gene a la molecule M, en la transformant en un radical, selon le schema : I I -C— -c— — C— H + R'l > — C. + RiH -C- -c- I I Ce nouveau radical, disons un macroradical si nous pensons au DNA, peut subir une transformation, soit en se decomposant avec formation d'une liaison double ou d'un radical, soit par addition d'oxygene en formant un radical peroxydyle etc. Dans d'autres cas, le radical primaire pourra attaquer une double liaison en formant un nouveau radical et s'y additionner conduisant ainsi a une polymerisation. Le role du protecteur P consiste a empecher le systeme de depasser le stade de formation du radical MV Ce but peut etre atteint soit en reagissant preferentielle- ment a M avec le radical initial R* selon la reaction : R* + PA > RA + P' a condition que P* soit un radical relativement inerte, incapable de reagir avec M en lui enlevant un atome ou en ouvrant une liaison double. Pour que la reaction MH + P* > M* + PH ne se produise pas, il faut qu'elle soit endothermique, c'est a dire qu'il faut que I'energie de liaison E(P— H) 118 DISCUSSION soit plus faible que I'energie de liaison E(M — H). Cette condition est assez facile a satisfaire, beaucoup plus facile que la condition que la reaction d'ouverture de double liaison soit endothermique. II se trouve que pour les mercaptans, Es_H < Ec-H) c'est a dire que les mercaptans cedent facilement des atomes d'hydro- gene a des radicaux possedant une valence libre sur un atome de carbone. Le radical RS* est incapable d'arracher a son tour un atome d'hydrogene. II peut par contre ouvrir une double liaison vinylique et amorcer une chaine de polymerisa- tion. Cette propriete des mercaptans est largement utilisee dans Tindustrie du caoutchouc synthetique oii Ton peut ainsi regler a volonte le poids moleculaire des chaines. Si la cellule, ou la partie de la cellule qu'il s'agit de proteger ne possede pas de composes avec des liaisons vinylique, ce qui je crois est une hypothese qu'il est permis de faire, le radical R'S* ne pourra que reagir avec un autre radical, par exemple en donnant R'S — SR' ou R'SR. Les mercaptans ne sont pas les seules substances satisfaisant a nos conditions, mais ils ont I'avantage de reconstituer la molecule M sous forme initiale, certains d'entre eux donnant des sous produits non toxiques (cystine par exemple). Qu'est-ce qui determine maintenant le choix de certains mercaptans, de preference a d'autres ? Plusieurs criteres interviennent probablement : (a) le solubilite dans le milieu entourant les entites a proteger (b) leur stabilite vis a vis du metabolisme (c) la non toxicite des sous produits. Bref, toute une serie de criteres que j'appelerais biologiques. En outre, certaines substitutions peuvent abaisser I'energie de liaison S — H et rendre I'hydrogene plus ' mobile '. Evidemment, et je tiens a insister la-dessus, le mode de protection esquisse ci-dessus n'est probablement pas le seul. Je ne rappellerai que la protection par les thio- carbamates dont il a ete question dans I'expose de Fox. Dans ce cas on pourrait penser, entre autre, a une ' reparation ' de la molecule coupee, le soufre jouant le role d'une ' colle ' et formant un pont entre les deux fragments. En effet, dans I'industrie du caoutchouc, on utilise de tels composes comme accelerateurs de vulcanisation ou meme comme agents vulcanisants ? Toutefois, nos connaissances sont actuellement trop limitees pour que cette suggestion soit autre chose qu'une hypothese tres hasardeuse. P. Alexander : In our joint investigation with Bacq concerning the mode of action of substance which protects mice against the lethal effects of ionizing radiations we reached the conclusion that these substances function by competing for HOj radicals. We were very conscious of the fact that out of the hundred or more substances studied the active SH compounds could act as transfer agents in the way in which Magat describes. However, this property is not shared by all the protective substances and in a detailed series of experiments, which are summarized in the paper in this symposium on page 49. Fox and I showed conclusively that their biological action cannot be ascribed to their ability to act as transfer agents. It is obviously dangerous to pick on one property of SH — compounds, which are capable of under- going many different types of chemical reaction, without considering the other active compounds. This is why we have always insisted that activity in model experiments can only provide information concerning biological mechanisms if the parallelism extends over many active compounds. In my extensive studies on the effects of ionizing radiations on polymers with my colleagues Fox and Charlesby, we have found a number of systems where protection occurs by a transfer mechanism. In these systems different compounds are placed in a different order of effectiveness from that found in vivo or in the synthetic systems where protection occurs by competition for free radicals. There is one other point in Magat's discussion on which I wish to comment and that is his suggestion that there is a general mechanism via peroxide formation by 119 CYSTEAMINE which ionizing radiations degrade polymers. We set out hoping to find such a mechanism and after having studied the effect of X-rays on aqueous solutions of about eight chemically different polymers, we find that almost every one shows a different behaviour. Some degrade only in the presence of oxygen while others degrade equally readily in its absence ; still others crosslink to give gels. The whole picture is very complex and it is not possible to deduce a general mechanism involving oxygen from experiments with one material. In particular the suggestion of Magat that DNA degrades according to this general pattern seems to be unfounded since the initial X-ray induced reaction is independent of oxygen. Further, both polystyrene in chloroform and DNA in water continue to degrade after the irradiation with X-rays is complete (after-effect) but the addition of an — SH compound immediately after irradiation hastens the decomposition of polystyrene (see paper by Fox, page 61) while Errera has shown that it reduces the ' after-effect ' of DNA. The same simple peroxide mechanism can obviously not apply to both systems. H. B. New^combe : In Magat's discussion, one of the points made by Patt appears to have been overlooked, namely that an effect can sometimes be demon- strated even when the chemical is added just after irradiation {e.g. in the case of cysteine and thymus leucocyte suspensions). At this time, the free radicals would have disappeared and it must be supposed that the protection must occur either through reaction with the induced peroxides or through an effect on the post-irradiation metabolism of the cells. R. Latarjet : L'effet final de la cysteamine est la resultante de divers processus dont les importances relatives dependent des conditions experimentales. En par- ticulier, a Faction protectrice /^r^'coce peut succeder une action tardive en sens contraire. En effet, la cysteamine peut favoriser Faction radiomimetique des peroxydes radio- formes. L'experience suivante en fait foi, effectuee par Brinton dans mon labora- toire, conformement a une prevision theorique du Fox. Des bacteries (E. coli, souche B) sont mises, pendant 10 min., en presence d'hydro- peroxyde de cumene a la concentration de 1,5 IQ-^* a 37° C. On les soumet alors a une breve et violente illumination qui augmente beaucoup Faction toxique du peroxyde (preuve que celui-ci agit par ses produits de decomposition). On elimine alors le peroxyde par dilution, et on etale les bacteries sur gelose en presence de diverses substances. Apres incubation, on denomblre les colonies bacteriennes. Voici les resultats d'une experience : Substance ajoutee apres le traitemenl par le peroxyde Nombre de colonies Remarque Neant (temoin) 361 389 Catalase 880 838 Restauration par action peroxy- dasique Diethyldithiocarbamate 602 638 Restauration par stabilisation des peroxydes Cysteamine 1 4 augmentation de l'effet toxique par decomposition des peroxydes Cette experience montre que, loin de proteger, la cysteamine peut agir. apres Firradiation, par un processus qui augmente tres notablement Faction cytotoxique de certains intermediaires chimiques produits dans les tissus par Firradiation. 120 DISCUSSION REFERENCES 1 Bacq, Z. M. et Fischer, P. Arch. Internal. Physiol. 1953, 61 417. 2 Van Cauwenberge et al. Arch. Internal. Physiol. 1953, 61 124. 3 Fischer, P. et Pirotte, M. Arch. Internal. Physiol. 1954, 62 76. * BAcq, Z. M.. Herve, a. et Fischer, A. Bull. Acad. Med. Belg. 1953 — Vie serie t.XVIII, 226. ^ Alexander, P., BAcq, Z. M. et al. Radiation Res., in press. ® Mitchell, J. S. Personal communication, 1954. 7 Bacq, Z. M., Nizet, a. et Herve, A. Arch. Internal. Physiol. 1952, 60 449. 8 Charlier, R. Proc. Soc. Exper. Biol. Med. 1954, 86 290. 9 Bacq, Z. M. Bull. Acad. Med. Belg. 1953, Vie serie, t. XVIII, 426. ^•^ Van Cauwenberge and Betz, H. Lancet, 1952, i, 1083. 11 Mandel, p. et al. C.R. soc. Biol. 1953, 236 2010 121 OBSERVATIONS ON THE EFFECT OF SPLEEN- SHIELDING AND THE INJECTION OF CELL SUSPENSIONS ON SURVIVAL FOLLOWING IRRADIATION * Leon O. Jacobson, Edna K. Marks and Evelyn O. Gaston Department of Medicine and the Argonne Cancer Research Hospital, The University of Chicago, Chicago, Illinois Our finding that spleen-shielding and spleen or embryo transplants enhance the survival of mice exposed to a lethal amount of total-body X-radiation (1025r) led to some further experiments in which we demonstrated that a suspension of spleen cells injected intraperitoneally also increases the survival of mice exposed to 800 r^'^. This latter observation was confirmed by Cole et aP. Lorenz'* found that the survival of mice exposed to 900 r was significantly increased if the animals were given intravenous injections of bone marrow suspensions after X-irradiation. These findings together with the observations that liver-shielding or the injection of mashed embryo suspensions protected against radiation-induced mortality prompted us to investigate the effectiveness of suspensions of cells from the liver of adult, baby, and embryonic mice. In addition, since the results from some of the experiments varied because the suspensions of cells from the several sources (spleen, bone marrow, and liver) were not standardized, it seemed timely to determine the optimum number of cells from each of these tissues that would provide maximum survival and the minimum number of cells necessary to efifect survival following lethal irradiation. Such information was needed to compare the 'potential' of cells from various tissues and to serve as a baseline for 'cell-type' studies of the various suspensions. This report gives the observations that we have made thus far. Observations Bone marrow — Lorenz'* aspirated bone marrow from four long bones (femora and radii) of mice by means of a 26-gauge hypodermic needle. The marrow was then suspended in buffered saline solution and given intravenously immediately after preparation to an irradiated mouse. We employed this technique in our earlier studies. The amount of marrow obtained under these conditions was found to vary considerably and thus the results were not consistent. In the studies reported here, mice that were to receive bone marrow suspensions were exposed to 900 r total-body X-radiation. Bone marrow for the suspensions was obtained from 10- to 12-week-old mice. Saline or Locke's solution was used as the diluent. Cell counts were made according * The contents of this paper have also been presented at the meetings of the 5th Inter- national Congress of Hematology, Paris, France, September 6-12, 1954. 122 LEON O. JACOBSON, EDNA K. MARKS AND EVELYN O. GASTON 15 o I V V 5 c I o s S •I S c: ^ ^ c o O lO C^ CT) CO 'O s2^ O 05 CM Tf CO — rf CO "* lO CO — o • S )^ -^ r-: ^ CO r^ w O t^ r^ — lt; o CO 00 CM CM to CM CM CM CO CM CO CM CM — CO — CO CO — — CO lO CM CM —< CO CM CM CM CO CM ^-, CO ^^ CM _^ C^I o ^^ ^^ ■-C CM CT> ,__, _ CM ^_ CM — ' CO 1—1 ^^ ^^ ■* ^« — ' W5 r^ CM ^^ , , a ■"^ 3 o — — ->! — CM <-; «> in CM C^4 CM >; ^^ >; -* — CM CO CM CO 5o 'it CO *— 1 ^- -f in CM CM e- ^^ -^ CM ^^ CO CO CO « •— ' ^ ~!3 — .— • T}< -o t-- CO "5^ ^^ Si c — CM CO — t^ Oi CM — CO CO ^ CO CO — 'J- lO CO iS Th r-~ - - in CO CM — '■^ CM CO m CO CM CO LT) — - CM CO to Tf in CM — — CO CM C--J — f-^ o ^ .^ i o CO rr; CM ^4 o CM ^ CO CO *— 1 o •^ o o CTi q> in ^ cb 6) 4" O) CT) CT) x^ -* 1 1 T 1 4- 1 O 1 -^ cp cp o o O r- « !>) o lO 6 in _!, CO t^ 'ij CM ^^ ^^ cb O o O) ^ <5 ^ CO CM CM CM in CM CM I ■o ^ <3 ^ CO o in ^ ^ t^ X o 17- o CO CO CO CO r-~ m CM CM CM CO CO in — CO -H JO CM CO in O t^ CO 123 OBSERVATIONS ON THE EFFECT OF SPLEEN-SHIELDING to routine laboratory methods to determine the number of nucleated cells per mm^. Since the suspensions taken from individual mice varied from a maximum of 10 X 10^ cells in 0-5 cm^ of diluent to a minimum of 0-07 X 10^ cells in 0-5 cm^ diluent, pooled marrow aspirations were made and the suspensions were standardized so that 0-5 cm^ contained the number of cells* we wished to administer intravenously to each irradiated mouse. The total number of nucleated cells injected per mouse used in these studies varied from 39-0 X 10« to 0-13 x 10". Although survival was found to be enhanced with as few as 0-5 X 10" cells per mouse, a suspension that provided each mouse with 5 to 10 X 10" cells proved to be the most effective (54-9 per cent survival) in decreasing mortality [Table I). With a suspension of 1 to 5-0 X 10" cells per mouse, 33-3 per cent of the animals survived the 28-day period of observation. As the number of cells was decreased, the per cent survival decreased corre- spondingly ; only 11-6 per cent survived when less than 1 X 10" cells were given to each mouse, and none survived when less than 250,000 cells were injected. It must be emphasized that suspensions containing more than 10 X 10" cells in 0-5 cm^ were no more effective in enhancing survival than those containing 1 to 5 X 10" cells. Of 196 mice that received more than 10 X 10" cells, 81 or 41 per cent survived the 28-day period of observa- tion. The incapacity of these concentrations to provide the protection afforded by lesser amounts is being studied. The results described above were obtained with marrow from adult (10- to 12-week) mice. When young mice (4- to 6-week) were used as donors, the results were more gratifying since fewer cells were necessary to obtain comparable results. There was 78-7 per cent survival when 7 to 8 X 10" cells were given {Table II). Studies are now in progress in which mice that had been irradiated with lethal amounts of X-rays were given less than 3x10" cells and as few as 50,000 nucleated cells. The data suggest that survival is effectively enhanced even when the total number of nucleated cells falls below 1 X 10". Effect of spleen-cell injections on survival of irradiated mice Studies of the effect of cells from the spleen on the survival of irradiated mice were also carried out in a way that was similar to that described for the bone marrow suspensions. In the original experiments, the spleen cells were obtained by inflating the extirpated mouse spleen with saline or Locke's solution. The cells that were thus extruded were injected intravenously into mice that had been subjected to 900 r. In later experiments, the spleens from adult mice were mashed gently in a mortar, or cut and pushed through a syringe, and suspended in Locke's solution. All of the cells that could be removed conveniently with a 26-gauge needle were used to make the suspension. In some instances, the cells were centrifuged and washed several times with Locke's solution before the final suspension was made. Since the spleens from baby mice are very fragile, it is necessary only to push them through a syringe to release the cells desired for injection, and centrifugation is not necessary. * Cells refers throughout the paper to the nucleated cells counted in an acetic acid (2 per cent) dilution. 124 a in o ^ 5 « ^ C O LEON O. JACOBSON, EDNA K. MARKS AND EVELYN O. GASTON O o CO r^ O Survival (No.) {% O O CO ^ 3 r^ to in r^ r^ r-~ CM r- X — ' CM CO CM »— • CM CM — CM lO CM CM CO CM CM CM c CM 5 CO o CT) CO •o lO ?£. t5 1 — ■^ ^^2 l^ -^ X ^Z CT) lO ■ '>C q; CO r-- -- i 4- CM O I I I I I in o — < m in CO in CO "^ cr> c^i I 00 CM U V o 1 o ~« 5 I o 2 "o >. o ■^ ^ "13 C 1? -s^ C^ 5 o I o C3 U -c; >~ to 5 -^ (J 0:; «^ CO 43 -^ ^ o- ^^ cc o — ■ oi •^ ^ .*-^ =^^ CO as l2 o CO c^i o o O '-^^ r^ O C CD CO CO CM ^- CO in c^i * * * CM ^ "*• '>o in o '>c '-C in o CO CM CM — — — — ' Ti---i — — CM— 'OCM in Tf- o '-^ CM CO '-^ — CM r~- O O r- in c in Th o o o r^ in CO o in CM in CTi r-~ CO CO •* CM C^ CO CO CO CO O O O CO CO CM -^ --< — CM •-C c^i -t- CT-. CO CO 'O CM CM "^ CO in bo c o CO CM O in r^ r-~ r^ CO — CM O CM — O c in CO •^ O I^ -^ o-*'co — --^comcoo I I I I I I I 1 I toooi^coincocMin in CO -^ ^ ^ 125 OBSERVATIONS ON THE EFFECT OF SPLEEN-SHIELDING In the original work by Jacobson et al^ in which splenic implants were found to enhance survival, four spleens from baby mice were required to effect increased survival of adult irradiated mice. When spleen-cell sus- pensions were injected intravenously, however, the cells from only one spleen were sufficient to enhance survival in several irradiated mice. To correlate the results of these studies with those from bone marrow experiments, cell counts were made on the spleen-cell suspensions. When 8 to 11 x 10^ nucleated cells from the spleens of 2-day mice were given intravenously, 70 per cent of the recipients survived the 28-day period of observation, with no deaths occurring before the 18th day after irradiation ; with 3 to 5 X 10^ cells, 7 of 12 or 58 per cent survived ; with 2 to 3 X 10^ nucleated cells, 8 of 1 1 or 71 per cent survived ; and with as few as 0-5 to 0-75 X 10^ cells, 70 per cent survived. As indicated in Table III, survival was generally in the range of 50 to 70 per cent regardless of the total number of cells injected. Cells obtained from adult spleen have thus far proved to be less effective than cells from the spleens of younger mice. Instantaneous deaths were frequent after the intravenous administration of cell suspensions containing 10 X 10^ or more cells*. This toxic action can be overcome to a great extent if the adult spleen cells are washed thoroughly by centrifugation in Locke's solution and if a few drops of heparin are added to the suspension made from the washed cells just prior to injection. The data in Table IV provide a comparison of the effectiveness of spleen cells obtained from mice aged 2 days, 4 to 6 weeks, and 10 to 12 weeks. In the latter group, the effect of washing the cells and the addition of heparin are also shown. Although spleen cells from adult mice had little, if any, effect on the survival of mice when 8 to 11 X 10® cells were given, 26 per cent of the irradiated recipients survived when 50 to 60 X 10® cells were injected intra- venously. The addition of heparin did not influence the 28-day survival. The effect of embryo cells on survival of mice — Since earlier studies showed that 35 per cent of mice survived when embryo mash was given intraperitoneally following a lethal exposure to X-radiation (1025r) and since less than 1 X 10® nucleated cells from 2-day mouse spleens were necessary to enhance survival, an effort has been made to determine the number of embryo cells that are necessary to bring about this protective action. Suspensions that were made from cells obtained by pressing the entire embryo through a tissue press or by grinding it in a mortar inevitably proved fatal to the irradiated recipient, which was injected intravenously. Portions of the soft tissue of the embryo (mostly liver) were tested. Suspensions from this tissue were made with Locke's solution and were given intravenously after irradiation. With 10 to 88 X 10® cells, 46-8 per cent of the mice survived 900 r. Fifty per cent survived with as few as 1 to 3 X 10® cells, and 26 per cent survived with 0-3 to 1-0 X 10® cells. With such a small amount as 0-1 to 0-3 X 10® embryo (liver) cells, 25 per cent survived 900 r {Table V). The comparative effectiveness of various tissue-cell suspensions is shown in Table VI. The data indicate that 5-0 to 10-0 x 10® cells per mouse are * Considerable difficulty is encountered when heparin is added to the suspension before the cells are counted since clumping of the white cells under these conditions is marked and accurate counts cannot be made. 126 LEON O. JACOBSON, EDNA K. MARKS AND EVELYN O. GASTON ^ O o en 3 -« ^ "S — C-l CM CO CM CM CM —^ — CO — CM ^^ — . CO m ^ -^ CO CM t^ t-~ ■* — CM CM CO ^O O — -f Oi ^ — ' ■>f CO O CO — . Tt- ^ CM O CO CTl CO o o CO CM ■* r^ CO ■*■ CM CO O CO 00 CO iri CO -r P 1111'' O (^ CO — CO '^ 6 6 o o o_ o" in o in o t-c o o en ^ ■2 o -a I CM ^ ^ o ^ til- r^ O CT) O O tJ- o m "^t- CM ^ CO to CO CO Tt- ^ h CO CM CM CM in CM I CM CO CM CM CM CM I 05 CO H S ■^ cn CO tn CM C-4 — — CO I^ C CM CO O — — CM CM C-l Total No. of cells {X 10«) 14-21 4-3-5-1 2-0-3-0 1-3-1-6 0-75-0-77 127 OBSERVATIONS ON THE EFFECT OF SPLEEN-SHIELDING as effective as higher concentrations. This is probably not, however, a true comparison since the bone marrow represents the effect of adult (10- to 12-week) tissue, whereas the liver and spleen were obtained from embryos or 2-day-old mice. The effect of cells from Ihe liver on the survival of irradiated mice — In previous experiments, it was found that cells obtained from embryonic mouse liver effectively enhanced the survival of mice exposed to 900 r. Therefore, studies were made on cells obtained from the livers of 2-day-old mice to determine whether such cells are equally effective when the liver is no longer an embryonic organ. The mice were killed by cervical fracture, and the liver was removed and mashed by pushing it several times through a syringe, using Locke's solution as the diluent. The cells were removed with a 26-gauge needle and cell counts were made. The suspension was then injected intravenously into the irradiated mice. Table VI. A comparison of the effect of intravenous injections of bone marrow, spleen, embryo and liver cell suspensions on the survival of mice exposed to 900 r Total No. of Survival (%) cells (X 10«) Bone marrow (10- to 12-week) Spleen cells (2-to 5-day) Embryo cells (16- to 20-day) Liver cells (2-day) 0-137-0-925 1-0-4-9 5 -0-9 -9 10-0-14-9 15-0-19-9 11-6 33-3 54-9 42-9 39-5 70-0 65-2 64-2 24-0 41-8 63-2 46-8 20-0 54-0 47-0 In a preliminary experiment, the cells were obtained from 2-day-old CF No. 1 mice and were given intravenously to LAFj females exposed pre- viously to 900 r. No deaths occurred during the first 21 days after irradia- tion ; however, 3 of 8 died before the 28-day period of observation was completed. In subsequent experiments, the CF No. 1 mice were used as both recipients and donors. Of the 97 mice that have been injected with cells from the livers of 2-day mice, 44 (43 per cent) survived. Mice that received 2 to 3 x 10® nucleated cells appeared to survive equally as well as those that were given 4-3 to 5-1 cells {Table VII). With total nucleated cells of the order of 0-75 x 10®, there was 20 per cent survival. A suspension of cells obtained from the liver has been photomicrographed {Figure 1, page 129) to show the various cell types that are present in the injection solution. The effect of injections of irradiated bone marrow cell suspensions on the survival of mice — In some experiments, mice were exposed to 600 r total-body X- radiation. In others, exposures ranging from 100 to 500 r were given. At intervals following exposure, marrow from the irradiated mice was removed and given intravenously to mice exposed previously to 900 r. An attempt was made to prepare a suspension that contained the same number of cells that would produce the maximum protection provided by normal non- irradiated marrow. 128 LEON O. JACOBSON, EDNA K. MARKS AND EVELYN O. GASTON ■T-* t i t3 a p; I ^ ^ t' ^ a .00 129 K OBSERVATIONS ON THE EFFECT OF SPLEEN-SHIELDING Suspensions of marrow made from mice at 0, 1, 3, and 6 hours after exposure to 600 r had no effect on survival when they were injected intra- venously into mice exposed previously to 900 r even though the number of cells obtained was comparable to that from normal non-irradiated mice. By 1 8 hours after total-body exposure to 600 r, the marrow of the donor mice had become so aplastic that 24 mice were required to provide enough cells to make up the desired concentration for injection into 6 irradiated mice. An equivalent number of donor mice was required to provide marrow 24 hours after 600 r. Not only had the factor in the marrow that is effective in enhancing survival been destroyed or inhibited, but some altera- tion in the marrow had also taken place that caused it to have a toxic effect on the recipient mice. Ten of 18 mice died within a few minutes following intravenous administration of this suspension, and none survived beyond the 10th day. The manifestation of this 'toxic' substance was not apparent during the first 18 hours after irradiation, and no studies have been made to determine how long it persists. A striking contrast, however, exists between marrow cells taken from a mouse 1 day after it has been irradiated and those taken 8 days after irradiation. Although it was necessary to sacrifice 36 or more animals to obtain enough cells to inject 4 animals with a suspension containing 9x10^ cells, all 4 survived the 28-day period of observation and no im- mediate deaths occurred. Later experiments revealed that survival, follow- ing the administration of 8-day (600 r) marrow, paralleled that of mice injected with normal marrow. With a suspension containing about 1 -0 X 10^ cells from either normal or 8-day marrow, 25 per cent of the irradiated recipients survived. Only 10 per cent survived when less than 1-0 X 10^ cells were given. Suspensions of bone marrow cells removed from mice 6 days after 600 r were injected into mice that had been exposed previously to 900 r. Indica- tions are that at 6 days some mechanism is already affecting survival. However, the diflficulty encountered in obtaining a sufficient number of cells from such aplastic marrow for a suspension containing about 8x10^ cells has made the extension of this study impractical. Other studies were made in which the donor mice received smaller amounts of radiation ( Table VIII, page 131). The marrow from donor mice exposed to 100 or 200 r was as effective as normal marrow in enhancing the survival of mice exposed to lethal amounts of radiation. Marrow removed as early as I hour after irradiation was as effective as that removed 3 days later. It appears that 300 r depresses but does not inhibit the activity of the 'factor' when the marrow is given 1 day or less after total-body irradiation. Exposures of 400 to 500 r destroy or inhibit the factor when the marrow cells are removed 1 day after X-radiation. However, these exposures, like 600 r, require many donors and give a 'toxic' effect that is expressed in a fairly high frequency of deaths immediately after the intravenous adminis- tration of the suspension. It has been demonstrated recently that the 'toxic' effect is present in the supernatant material * of the bone marrow suspensions. Centrifugation * Centrifuged at 2,500 rpm for 5 minutes. Supernatant was not completely cell-free. 130 LEON O. JACOBSON, EDNA K. MARKS AND EVELYN O. GASTON S 1= o o o o fc ^ ^ ^ ^ t5 3 :| u"! m o o in ^^ 00 lO t~~ t£) lO lO ^ - - — X r^ CO CM o in ^ g oi CO to in ^^ — CO (O — — C^) cs cs CO — > cs in r-l CS CS — H in CS CO — • -"J- — CS CS CO — — Tt- in CS CS CO CO CO CO -- (O r^ -~ ^ CO O -H — CO CS CO o (O CO CO CS CO to CS CJ o^ CO CO o^ ^^ CO ^^ I I I I I I 1 to (O 0-2 o Disrupted cell suspension incubated pvitl? DNA -ase Disrupted cell suspension incubated- no enzyme ^ Cell suspension-untvastied- ^ incubated ivitfi DNA I i\ ase 25% for 30 mn. ^ 220 260 300 IVai^elength S'^O 220 Ta[L 260 300 Wa'/elength SfO Figure 1. Effect of deoxyribonuclease treatment on unwashed mouse spleen cell suspension Figure 2. Effect of deoxyribonuclease treat- ment on disrupted spleen cell suspension suspension, is in striking contrast to that of the unwashed cell suspension incubated with DNA-ase. The optical density at 260 m;^ {i.e. 0-758) corresponds to a Hberation of 39 per cent of the total cellular DNA in the disrupted cell suspension by the DNA-ase. A study was next made of the effect of DNA-ase on a washed spleen homogenate preparation, the cell count of which was approximately equal to that of the cell suspension used above. The spleens obtained from one- week-old mice and weighing 280 mg were homogenized in a Potter ground- glass homogenizer, washed twice with cold 0-14M NaCl, rehomogenized, and resuspended in 10ml 0-14 M NaCl. Two homogenates were prepared concurrently ; one contained 6-9 X 10*5 cells and/or cell bodies per ml : the other 7-9 X 10*= per ml. Both homogenates were incubated at 25°C for 30 minutes, one (A) with added DNA-ase, the other {B) without enzyme. After incubation, the flasks were chilled, the homogenates centrifuged, and the clear supernatants withdrawn for analysis. The supernatant from B 143 NATURE OF SPLEEN-BONE MARROW RADIATION RECOVERY FACTOR gave a negative Dische reaction for DNA ; while the supernatant from A was found to contain 345 [xg DNA per ml, or 3-27mg DNA in the whole supernatant. This represents a solubilization of 43 per cent of the total DNA present in the homogenate. The ultraviolet absorption data on these supernatants, are presented in Figure 3. The high specific absorption at 260 mij. of the supernatant from the DNA-ase treated homogenate is again noteworthy. The differential effect of DNA-ase on a fresh, non-disrupted spleen cell suspension and on a spleen homogenate containing the same number of whole cells, is evident from Figure 4. The experimental data support the view that the DNA as present in fresh, intact, non-homogenized spleen cell suspension is not available as substrate 700 5 Homogenate from 280 mg spleen e-9 — X W^ ce//s/Tr\X incu- bated Witt) DNA-ase. 25°Zfor 30 min. _J \ \ !_ ttomoffenate from 280 mg spteen 7-9 _ x10^ cetts/-mX I'ncu- 6a ted — no ens/me 300 3W 380 Woyelenqtti Ta^i. Figure 3. Liberation of soluble deoxyribonucleic acid from washed spleen homogenate by deoxyribonuclease (J-/ absorption after' incubation witti DNA - ase 25 °C for 30 mm. Suspending medium ^ 0-n M NaCl~ I I -IVostied homogenate' from 280 mg spleen 6-9x10^ c 20 Reg. hepatique jj 25 16,3 18 (90%) JJ 20 Inj. MEA aiant — 5 12,7 5 (2504) >? 40 Controles — 95 23,5 0 {apres \\].) 850 40 Reg. hepatique + inj. MEA avant 8 10 4,8 37 (92,5%) >5 40 Reg. hepatique 35 90 21,1 3 (7,5%) ?3 40 Inj. MEA aiant 12,5 7,9 5 (12,5%) 850 20 Reg. sterno-costale + inj. MEA avant . . 4 35 14,5 15 (75%) ?j 20 Reg. epigastrique + inj. MEA avant . . 51 35 14,25 16 (80%) ; J 20 Reg. sus-ombilicale + inj. MEA avant . . J? 20 9,4 14 (70%) >» 20 Reg. jianc gauche -^ inj. MEA avant . . , , 20 12,25 6 (30Oo) 3 J 20 Reg. sterno-costale . . 53 100 19,25 0 {apres 5 j.) )5 20 Reg. epigastrique 53 90 18,6 0 {apres 6 j.) >> 20 Reg. sus-omUlicale . . 53 70 18.5 7 (35%) J5 20 Reg. flanc gauche 53 95 25,5 0 {apres lOj.) 5) 20 Controles 100 33.5 lO {apres 8 \.) injectes de MEA avant I'irradiation. Cela s'explique par le fait que si tous ces animaux out regu 10 mg de MEA, les premiers pesent plus que les seconds et ont done re9U une dose de MEA proportionnellement moindre. Au cours de ces recherches, nous avons pu faire deux observations impor- tantes, pensons-nous, au point de vue de la comprehension des mecanismes d'action tant de la MEA que de la protection mecanique de I'intestin. Nous avons note que les animaux injectes d'une dose suffisante de MEA avant I'irradiation presentaient peu de phenomenes intestinaux (diarrhees) et une perte de poids moins importante pendant les premiers jours qui suivent I'irradiation. Ce sont la, deux raisons parmi celles qui leur permettent de survivre assez longuement apres I'administration de la dose mortelle de R.X. Ces animaux reprennent du poids des le 5me jour. La majorite meurt neanmoins, apres une dose de 850 r, en presentant une aplasie marquee de la moelle osseuse. La quasi totalite de ces rats presentent une deuxieme chute ponderale. Nous avions note precedemment deja^" que des animaux irradies de 700 r chez qui on protege un volume suffisant d'anses intestinales ne presentent qu'une perte de poids peu importante et peu ou pas de diarrhees, qu'ils reprennent du poids, mais que s'ils n'ont pas eu concomi- tamment un volume suffisant de moelle osseuse protegee, ils presentent une deuxieme chute ponderale et meurent generalement. 163 DU MECANISME PHYSIOPATHOLOGiqUE Nous avons represente dans la Figure 2 les courbes de poids d'animaux irradies par 850 r avec leur region hepatique protegee mecaniquement, avec ou sans injection prealable de MEA, celles des animaiix qui n'ont subi que I'injection de MEA prealablement a I'irradiation et celles des animaux irradies controles. Leur lecture permet de voir que les animaux injectes de MEA seul se comportent comme des rats irradies par 700 r, dont I'in- testin aurait ete protege mecaniquement. lis presentent, en effet, le Seme jour apres I'irradiation, une premiere chute de poids tres peu importante qui est suivie, apres une reprise nette de poids, d'une seconde chute ponderale beaucoup plus importante se situant entre le 13eme et le 17eme jour; nous croyons que cette derniere chute ponderale est due a I'absence de protection de moelle osseuse. De fait les animaux, qui, outre I'injection de MEA avant I'irradiation, ont eu la region hepatique protegee mecanique- ment, qui ont done eu quelques hemi-vertebres dorsales et quelques segments tio 9Z,5%surv 6.i5% costaux proteges, ne presentent guere cette 2eme chute ponderale. A 850 r, quoiqu'en protegeant mecaniquement la region hepatique, nous protegeons quelques anses intestinales ; nous ne reduisons pourtant guere la premiere chute de poids et nous n'empechons pas une importante deuxieme chute ponderale. Nous croyons que cela est du au fait que chez des animaux ainsi proteges nous ne protegeons pas suffisamment d'intestin. En effet, quand a 850 r on protege plus d'anses intestinales et un volume analogue de moelle osseuse comme c'est le cas par exemple en protegeant la region sus-ombilicale ( Tableau IV), on reduit forte-ment la premiere chute ponderale sans, toutefois, encore supprimer entierement la seconde. L'etude histologique des intestins des animaux injectes de MEA avant une irradiation de 700 r et ceux dont I'intestin a ete protege mecaniquement pendant une irradiation de 700 r permet de constater que 24 heures apres I'irradiation, les lesions intestinales de ces animaux sont les memes que celles provoquees chez les controles. Mais les rats dont la region sus- ombilicale a ete protegee ou qui ont ete injecte de MEA presentent respec- tivement apres la 48eme et 72eme heure une activite mitotique intense au 164 J. MAISIN, H. MAISIN ET A. DUNJIC niveau de leurs glandes de Lieberkuhn suivie d'une reparation tres rapide de leurs lesions, alors que, chez les animaux controles, cette activite mitotique ne debute qu'a partir du 4eme-5eme jour. Nous decrivons ces lesions en detail dans une autre communication de ce symposium^^. II se pourrait done que I'absence de diarrhee chez les animaux injectes de MEA avant I'irradiation et chez ceux qui ont eu I'intestin protege mecaniquement soit due a une regeneration plus rapide de I'intestin. Ces observations nous ont pousse a rechercher I'influence de Tinjection intraperitoneale de moelle osseuse apres I'irradiation chez des animaux injectes, avant I'irradiation, de MEA^''. Tons les animaux utilises ici ont un poids moyen de 140 g et ont ete maintenus a jex'm de la fa^on habituelle. Dans ce but, nous avons injecte une suspension de moelle osseuse dans du chlorure de sodium a 9 0/00, chez des rats, immediatement apres, 6 heures et 24 heures apres I'irradiation (groupes I, V et VII du Tableau V). Comme controles, nous avons egalement irradie des animaux uniquement injectes de moelle osseuse directement et 6 heures apres I'irradiation (groupes IV et VI). D'autres rats injectes de MEA avant I'irradiation ont recu de la moelle de jeunes rats de 5 a 6 semaines qui avait ete maintenue a 0°C (groupe VIII) ou a — 40°C (groupe IX) pendant 18 heures. Chaque animal irradie regoit la moelle osseuse des femurs, tibias et humerus d'un rat de 5-6 semaines et pesant environ 50 g. Nous avons injecte a un autre groupe d'animaux, prealablement injecte de MEA, la moelle osseuse des femurs, tibias et humerus de deux jeunes rats de 5 a 6 semaines (groupe II). Nous avons egalement injecte, a des rats prealablement injectes de MEA, la moelle d'un femur, d'un tibia et d'un humerus d'un rat adulte (groupe III). Finalement, une derniere serie de rats injectes de MEA avant I'irradiation a ete injectee de la moelle d'un femur, d'un tibia et d'un humerus, d'un jeune cobaye de 5-6 semaines pesant environ 150 g (groupe X). Etant donne que I'injection de MEA avant I'irradiation semble jouer le role d'une pro- tection mecanique pendant I'irradiation, nous avons voulu completer cette serie d'experiences, en injectant de la moelle osseuse de jeunes rats chez des rats chez cjui nous avons protege mecaniquement de I'intestin pendant I'irradiation. Nous I'avons realise en protegeant les deux flancs des rats par deux ecrans de plomb de Acva^ (groupe XI). Nous avons evidemment egalement irradie des animaux chez lesquels nous n'avions protege que les deux flancs, comme controles (groupe XII). Nos resultats sont rassembles dans le Tableau V. A la lecture de ce tableau, on pent conclure, que I'injection de moelle osseuse directement apres et 6 heures apres I'irradiation permet d'obtenir des survies considerables apres 30 jours pour autant que Ton injecte les animaux de MEA prealablement a I'irradiation. L'injection de moelle a elle seule ne donne aucune survie apres 8 jours et I'injection de MEA seule ne donne que 6,25 pour cent de survie apres 30 jours. Nous permettons done a un nombre interessant d'animaux dont la plupart auraient succombe, s'il n'avait subi qu'une injection de MEA prealablement a I'irradiation, de franchir la periode critique pendant laquelle s'installe I'aplasie medullaire. Rappelons qu'a 850 r, la protection mecanique de moelle osseuse seule {Tableau IV) ne permet aucune survie. Les resultats obtenus dans le groupe II semblent montrer qu"il est inutile d'injecter des quantites de moelle 165 DU MECANISME PHYSIOPATHOLOGIQUE superieuies a celles que nous avons injectees dans le groupe I. Un delai de 24 hevires entre I'irradiation des animaux prealablement injectes de MEA et I'injection de moelle osseuse semble trop long car la survie devient moins Tableau V Nombre de rats Conditions expert men tales Dose de R.X. administree : 850 r en tons cas Animaux diar- rheiques % Moyenne des maximum de perte de poids exprimee en % de la moyenne des poids initiaux Nombre de rats en vie apres 30 jours 59 Gr. I. Inj. intraperit. moelle osseuse un jeune rat directement apres irradiation + inj. MEA avatU . . 23,38 13,1 32 (54,2%) 19 Gr. II. Inj. intraperit. moelle osseuse deux jeunes rats directement apres irradiation + inj. MEA avant . . 10,5 11,7 9 (47,3%) 9 Gr. III. Inj. intraperit. moelle osseuse rat adulte directement apres irradia- tion + inj. MEA avant 44,4 13,9 2 (22,2%) 10 Gr. IV. Inj. intraperit. moelle osseuse jeune rat, directement apres irradia- tion 100 24,4 0 {apres 6j.) 29 Gr. V. Inj. intraperit. moelle osseuse jeune rat, 6 heures apres irradiation + inj. MEA avant 25,1 13,6 12 (41,4%) 10 Gr. VI. Inj. intraperit moelle osseuse jeune rat, 6 heures apres irradiation 100 31,2 0 {apres 8j.) 20 Gr. Wl.Inj. intraperit. moelle osseuse jeune rat 24 heures apres irradia- tion + inj. MEA avant 40 14 6 (30%) 20 Gr. VIII. Inj. intraperit. moelle os- seuse jeune rat maintenu « 0° C durant 18 heures directement apres irradiation + inj. MEA avant . . 30 12,7 6 (30%) 29 Gr. IX. Inj. intraperit. moelle osseuse jeune rat maintenu a —40^ C. du- rant 18 heures directement apres ir- radiation + inj- MEA avant . . 17,2 13,4 4 (13,8%) 40 Gr. X. Inj. intraperit. moelle osseuse jeune cobaye directement apres irra- diation + inj. MEA avant 35 15,G 4 (10%) 20 Gr. XI. Protection region flanc droit et gauche (8 cm') + inj. intraperit. moelle osseuse jeune rat, directe- ment apres irradiation 30 26,6 3 (15%) 20 Gr. XII. Protection flanc droit et gauche (8 cm^) . . 35 33.6 0 {apres 9j.) 80 //;/. MEA avant 23.8 13,2 5 (6,25%) 40 Controles . . . . . . . . 100 33,5 0 {apres 8j.) interessante. La conservation de la moelle a 0° C diminue egalement I'efficacite de la moelle injectee. Les tres basses temperatures sont nefastes a la conservation d'une moelle encore active, les resultats obtenus ne different guere de ceux obtenus chez des rats uniquement injectes de MEA. 166 J. MAISIN, H. MAISIN ET A. DUNJIC Nous croyons etre en droit de pouvoir dire a I'heure actuelle que ces resultats ne sont pas necessairement dus a la pullulation sur place des cellules iujectees. En efFet, nous n'avons pu trouver trace dans le peritoine, de la moelle injectee, 2, 3 et 6 jours apres cette injection. Ces observations ont portes chaque fois sur 3 animaux. Les cellules injectees ont-elles ete absorbees comme telles ou sont-elles detruites sur place ? En d'autres mots sont-ce les cellules injectees ou un facteur qui est responsable de la survie de certains animaux ? Nous ne sommes guere encore en mesure de repondre a ce dilemme. Remarquons, toutefois, cjue les survies obtenues a la suite d'injection de moelle de cobaye ne sont pas demons- tratives. Ces derniers resultats ne plaident done pas pour I'existence d'un fac- teur. D'autres travaux sont cependant necessaires avant de pouvoir conclure. Pour terminer, nous constatons cjue la protection pendant I'irradiation des anses intestinales par deux ccrans de plomb de 4 cm- places au niveau de chacun des flancs chez des animaux injectes de moelle osseuse apres I'irradiation, permet comme on etait en droit de s'y attendre a un certain pourcentage d'animaux de survivre. La difference de survie entre ces rats et ceux qui ont seulement eu les anses intestinales des deux flancs protegees est significative. La survie des animaux dont I'intestin a ete protege de cette fagon est neanmoins beaucoup moins interessante que celle des animaux chez qui Ton a remplace cette protection par une injection de MEA. Peut-etre ne protegeons-nous pas suflfisamment d'anses intestinales ? La perte maximum de poids semble plaider en faveur d'une telle hypothese. Nous avons essaye de repeter une experience semblable en protegeant chez 20 animaux toute la masse des anses intestinales, exteriorisee dans une boite de plomb, mais sans succes. En effet, a 850 r, ces animaux ne resistent pas au choc operatoire et meurent tons endeans les 9 jours. Les courbes de poids des animaux injectes de MEA avant I'irradiation et apres celle-ci de moelle osseuse sont, chaque fois que Ton injecte de la moelle de jeune rat dans un delai de 6 heures et quelque soit la quantite injectee, semblables a celles d'animaux chez qui Ton aurait protege de la moelle osseuse mecaniquement apres les avoir injectes de MEA. Remarquons que la courbe de poids des animaux qui ont regu, outre, I'injection de MEA une injection de moelle de cobaye se comporte d'une fac^on assez semblable a celles des rats uniquement injectes de MEA. L'allure de la courbe de poids des animaux injectes de moelle de jeune rat conservee a — 40°C n'est guere meilleure. Les courbes de poids des animaux injectes de moelle de rat adulte ou de moelle de jeune rat conservee a 0°C sont intermediaires entre ces dernieres et celles de ceux injectes endeans les 6 heures apres I'irradiation. La courbe de poids des animaux a intestin protege est la plus mauvaise. Elle se caracterise surtout par le fait que la premiere chute ponderale est tres importante, elle atteint 26,6% et elle reste basse jusqu'au 16eme jour, comme si le volume d'intestin protege etait nettement insuffisant, hypothese que nous avons deja formulee plus haut. RESUME Nous avons notamment montre dans la deuxieme partie de ce travail que I'injection de MEA avant I'irradiation diminue les phcnomenes gastro- intestinaux et la perte de poids concommitante a ceux-ci, comme si on 167 DU MECANISME PHYSIOPATHOLOGiqUE protege I'intestin de ces animaux mecaniquement. Ces animaux presentent d'ailleurs, tout comme ceux chez qui on n'aurait protege que I'intestin mecaniquement, une deuxieme chute ponderale et une survie finale peu importante ; a 850 r il n'y en a que 6,25% qui survivent 30 jours. Pour augmenter ce taux de survae et le faire passer a 75 % par exemple et supprimer la deuxieme chute ponderale, il faut proteger concomitamment un volume suffisant de moelle osseuse. Nous montrons par ailleurs que I'injection de MEA avant une irradiation de 850 r permet d'obtenir un taux de survie atteignant de 45-60% apres 30 jours chez des animaux injectes d'une sus- pension de moelle osseuse homologue endeans les 6 heures apres I'irradiation; ces animaux ne presentent pas de deuxieme chute ponderale. L'injec- tion de moelle osseuse seule ne permet pas aux animaux de survivre au dela de 8 jours. Les resultats obtenus en injectant de la moelle de cobaye a des rats prealablement injectes de MEA, sont peu concluants. Nous tenons a remercier notre laborantine Mademoiselle A. Guillaume de son assistance technique devouee. La mercaptoethylamine utilisee au cours de ce travail provient des Laboratoires Labaz a Bruxelles. REFERENCES ^ Jacobson, L. O., Marks, E. K., Robson, M. J., Gaston, E. et Zirkle, R. E. J. Lab. din. Med. 1949, 34 1538. 2 Mandart, M., Lambert, G., Maisin, H. et Maisin, J. C.R. Soc. Biol. Paris, 1952, 146 1647. ^ Maisin, J., Mandart, M. et Lambert, G. Communication faite au premier Congres des medecins electroradiologistes de culture latine (Bruxelles, juillet 1951). J. beige Radiol. 1952, 35 337. * Gershon-Cohen, J., Hermel, M. B. et Griffith, J. Q..,Jr. Science, 1951, 114 157. '' Maisin, J., Dunjic, A., Van Lancker, J., Lambert, G. et Passau, L. C.R. Soc. Biol. Paris, 1953, 147 1520. "Mandart, M., Lambert, G. et Maisin, J. ibid, 1952, 146 1305. ' Maisin, J., Van Lancker, J., Dunjic, A., Lambert, G. et Passau, L. ibid, 1952, 147 1517. * Mandart, M., Lambert, G. et Maisin, J. ibid, 1952, 146 1645. 9 Mandart, M., Lambert, G. et Maisin, J. ibid, 1952, 146 1392. 10 Maisin, J., Dunjic, A. et Maisin, H. ibid, 1954, 148 611. 11 Maisin, J., Maisin, H. et Dunjic, A. ibid, 1954, 148 743. 12 BAcq, Z. M., Herve, a., Lecomte, J., Fischer, P. et Blavier, J. Arch. int. Physiol. 1951,59 442. 13 BACCi, Z. M. et Herve, A. Bull. Acad Med. roy. Belg. 1952, 17 13. 1* Lambert, G., Maisin, J. et Mandart, M., C.R. Soc. Biol. Paris, 1952, 146 1434. 15 Maisin, H., Wolfe, R., Tobias, C. A. et Lawrence, J. H. Observation non publiee, 1953. 1® Maisin, J., Maisin, H. et Dunjic, A. Communication faite a la Soc. de Biologic du 29 mai 1954. C.R. Soc. Biol. Paris, 1954, 148 1293. 1' Maisin, J., Maisin, H. et Dunjic, A. Observation non publiee, 1954. 1* Maisin, H. et Fievez, C. Symposium de Radiologic, Liige, 1954. Butterworths, Londres, 1955. DISCUSSION C. Burg : On a I'impression que le probleme des substances protectrices presen- terait un aspect different s'il etait envisage, non pas vis-a-vis d'une dose unique, 168 DISCUSSION lethale de rayons X, mais par rapport a une irradiation en quelque sorte chronique ; mais finalement lethale. Sans nier I'interet du premier aspect de la question, on peut se demander si pour I'etude du mecanisme d'action des agents de protection, il ne serait pas plus simple, malgre les apparences, de s'adresser au second cas. En effet, I'effet brutal des radiations declanche une intervention de toute la serie des glandes endocrines et met probablement en jeu une serie de processus surajoutes, qui peuvent compliquer singulierement la question. B. JoLLES : I should like to ask Maisin whether he noted if any of the deaths of the animals in his experiments with regional shielding fell in the '3-5 days ' category of deaths described by Rajewski. I ask this question because in a series of experi- ments with sieve irradiation when studying survival curves of animals irradiated with open and sieve fields (double the open field dose through a 50 per cent sieve) it was noted that while the number of survivors in the ' sieve ' group was greater than in the ' open ' field group, death when it did occur in the sieve animals, took place at an earlier date than in the open field group. Betz : Les experiences que Maisin vient de nous exposer sont interessantes en ce sens qu'elles montrent la multiplicite des facteurs reglant la regeneration hema- topoietique du rat irradie. J'aurai I'occasion de presenter ici meme des resultats qui plaident dans le meme sens. S'il est difficile de dire comment agit la protection de I'intestin, je voudrais ajouter une hypothese a celles formulees par Maisin. Nous avons montre que I'hypercorticisme de I'animal irradie exerce une action inhibitrice sur la regeneration hematopoietique. On peut penser qu'en protegeant I'intestin, on diminue considerablement I'hyperactivite du cortex surrenalien. On sait en effet que la reaction surrenalienne est particulicrement intense lorsque la masse intes- tinale est comprise dans le champs de I'irradiation. 169 LABORATORY STUDIES AND CLINICAL TRIALS OF SOME CHEMICAL RADIO-SENSITIZERS J. S. Mitchell Department of Radiotherapeutics, University of Cambridge This paper deals mainly with work completed and in progress during the past year and is intended to supplement the review of the subject which I gave in 1953 at the 7th International Congress of Radiology (Mitchell^). The aim of this investigation is to try to improve the results of radio- therapy of some types of cancer by the ancillary use of chemical agents designed to act as radio-sensitizers. It is emphasized that in general these chemicals when acting alone are not chemotherapeutic agents. In addition to its practical importance, the possibility of chemical radio-sensitization appears to be of theoretical interest. The first compound studied, which is still perhaps the most interesting, is tetra-sodium 2-methyl-l : 4-naphthohydroquinone diphosphate (' Syn- kavit ' ; Compound I). Clinical trials of its use have been in progress since November, 1946, and I have employed it in the treatment of about 1070 patients mainly in conjunction with radiotherapy. I wish to emphasize the more recent clinical studies which are in progress and include the design of a clinical trial for evaluation of a radio-sensitizer. In August, 1953, a special clinical trial was started to examine the combined use of intravenous Com- pound I (Synkavit), oxygen administration before and during irradiation and X-ray therapy, in the treatment of some patients with advanced cancer. The results of various aspects of this work have been published by Mitchell and Simon-Reuss^ in 1947, Mitchell^"^ in the years 1948-52, Mitchell and Simon-Reuss^- ^° in 1952, Hughes and Simon-Reuss^^ in 1953. In addition, a rmmber of papers dealing mainly with mitotic inhibition have been published on the following groups of compounds and related aspects of the problem : Certain quinones by Friedmann, Marrian and Simon- Reuss^^ (1948),svilphydryl addition compounds of some quinones and related compounds by Friedmann, Marrian and Simon-Reuss^^ (1948), maleimide and related substances by the same authors'* in 1949, the reactions of sub- stituted maleimides with thiols (Marrian'^ in 1949), the condensation of N-substituted maleimides with thiourea (Marrian*^ in 1949), the action of 1 : 4-naphthohydroquinone diphosphate (Friedmann and Bailey*'''), unsaturated imides with special reference to their reaction with sulphydryl groups (Friedmann, Marrian and Simon-Reuss^^, 1952), halogen deriva- tives of the 1 : 4-naphthoquinone group and maleic acid series (Friedmann, Marrian and Simon-Reuss*^, 1952), a spectrometric investigation of the interaction of glutathione with maleimide and N-ethylmaleimide (Fried- mann 2*^, 1952), and the action of X-rays on the glutathione 1 : 4 naphtho- quinone reaction (Friedmann-^ 1954). As a different method of approach, studies have been reported on the antagonization of the antimitotic action of 170 J. S. MITCHELL tetra-sodium 2-methyl-l : 4-naphthohydroquinone diphosphate (Com- pound I) by nucleotides, nucleosides, purines and pyrimidines and some other compounds by Mitchell"' in 1950 and 1951. The report by MitchelF has been criticized by Gellhorn and Gagliano^- (1950). Berkmax" (1951, 1953} published clinical results similar to those of Mitchell. Clinical and laboratory studies have been published by Jolles^^ (1952). Experiments on the Ehrlich mouse carcinoma have been described by Dittrich and Schmermund" (1953). An account of six years' clinical studies and attempts at quantitative clinical assessment of Compound I as a radio-sensitizer in the radiotherapy of malignant tumours has been published (Mitchell, 1953). Recently, oxidation-reduction potential and pH measurements have been made m vivo using the Walker rat carcinoma 256 and the Jensen sarcoma to study the effects of oxygen, Synkavit and X-rays alone and in combination (Cater and Phillips 2*5). J^ew Compounds The attempt to devise compounds which incorporate radioactive atoms and may concentrate in malignant tumours has led to the development of tetra- sodium 6-iodo-2-methyl-l : 4-naphthohydroquinone diphosphate ('6-iodo Synkavit 'j. The synthesis of this compound was carried out by Dr. K.J. M. Andrews, of Roche Products, Welwyn Garden City, by the kind arrange- ment of Dr. A. L. Morrison. The tissue-culture studies made on chick fibro- plasts by Mrs. Simon-Reuss show that as expected the introduction of the iodine atom in the 6-position slightly reduces the activity as measured in terms of the concentration producing 50 per cent mitotic inhibition by a factor of about 1 -5 but otherwise produces cytological effects very similar to those of Compound I. The idea which I had of incorporating a ' radio-mimetic ' group and a radio-sensitizer in the same molecule as a related method of improvement upon Synkavit, has led to the development of 2-ethylenimino- 1 : 4-naphtho- quinone. After reading the paper by Domagk, Petersen and Gauss" (1954), this seemed a natural step. O O 2-ethylenimino- 1 : 4-naphthoquinone The compound 2-ethylenimino- 1 : 4-naphthoquinone and the corre- sponding known open-chain compound 2-^-hydroxyethylamine-l : 4- naphthoquinone (Fieser, L. F. et al'-^, 1948), have been synthesized in this Department by Mr. D. R. Maxwell, with the advice of Dr. D. H. Marrian. Animal experiments in progress using the Walker rat carcinoma suggest that 2-ethylenimino- 1 : 4-naphthoquinone has some action in producing tumour retrogression by itself, in addition to radio-sensitization when used in combination with therapeutic doses of X-rays. The tissue-culture studies by Mrs. Simon-Reuss using chick fibroblasts show that 2-ethylenimino- 1 : 4- naphthoquinone produces 50 per cent mitotic inhibition after 24 hours in concentration approximately 4 X 10^ M with a striking accumulation of 171 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS cells in prophase and metaphase especially in lower concentrations. Similar changes are found with the Ehrlich ascites tumour. Tissue-culture studies With regard to the general technique with tissue-culture experiments, experimental developments during the past few years to study mitotic inhibition, cytological effects generally and radio-sensitization by chemical agents have proved satisfactory. Compound I and Compound XXVIII are the usual reference substances. Probit analysis has been used. The study of the combination of the action of X-radiation and Compound I by the sum- mation method has demonstrated potentiation of both mitotic inhibition and chromosome fragmentation (similar results have been obtained in some experi- ments on the roots of Allium Cepa by Mr. K. C. Bora ; it has also been found that in this material the compound 2-ethylenimino-l : 4-naphthoquinone pro- duces gross inhibition of the growth of roots in concentration 10~^M). The problem of the thermal instability of Compound I which appeared to account for some erratic results in some animal experiments and clinical trials, appears to have been solved by the finding that this thermal instability is associated with the presence of an easily removable impurity in the aqueous solutions in the ampoules in the absence of oxygen (Dr. A. L. Morrison). With the purified preparations of Compound I, it now seems possible to obtain consistent results without the necessity to store the ampoules at 3° C. An interesting experiment by Mrs. Simon-Reuss is reaching completion and will be reported in detail elsewhere. The influence of Synkavit and its effects as a radio-sensitizer on chick fibroblasts in culture have been followed after subcultivation for up to 17 passages. A pure strain of chick fibroblasts was divided into batches of cultures ; one batch was treated with Synkavit for 24 hours in concentrations 2, 3, 4 x 10~^M respectively in different experiments. The other cultures were kept for controls for testing the effects of Synkavit and of irradiation. The whole strain was subcultured every 48 hours, washed in Tyrode solution and transferred to fresh medium con- sisting of plasma and embryo extract only. At various intervals six cultures were taken from each group and the group formerly treated with Synkavit was irradiated at 18 hours and a control group was similarly irradiated. Further, one of the treated groups and one of the untreated groups were kept as controls. These cultures were fixed and stained and counted after 24 hours. The experiments lasted for 16, 16 and 17 passages, respectively for 5-54- weeks. The cultures originally treated with Synkavit alone showed no abnormality and no mitotic inhibition and were in no way diflferent in appearance from the controls. The untreated cultures irradiated with 150r showed 22-28 per cent mitotic inhibition throughout the whole experiment. Those treated originally with 2 X 10~^M Synkavit and irradiated with 150r showed mitotic inhibition starting off' with 75 per cent for three passages, then falling to 60 per cent at the sixth passage and 26 per cent at the tenth passage. The cultures originally treated with 4 X 10~^M Synkavit showed no inhibition and no cytological abnormality. 300 r produced 40 per cent inhibition in the controls, but 300 r administered to the subcultures of the strain originally treated with Synkavit showed 94 per cent mitotic inhibition persisting for several subcultures with 80 per cent at the fourteenth passage 172 J. S. MITCHELL and 60 per cent inhibition at the sixteenth passage. It appears from these experiments that initial treatment of the cultures leads to a persistence of radio-sensitivity in the subcultures. To assess the part played by retained Synkavit in the cultures, Mrs. Simon-Reuss has carried out independent experiments in which it has been found that radio-sensitization persists down to a concentration between 10"^ and 10" i« M Synkavit. Some experi- ments are in progress with ^*C labelled Synkavit. Possible mechanism of radio-sensitization Compound I appears to be selectively concentrated by the tumour cells with a high differential absorption ratio. The phosphate groups are probably essential for its passage through the cell membranes. Inside the tumour cell it appears to be concentrated in the perinuclear region perhaps in the mitochondria. The parent 2-methyl-l : 4-naphthoquinone is likely to be formed in situ and react specifically with sulph)dryl compounds. In this way, selective radio-sensitization of the tumour cells by Compound I is envisaged as the converse of — SH protection against ionizing radiations. Then increase in the oxygen tension in the tumour cells is likely to be associated with optimum use of the available oxygen for increasing the effects of the radiation. It is of interest that in the measurements by Drs. Cater and Phillips of oxidation-reduction potentials in vivo in the Walker rat carcinoma it was found when the animal breathed oxygen the potential taken up by the platinum electrode relative to the silver-silver chloride electrode increased by 10-50mV, while intravenous injection of Synkavit (5-lOmg per 200g rat) caused an immediate fall of 30 to 200 mV followed by gradual recovery. The observation that the mitotic inhibition produced in chick fibroblast cultures by Compound I is abolished by an equimolecular concentration of guanosine, but not by isoguanine riboside, guanylic acid, guanine or any other related compound suggests a chemical specificity in the action of Com- pound I. It is not impossible that like some 2-OH-3-alkyl-naphthoquinones (Ball, Anfinsen and Cooper^^, 1947, see Potter and Reif'o, 1952), it pre- vents the reduction of cytochrome C and interferes with some process of phosphorylation. A problem is raised by the observation that tetra- sodium 2 : 3-dimethyl-l : 4-naphthohydroquinone diphosphate, Compound XXVIII (Mitchell and Simon-ReusV- ^o, 1952, and Mitchell^, 1953) is a good, radio-sensitizer for both chick fibroblast cultures and the Walker rat carcinoma and does not react in vitro with sulphydryl compounds. Animal experiments The remarkably low acute and chronic toxicity of Compound I has been confirmed in experiments on rats, mice and rabbits. In rats, the repeated intramuscular administration of the compound in large doses increased the mortality following exposure of the whole body to X-radiation. Experi- ments by Jolles^* (1952) in the rabbit and guinea-pig suggests species differences. Further experiments are in progress. It has recently been found that using similar methods 2-ethylenimino-l : 4-naphthoquinone shows no acute toxic effects after intravenous administration in doses in the region of 2-5 mg per kilo in rats, mice and rabbits. 173 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS The earlier therapeutic experiments with Compound I were sufficiently suggestive to provide a basis for clinical trials, but were far from satisfactory because of the rather small number of animals and the high rate of spon- taneous retrogression of the tumours. This difficulty appears to have been overcome by maintaining the Walker rat carcinoma as ascites tumour and in recent experiments almost invariably using solid tumours at the second passage from the ascites tumour. I wish to thank Professor A. Haddow for his help in supplying animals and tumours, with which to start these experiments. Two large-scale therapeutic experiments with tetra-sodium 2-methyl 1 : 4-naphthohydroquinone diphosphate (Compound I) and tetra-sodium 2 : 3-dimethyl-l : 4 - naphthohydroquinone diphosphate (Compound XXVIII), on the production of permanent retrogression of the Walker rat Figure I. Arrangements for roentgen irradiation of experimental tumour in rat. The rat is wrapped in a flexible lead plate with the tumour projecting through an elliptical aperture. carcinoma 256 have now been completed. The details of the experiment with Compound I are summarized in Table I. The results for Compound XXVIII (Mitchell ^ 1953) are compared with those of Compound I in Table IL Experimental Methods— The main features of this type of animal experiment with the Walker rat carcinoma 256 in the rat, are given in Tables I and //. The following experimental details may be added. For the irradiation of the tumours in groups C and D the rat is wrapped up in a lead plate of thickness 1-3 mm with the tumour projecting through an elliptical aperture in the plate of dimensions 4 X 3 -3 cm. A con- venient size for the lead plate is 21 cm X 17 -5 cm transversely ; the elliptical aperture reaches to about 1 cm distance from the middle of the longer edge. The experimental arrangement with the rat held in position in this way for irradiation is shown in Figure 1. The selection of the tumour size in relation 174 J. S. MITCHELL < CO ^ -1 lO frt W5 si -" O E d h -n c rt 5 :: fi u -a -*-> '■5 •- Ji u nj 0 ..ri CD H in c c (3 "■ ■^ CM f-* be ^ - ^ c t/: ■*-* u Tj rt V rt % h ,-.-> C / n L-< ^ n1 tr J2 •- C rt ii X ? ^ -^ 5 •<5 CM _^ P 3 CO _- , c s a ^ o S U p 'o i -p *- s o a ■p rt C. >■ c ^ 0^ p -^ ■p « c = .p 3 M br. >rBo -p o o p jz B ^^ p " _P CO ^ _ -P -p >- i o C t^ 1- P — p « S -o p: ^ 4J 'g P --^ ■ .s ^ p f V > O T3 a p o p > O C V a. Q P ,0 -o 1; w y: lO •is ^ P :/: P 3 4- O _ P c« Ki - •- o 3 ^ M ^- 5 ^^ c o 0/ c3 ^.SPc U o p I- J2 O CO V > ^&- ..^S OT3 2 2 — -•22 > 03 ^ 3 i^ tfi c — C ** ^ '^ j£ <^ 5 rt o :» a oj o S '^ T3 . O ■ o M 5 '-' « W tft X CO o ii < cs U S 2 « 5 s 8 C ■2 s -o CO X X 1 CO M 1— 1 . o 2t-" -CC — oT ^ t>0 00 ■- Ol CO o « bc -rr N •« ^ 00 ■a X X CI S" .. be 05 CO f-l — S"x •a t* CO T3 00 99 rH ■a T3 OS X X CO M (M 3«' 00^" tS" 00 ■- "^«- ^y ao f— t o i-( t^ X T-i tJJ .- X to ^ o t^ i-H C5 00 X "* CO "^ o CO ■o. = s; 2x i' X t^ (M O so 6C ■ * lO &£ t^ o (M (N 9» ^ =-< oc X -a CO X S X lis -^ T) X! ■* 00 T3 I— t CO CO X X X CO en r* J >. i-H M* CO ^ ■a 5S.X "9, t^CT !N X CO ■0 M i-t .*. r-( 60 .- ho m M 1—1 r-t to CD 05 I— 1 S e; 'w •0 T3 (T^ 0 ca C^ r-t X X X 0 C'=> S,"* in -I d ." 0 •- ho 60 to ta CD f-H 0 00 CI CO IM X 00 GO o .. 60 CO X 60 ■0 1 CO •V TJ -o I-H rH CO (N f-H j M X OCO X X CO CO -~--|> CO i^oo o"'"' CO CO 60 60 a-. bD tuO 00 '^ CO 1— 1 10 S =c >* ^ cs ■d ■* -a •^ 'L3 r-t 0 1—4 ca •0 i-t "^ CO 1-4 X X X X <\ S.X 0 rl OS 00 HLos p 1-t r-1 .- 0 •- 0 _ 0 .^ 0 60 60 60 OS bo I-H CO I— I CO s^ so ~~ao — ■'^ be O 00 T3 CI 91 X 00 -t- t^ CO 60 ri IS i-( CO eo CO CO 10 lO lO 10 10 10 »£5 X X X X X X ^ — I-H '"' cs [i< c nl ■5- ►^ o a- c2 o c< Oi rn a K! l-i 0 hn **-• 0 .9 ■s -1 • •dJ X > X -^ -4 CD > CO > CO > CO ^ti t/j (rt u I-- GO T— ( f-( I— 1 t-H r-< V u •-^ IS S '-' 1- -s 0 br. c CO "^ 0 « lium 2 : rcinoma the case ng group -3 c o a o IS S -Q. o =; rl '-^ 11 Q I— o CO o 00 i-t o I— CO CO Oi co_ CO co O CO CO o" T3 W .-. i-. CO .9- -TS -a o u 2.S tetra er ra 0cm e foil 13 e u o S '^ o GO I-- C3> Q^ t^ o CI O CO CO CO 1-1 and Walk an 4. in th -3 ? 3 CO CO o r-t ■5i" :d CO rH OS i-H o CO M -^ ^2 V u 0 X s-2 hosphate (Compound nent retrogression of t administration. I 3-2 cm (or not more ited to batches of 5 to u J2 •ri U 3 O CO 0 GOOr with field 5-0 "emale animals. -2 5 S 3 oa en o CO O a CO I— 1 o CO rH O CO CO CO o S. ^ CO o CO r-t GO »— 1 CO to ■^ CO CO lo o ^- =«•&. CC rH ?— I CO co" c^ 1— 1 o O^ o o o -c "5- Table II ■1-1 : 4-naphthohydroquinone dip III) on the production of perma: nous and intramuscular routes of than 2-0 cm and not more than elected weight and sex were alloc; 4J 0, 1500 o f male an u. U u u l-< u U u t-l u u tH lO o i ° s o o o o o o o o o o o o be ro .S M-S o o o ITS o o o o o o 5^ 0 3 >^ "3 3 3 3 ;e either 1100, 1250, 1! C. : comparable numbers 111 <3 ■ I— 1 1— t rH l-H rH IN r-( CO in CD 1— 1 1— t I-H I-H ^H o 'c a o 2 1 .2 g daily for 2 to 8 days before irradiation, ,g on day of irradiation at 30 minutes be- beginning, and subsequently 10 mg daily 'arying intervals from 2 days after irradia- up to 28 days after retrogression of the 3ur in the earlier experiments. a; (/I 3 o I .2^ "3 = t: o — ' he oE >.o nJrH 7 daily injections each of 10 mg before day esponriing to irradiation, then 10 mg on day and usually on the two following days nts with tetra-sodium 2-methv iphosphate (Compound XXV omparison of effects of intrave maximum dimensions not less fter transplantation in rats of s ;? 3 53 > _B >- -0 amuscular injections, ith central tumour dos X-ray treatment as in are pooled and includ( ■si G t/1 "S'S 5.2 ■g-s s l-< c 3 c E tx O to u 1 E 6 o 1 3 3 E to O CO *J to be E o o "o « S o 2 0.2 .2 E E E s •a le dose of 10 to 25 mg on thi with 2 to 3 injections each eding days. t/1 s XI 3 o 3 > 2 c aily intr only, w Bwith : ch class E U5 tic S 10 m 15m fore for \ tion tumi "5; C o o 5 t rt CO o£ S u T3 0 be _>^ ~ ^ s ?^ s s is "e , r 'J Tj IJ cii ^1 t/) 3 (A 3 O c o > 2 c 1 of experi oquinon ation an tumours ) growin c "2 — : 3 ^ 2 or by repeate C. X-ray treatm D. Compound a; The experiments in § g > 2 c 3 1 C 1— 1 Summary naphthohydr radio-sensitiz Measured Compound I A. Cont B. Com S s 5 s - > X X ^J 177 N LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS that radio-sensitization is obtained for intravenous injection either immedi- ately before or at 30 minutes before starting the X-ray treatment. The difference between groups C and D for intravenous injection is regarded as significant. For the pooled intravenous doses x""[i]c = 5-953, P = 0-014 and for the single intravenous doses only, X ^[i]c = 5-133 and P = 0-023. These experiments suggest that Compound XXVIII when administered by intravenous injection in doses approximately twice those of Compound I is an effective radio-sensitizer for the Walker rat carcinoma 256, though the findings suggest that Compound XXVIII is slightly less effective as a radio-sensitizer than Compound I. However, there is no very convincing difference in effectiveness between the two compounds in these animal experiments. Distribution studies — Distribution studies of Compound I in the rat with the Walker carcinoma 256 have demonstrated increased fluorescence of a deri- vative of the compound with Wood's light (wavelengths 3,650, 3,655 and 3,663 A) in the actively growing parts of the tumour and some other tissues, including sternal marrow, lymphoid tissue, testis, ovary and kidney after intramuscular injection. After intravenous injection with small doses there is much more selective concentration of the compound in the tumour. With larger intravenous doses there is spill-over into the other organs mentioned. The compound responsible for the yellow fluorescence appears to be 2-methyl-l : 4-naphthoquinone-2 : 3-oxide, as confirmed by the fluorescence spectrum. These distribution studies were summarized in Table II of Mitchell^ (1953). Other compounds which accumulate in the actively growing parts of the Walker rat carcinoma 256 are the tetra-sodium salts of 2 : 3-dimethyl-l : 4-naphthohydroquinone diphosphate (Compound XXVIII), and 2-methyl-3-bromo-l : 4-naphthohydroquinone diphosphate (Compound IX). In the case of Compound IX, there is a considerably lower differential absorption ratio in the tumour, as shown by fluorescence, than in the case of Compound I, and this has been confirmed by Mr. D, R. Maxwell by radioactive tracer studies with Compound IX labelled with »2Br. Distribution studies using Compound I labefled with ^^C in the methyl group are in progress. Clinical trials of tetra-sodium 2-rnethyl-l : 4-naphthohydroquinone diphosphate as a radio-sensitizer Attempts to evaluate tetra-sodium 2-methyl-l : 4-naphthohydroquinone diphosphate (Synkavit, Compound Ij as a radio-sensitizer in the radio- therapy of maHgnant tumours have been in progress since November 1946. A summary of the results as assessed on 31 May, 1953, has been published (Mitchell 1). The investigation is continuing. It is emphasized that in general the compound alone has no therapeutic eflfect on malignant tumours. The preliminary general survey of the use of Compound I in the treatment of patients with various types of malignant tumours other than carcinoma of the bronchus with follow-up for at least 5 years, suggests that the proportion of cases showing unexpectedly good clinical response is greater following radiotherapy combined with the compound administered by intravenous injection than following radiotherapy combined with the compound 178 J. S. MITCHELL administered by intramuscular injection. Of particular interest are the results obtained in carcinoma of the mouth and carcinoma of the ovary. Of interest is the frequent clinical observation of focal pain and sensations in the region of the tumour after intravenous injection of large doses of Compound I. The preliminary clinical studies of the influence of the ancillary use of the compound on the svuvival times of inoperable cases of carcinoma of the bronchus treated by X-ray therapy have been discussed in detail. The evidence obtained was sufficiently suggestive to justify further work, but the methods used were clearly inadequate. It is suggested that the design of a clinical trial for quantitative evaluation of any proposed method of treatment of cancer is one of the most important problems at the present time. The most important single feature of the design is random allocation of patients to two or more alternative forms of treatment. At the same time it is absolutely essential to provide for each individual patient that form of treatment which is the best according to present knowledge at the beginning of the investigation. Accordingly, it is often necessary to plan to depart from what might be termed the theoretically ideal form of experiment with deliberate sacrifice of some information. In the present example, cases were allocated at random with the aid of a table of random numbers to one or two alternative forms of treatment, X-ray therapy combined with the compound administered by intravenous injec- tion and X-ray therapy combined with the compound administered by intramuscular injecdon, the latter being regarded as the control group. This type of design of a clinical therapeutic trial must be justified a posteriori. This has been done, and includes consideration of all the various factors which may be relevant. To date, this trial includes 173 patients but the follow-up must be continued. Already for the first 91 cases the two groups are substantially identical in composition and there is evidence of a small but definite improvement in survival of the male patients with reasonably certain diagnosis (excluding those treated surgically) in the group treated with X-ray therapy combined with intravenous compound in relation to those in the group treated with X-ray therapy combined with intramuscular compound. Statistical details have been published (MitchelP, 1953, especially Table II). In general terms, in comparable groups of cases of inoperable carcinoma of the bronchus, the mean survival time after the first X-ra\' treatment was only about 4 months with X-ray therapy only, about 6 months with X-ray therapy combined with intramuscular compound and about 1 1 months with X-ray therapy combined with intravenous com- pound. The improvement in this last group is certainly in accordance with clinical impressions and indicates a small prolongation of useful life. The mean interval from the first symptom to the first X-ray treatment is about 7 (6 8) months in all the groups. One unsolved problem to which much attention is being devoted is to decide when to stop a clinical trial such as the present one. It is concluded that the results obtained so far indicate that intravenous administration of Compound I has a small but useful eflfect as a clinical radio-sensitizer. Even though large eff"ects have not been produced, the results provide evidence that it is possible to improve the response of some 179 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS human malignant tumours to radiotherapy by means of chemical radio- sensitizers. Combination of Compound I, oxygen and X-ray therapy — Since August, 1953, a clinical trial has been in progress of the combined use of intravenous Com- pound I (Synkavit), oxygen administration before and during irradiation and X-ray therapy in the attempted treatment of patients with advanced cancer. The compound has been given in the usual manner with maximum unit doses in the region of 100 to 150mg at 30 minutes before starting the X-ray treatments at each visit. The oxygen is administered at atmospheric pressure by means of either a B.L.B. mask or an anaesthetic mask, with a flow-rate meter at about 6 litres per minute, starting usually 20 minutes before and continued throughout irradiation. Every endeavour has been made to employ the normal methods of X-ray therapy, often with large fields, and to avoid any modification of the techniques as a result of the combined treat- ment. So far 34 cases have been treated, mainly advanced cases of car- cinoma of the stomach, colon, and ovary and inoperable cases of carcinoma of the bronchus after thoracotomy. It is of course too early to assess the results. With the combined treatment, both the skin reactions and the general reactions appear to be no more severe than would be expected with the X-ray therapy only. I wish to thank Dr. S. D. Sturton for his help in the initial part of this investigation. These trials are being continued, together with studies of the combination of oxygen and X-ray therapy. A randomized trial has been started using four groups. X-ray therapy combined with intramuscular Synkavit, oxygen combined with X-ray, intravenous Synkavit combined with X-ray and intravenous Synkavit, oxygen and X-ray. Experiments with animal tumours in these fields are being carried out by Dr. D. B. Cater and Dr. A. F. Phillips. SUMMARY The aim of this investigation is to try to improve the results of radiotherapy of some types of cancer by the ancillary use of chemical agents designed to act as radio-sensitizers. This paper deals mainly with recent results but also refers to the previous studies carried out since 1946. The first compound studied, tetra-sodium 2-methyl-l : 4-naphthohydroquinone diphosphate (Synkavit ; Compound I), is stiU perhaps the most interesting. Clinical trials of its use have been in progress since November, 1946, and about 1 ,070 patients have been treated by the combination of radiotherapy with this compound. In the laboratory studies we have used : — (7) tissue cultures mainly of chick fibroblasts, for the primary sorting test and for studies of the relation between chemical structure and biological action. {2) animal experiments with rats mainly, but also using mice and rabbits, for : — {a) tests of chemical toxicity and radiotoxicity in the case of compounds incorporating radio-isotopes. ib) studies of radio-sensitization of normal tissues with total body radiation. 180 J. S. MITCHELL {c) Studies of the distribution of the chemicals in the tumour and normal tissues, using mainly both fluorescence methods and radioactive labelling. (d) radiotherapeutic experiments to study radio-sensitization in trans- planted tumours, mainly the Walker rat carcinoma 256. New compounds examined recently include tetra-sodium 6-iodo-2- methyl-1 : 4-naphthohydroquinone diphosphate and 2-ethylenimino-l : 4- naphthoquinone, the latter compound appearing to be of particular interest. The problem of the thermal instability of Compound I appears to have been solved by the finding that this thermal instability is associated with the presence of an easily removable impurity so that stable aqueous solutions of Compound I can now be prepared. An interesting experiment by Mrs. Simon-Reuss is summarized ; it appears that initial treatment of chick fibroblast cultures with Compound I leads to a persistence of radio-sensitivity in subcultures even after 16 passages. The possible mechanisms of radio-sensitization are discussed ; the selec- tive sensitization of the tumour cells is envisaged as the converse of — SH protection. Two large-scale therapeutic radio-sensitization experiments with Com- pound I and Compound XXVIII have now been completed and are reported in detail. With both compounds, significant radio-sensitization is observed when the compound is administered by the intravenous route but not when the compound is given by the intramuscular route. The clinical trials of tetra-sodium 2-methyl-l : 4-naphthohydroquinone diphosphate (Compound I) as a radio-sensitizer are summarized. The pre- liminary general survey in the treatment of patients with various types of malignant tumours other than carcinoma of the bronchus, with follow-up for at least 5 years, suggests that the proportion of cases showing unex- pectedly good clinical response is greater following radiotherapy combined with the compound administered by intravenous injection than following radiotherapy combined with the compound administered by intramuscular injection. Of particular interest are the results obtained in carcinoma of the mouth and carcinoma of the ovary. The preliminary clinical studies of the influence of the ancillary use of the compound on the survival times of inoperable cases of carcinoma of the bronchus treated by X-ray therapy were very suggestive, but the methods of investigation used were inadequate. It is suggested that the design of a clinical trial for quantitative evaluation of any proposed treatment of cancer is one of the most important problems at the present time. The most important single feature of the design is random allocation of patients to two or more alternative forms of treatment. At the same time it is absolutely essential to provide for the individual patient that form of treatment which is the best according to present know- ledge at the beginning of the investigation. A clinical trial of Compound I as a radio-sensitizer in the treatment of inoperable cases of carcinoma of the bronchus is discussed ; so far it includes 1 73 patients. In general terms, in comparable groups of cases of inoperable carcinoma of the bronchus, the mean survival time after the first X-ray treatment was about 4 months with 181 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS X-ray therapy onl)-, about 6 months with X-ray therapy combined with intramuscular compound and about 11 months with X-ray therapy com- bined with intravenous compound. It is concluded that the results obtained so far show that intravenous administration of Compound I has a small but useful effect as a radio-sensitizer. Since August, 1953, clinical trials have been in progress of the combined use of Compound I, oxygen administration before and during irradiation, and X-ray therapy in the treatment of patients with advanced cancer. After preliminary studies a randomized trial has been started. ADDENDUM ANIMAL EXPERIMENTS WITH THE WALKER CARCINOMA 256 IN THE RAT (1) Distribution Studies With reference to the fluorescence method for study of the distribution of Compound I in the rat with the Walker carcinoma 256, further measurements of the fluorescence spectrum with Wood's light (wavelengths 3,650, 3,655 and 3,663 A) and of its behaviour with pH confirm that the compound responsible for the fluorescence of the tissues is probably 2-methyl-l : 4- naphthoquinone-2 : 3-oxide. Cut surfaces of tissues require about 6 hours at room temperature in the presence of air for full development of this fluorescent derivation of Compound I but the fluorescence develops within 15 to 30 minutes in the presence of alkali and hydrogen peroxide ; varying concentrations between N/100 and 3N NaOH and 10 to 100 volume H2O2 have been used, the fluorescence developing most rapidly at the highest concentrations. The fluorescence does not appear when the tissues are kept in air at — 20°C and — 4°C, or are kept in nitrogen at room temperature for 15 days, though the fluorescence starts to develop within 10 minutes after the admission of air. In continuation of these investigations, the method of fluorescence micro- scopy has been used to obtain information about the localisation of Com- pound I or the compounds derived from it within the tumour cells. In Figure 2 the appearances with fluorescence microscopy and the same area of the tumour stained with toluidine blue are compared for a region in the growing edge of the Walker carcinoma after treatment of the rat with large intramuscular doses of Compound I. The essential features of the technique are summarized in the legend to Figure 2. It must be mentioned that the development of the fluorescence was accelerated by running N/100 NaOH followed by 30 volume HPa and then further N/100 NaOH under the coverslip. The upper left-hand part of the plates shows an area of actively growing tumour ; below the margin of this is an area of mainly necrotic and partly relatively inactive tumour. Comparison of the appearances with fluorescence microscopy and toluidine blue staining of the actively growing parts of the tumour show that in the proliferating cells not in mitosis the fluorescent derivative of Compound I is concentrated mainly in the peri- nuclear region of the cytoplasm of the tumour cells, with a paler peripheral region and some fluorescent granules in the cytoplasm. 182 J. S. MITCHELL 2. Therapeutic Experiments In tlie therapeutic experiments- to test radio-sensitization by tetra-sodium 2-meth\l-l : 4-naphthohydroquinone diphosphate (Compound I) of the Table III. — Clinical Therapeutic Trial of Compound I as a Radio-sensitizer Summary of Results to 30th June 1954 Treatment of inoperable cases of carcinoma of the bronchus with randomized allocation to alternati\e treatments. X-ray therapy combined with intravenous Compound I (X-J-I-VS) and X-ray therapy combined with intramuscular Compound I (X^I-MS) which is the control group. Results assessed 30th June 1954. Cases No. 1-151 inclusive in which the first X-ray treatment was given between 1st April 1951 and 31st December 1953. Treatable less advanced male cases Selected from these cases are those with no evidence of extra-thoracic spread, metastases in ribs or the syndrome of superior mediastinal obstruction at the first X-ray treatment and those in which the minimum tumour dose was not less than l,200r. For the present purpose cases treated surgically at any stage, cases regarded as operable but not treated surgically on account of poor general condition and female patients are excluded. Survi\al is estimated in months to the nearest month from the first X-ray treatment and also from the first symptom. Number of cases sursiving 8 months or more after first X-ray treatment/ Total number of cases in each group Group X+I-VS X+I-MS (a) Histology positi\e for carcinoma of the bronchus (one p.m. and sputum, all others bronchial biopsy) . . 7/11 3/15 (b) Conclusive e\idence of malignant cells in sputum and/or bronchial aspirate, or probable malignant cells in sputum and/or bronchial aspirate, together with subsequent development of definite metastases 5/7* 0/6 (c) Reasonably certain diagnosis with typical broncho- scopic and radiological appearances and clinical course, including subsequent development of definite metastases . . . . . . . . . . 3/3 0/4 15/21 3/25 Pooled groups (a), (b) and (c) * In this group is included one case surviving 4 months from the first X-ray treatment and 10 months from the first symptom who was given intramuscular compound because of incorrect suspicion of cerebral metastases ; for calculation of mean survival times, this case is transferred to the group X+I-MS. Mean survival times {to date) from first X-ray treatment : 10-2 months for X— I-\"S, and 4-8 months for X+I-MS. The difference is significant ; for 44 d.f., t = 4-448, and P-::0-001. from first symptom : 15-8 months for X-f-I-VS, and 11-2 months for X+ I-MS. The difference is significant ; for 44 d.f.. t=2-520 and P=0-0163. It is to be noted that at the date of assessment, 4 patients were still li\ing in the series X— I-VS and one patient was li\ing in the series X-rl-MS. Walker carcinoma 256, in the rat, a number of experiments have been carried out which are not included in this paper and which will be reported else- where, together with further experiments in progress. The increased proportion of tumours showing permanent retrogression in Group D of Table I (page 175), that is with intravenous Compound I and 183 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS Figure 2. Comparison of fluorescence microscopy and toliiidine blue staining of an area in the growing edge of the Walker rat carcinoma after treatment with large intramuscular doses of Synkavit. Fluorescence microscopy with Wood's light. Frozen section. Fluorescence developed with N/100 NaOH and 30 volume HjOj. 184 J. S. MITCHELL Original magnification X 570. <^e«j- 1 mm water immersion objective. Rat received 23 daily doses each of lOmg of Synkavit, with lOOmg at 20 minutes before death. The compound responsible for the yellow fluorescence appears to be 2-methyl-l : 4-naphthoquinone- 2 : 3-oxide. 185 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS central tumour dose l,100r, would be given by a central tumour dose of X-radiation alone of at least 1,400 r, and probably about 1,500 r. In the early stages of this work no effect appeared to be obtained with intravenous Compound I given immediately after the end of the X-radiation of the tumoiu', but this experiment will be repeated. Table IV. — Clinical Therapeutic Trial of Compound I as a Radio-sensitizer (Contd.) Number of cases surviving 8 months or more after first X-ray treatment/ Total number of cases in each group Group X+I-VS X+I-MS (d) Verified as in (a) and (b) but minimum tumour dose less than 1,200 r 0/4 0/5 (None sur\ived (None survived more than two more than two months) months) (e) Male patients with extra-thoracic spread and/or metastases in ribs at first X-ray treatment, or with syndrome of superior mediastinal obstruction .. 5/26 3/16 (el) Excluding cases with minimum tumour dose less than 1,200 r 4/21 2/11 (f) Male patients treated surgically at some stage . . 2/7 7/7 (g) Male patients with diagnosis of carcinoma of the bronchus not verified : (gl) diagnosis of carcinoma of bronchus i/«/i/oiY'(f .. 0/5 3/5 (g2) diagnosis of carcinoma of bronchus unlikely O'" disproved 1/5 6/6 (h) Female patients, all groups except bronchial adenoma 2/9 2/4 (i) Bronchial adenoma .. .. .. .. .. 0/2 0/1 (j) No X-ray therapy ; compound only ...... 0/1 (k) Operable but not treated surgically on account of poor general condition . . . . . . . . 2/2 For the unselected inoperable male patients treated with effective doses of X-radiation in the pooled groups (a), (b), (c) and (el), the proportions surviving 8 months or more after the first X-ray treatment in the series X+I-VS and X+I-MS are respectively 19/42 and 5/36 : x'>[i] = 8-64, so that P= 0-003. Survival from the first symptom is a rather less sensitive and less objective test. The pro- portions survi\ing 1 1 months or more after the first symptom are respectively (after transfer of the case marked * to the series X+I-MS) for the series X+I-VS, 26/41 and for the series X+I-MS, 14/37 ; x^c[i] = 4-12, so that P=0-0043 and this difference between the two series can be regarded as significant, even after the inclusion of the unfavourable cases in group (el). With reference to Table I, the animals were killed when the maximum dimension of the tumour reached 5 • 0 cm or when the animal was look- ing ill and cachectic before that size of the tumour was reached. Under these conditions, there were very few animals with metastases detectable macroscopically, CLINICAL TRIALS OF TETR A-SO D lU M- 2 - METH Y L- 1 : 4- N APHTH O- HYDROqUINONE DIPHOSPHATE, COMPOUND I, AS A RADIO-SENSITIZER The results in this clinical trial as assessed on 30th June 1954 are summarized in Tables III and IV. These tables are a revision of Table II of my earlier report^ ; the basis for the classification and assessment of the results is sub- 186 J. S. MITCHELL staiitially the same in Tables III and IV as in that paper. It is to be noted in groups (a), (b) apd (c) of Table III that at the date of assessment 4 patients are still living in the series X + I-VS and one patient is living in the series X-f-I-MS, and it is almost certain that the final mean survival time in the group X+I-VS will be substantially greater than the value given of 10-2 months from the first X-ray treatment. As this clinical trial with random- ized allocation proceeds and the number of cases included increases, the difference between the results in the series X-f I-VS and X-f-I-MS increases in significance. In addition to the statistical estimation of survival times from the first X-ray treatment and from the first symptom, I think it is of the greatest importance to assess the detailed progress of the course of the disease in the individual patients and, in particular, to try to obtain an estimate of the ^/o. 17. H..... Ro (esjrs) X+ iMS Co. left main bronchus - Inoperable Performance si at us WO s Loss of sirenqfh Welghl It. 182 W 12 Tnon+hs Dyspnoea Tl1-6-51 D folloiving M. T.D. Influenza 3000 r In 39 days 15t W 1U-1 me Jio Esflmaled prolongation of life 0 months (possibly -2 months) Figure 3. effect of the different types of treatment. I have seen nearly all the patients referred to in Tables III and IV personally but it is essential to try to present the results for the individual patients objectively. A useful method of approach is by the use of the Performance Status defined by Karnofsky ef al^^ (Table 3). Selected examples for two patients who appeared to be reasonably txpical are given in Figures 3 and 4. The former is for a case treated with X-ray therapy combined with intramuscular Compound I and the latter for a case treated with X-ray therapy combined with intravenous Compound I. By extrapolation of the curve of deteriorating Performance Status before treatment, an attempt is made to estimate the duration of life in the absence of the treatment given and from this to estimate the prolonga- tion of life, and also prolongation of useful life, as a result of the form of treatment administered. In Figure 3 in the case treated with X-ray therapy combined with intramuscular compound, it appears unlikely that the treatment resulted in any prolongation of life but that there was possibly a slight palliation of symptoms, although even this conclusion is by no means 187 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS certain. With regard to the case shown in Figure 4 and treated by X-ray therapy combined with intravenous compound, the graphical presentation indicates a substantial prolongation of life and also of useful life as a result of the treatment ; this conclusion supports the clinical findings in the case of this patient throughout the course of his illness. Other compounds With regard to the investigations of 2-ethylenimino-l : 4-naphthoquinone both in animal experiments and clinical trials (see page 171), the low solu- bility of this compound presents considerable difficulty and it appears to be desirable to attempt the difficult problem of preparing the corresponding phosphorylated compound, 2-ethylenimino-l : 4-naphthohydroquinone di- phosphate. It is of interest that a focal reaction in the region of the tumour was observed several times in a patient with advanced carcinoma of No. 38. A Sm (72jns) X +il/8 Ca. bronchus R.U.L. Mediastinal glands -inoperable Performance si a I us Limiled mobility rt i^ocal cord 100 -12 -10 -8 -6 Weight lb. f 96 Tired easily -2 0 T10-12-S3 X+zVS M.T.D. 1S00r in 8 days /7« fSI 8 70 12 n 16 18 ^^ -months D Pain in rt. side chest and shoulder becoming severe 177 176 Steady loss of iv eight Estimated prolongation of life 12 months Figure 4. the floor of the mouth after intravenous, injection of about lOmg of 2- ethylenimino-1 : 4-naphthoquinone in 170 ml of normal saline. It is suggested that further study of this compound and related compounds is desirable. REFERENCES ' Mitchell, J. S. Brit. J. Cancer, 1953, 7 313 ; Acta radio/. Stockh. Supp. 116 341. 2 Mitchell, J. S. and Simon-Reuss I. Nature, Land. 1947, 160 98. 3 Mitchell, J. S. Brit. J. Cancer, 1948, 2 351. Experientia, 1949, 5 293. Ann. Rep. Brit. Emp. Cancer Campaign, 1949, 27 214. ibid, 1950, 28 213. ibid, 1951, 29 192. ibid, 1952, 30 238. and Simon-Reuss, I. Brit. J. Cancer, 1952, 6 305. 188 * Mitchell, J. S. 5 Mitchell, J. S. " Mitchell, J. S. ' Mitchell, J. S. ^ Mitchell, J. S. ^ Mitchell, J. S. DISCUSSION 1" Mitchell, J. S. and Simon-Reuss, I. Brit. J. Cancer, 1952, 6 317. " Hughes, A. and Simon-Reuss, I. ibid, 1953, 7 142. 12 Friedmann, E., Marrian, D. H. and Simon-Reuss, I. Brit. J. Pharmacol. 1948, 3 263. 13 Friedmann, E., Marrian, D. H. and Simon-Reuss, J. ibid, 1948,3 335. 1^ Friedmann, E., Marrian, D. H. and Simon-Reuss, J. ibid, 1949,4 105. 15 Marrian, D. H. J. Chem. Soc. 1949, 1515. i« Marrian, D. H. ibid, 1949, 1797. 17 Friedmann, E. and Bailey, N. T. J. Biochim. Biophys. Acta, 1950, 6 274. IS Friedm.\nn, E., Marrian, D. H. and Simon-Reuss, I. ibid, 1952, 9 61. 19 Friedmann, E., Marrian, D. H. and Simon-Reuss, I. ibid, 1952, 8 504. 20 Friedmann, E. ibid, 1952, 9 65. 21 Friedmann, E. Brit. J. Radiol. 1954, 27 137. 22 Gellhorn, a. and Gagll\no, T. Brit. J. Cancer, 1950, 4 103. 23 BERKM.A.N, A. Tiirkiye Tip Enciimeni Arsive {Istanbul), 1951, 2 1. Berkman, a. Seventh Int. Congress Radiology Abstract TWO, 1953. 2* JoLLES, B. Ann. Rep. Brit. Empire Cancer Campaign, 1952, 30 324. 25 DiTTRiCH, W. and Schmermund, H.-J., Strahlentherapic, 1953, 90 88. 2 6 Cater, D. B. and Phillips, A. F. Nature, Land. 1954, 174 121. 2' Domagk, G., Peterson, S. and Gauss, W. Z^- Fd. Krebsforsch. 1954, 59 617. 28 Fieser, L. F., et al. J. Amer. Chem. Soc. 1948, 70 3215. 29 Ball, E. G., Anfinsen, C. B. and Cooper, O. J. biol. Chem. 1947, 168 257. 30 Potter, V. R. and Reif, A. E. ibid, 1952, 194 287. Fisher, R. A. Statistical Methods for Research Workers, Oliver and Boyd, Ltd.. Edinburgh, 1925-46. Fisher, R. A. The Designof Experiments, Oliver and Boyd, Ltd., Edinburgh, 1935-46. Medical Research Council. Brit. Med. J. 1948, 2 769. Medical Research Council, ibid, 1950, 2 1073. Hill, A., Bradford, Principles of Medical Statistics, Pub. by Lancet, Ltd., London, 1952, Fifth Edn., pp. 5, 233. 31 KLA.RNOFSKY, D. A., BURCHENAL, J. H., ArMISTEAD, G. C, Jnr., SOUTHAM, C. M., Bernstein, J. L., Graver, L. F. and Rhoads, C. P. Arch. Int. Med. 1951, 87 477. DISCUSSION S. Neukomm : For four years, our research team has devoted part of its activity to the study of the metabolism of Synkavit, injected in rats, and its mode of action in vitro on fibroblast from the heart of mice. We used Synkavit labelled with radio- active phosphorus*. This substance undergoes a more or less rapid hydrolysis in the organism, according to the nature of the organs in which it is fixed. This hydrolysis brings about the liberation of the phosphoric groups (PO4), which then follow the general metabolism of the PO4 ions. From then on, the destiny of the basic molecule (2-methyl-l, 4-naphto-hydroquinone) can no longer be followed. Still, it is most probable that this molecule remains locally fixed in the cells in an oxidized form, as Mitchell's report seems to show. With reference to the distribution of Synkavit in the various organs of rats, our results confirm and extend Mitchell's statements. Synkavit is mainly fixed and accumulates (7) in the organs which have intense cellular proliferation centres and * Neukomm, S., Peguiron, L., Lerch, P., and Richard, M. Arch, internal. Pharmacodyn Therapie, XCIII, 1953, 373. Peguiron, L. and Neukomm, S. Acta Anat. 1954, 21 46. 189 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS (2) in Storage, detoxication or elimination organs. There is a close correlation between the capacity of the cells to absorb Synkavit, and the rate of renewal of deoxyribo- nucleic acids. Further, the retention of the product is greater when the proliferation is more intense. It has been clearly shown that Synkavit does not provoke any important morpho- logical lesions in mitosis. It is a weak antimitotic. By histophotometric measure- ments of fibroblast nuclei cultivated in vitro and stained by the Feulgen reagent, we have established that Synkavit decreases the proportion of cells containing high con- centration of deoxyribonucleic acid (pre-prophasic nuclei). With the concentrations we have used the proliferation of the cultures is not hindered. The observed effect is thus not bound to a decrease in the number of mitosis, but rather to change of the time of synthesis of the deoxyribonucleic acid, leading to a delay of prophase. Our research shows that the quantity of Synkavit necessary to obtain this effect is extremely low. This corroborates and reinforces the idea that the basic molecule of Synkavit remains fixed in the cell and intervenes in the metabolism of the deoxyribonucleic acid. The damage caused to the fibroblast cultures immersed in Synkavit solutions containing proteins is only half that found in solutions (Tyrode) that do not con- tain any proteins. On the other hand, exposure of cultures for 50 minutes to Syn- kavit in Tyrode solution shows damages equal to that caused by Synkavit directly introduced in the culture medium ; in other words, with equal concentrations, the effect of Synkavit develops in 50 minutes in Tyrode and in 48 hours in plasma. Other experiments (cultures treated with Synkavit compared with non-treated cultures, influence of the number, the duration and the volume of the wash-liquid after exposure of the cultures to Synkavit) show that Synkavit is for the greatest part adsorbed on the proteins or on the cellular membranes.* These data help to explain the important difference in the therapeutic eff^ect obtained by administering Synkavit by the intramuscular or intravenous route. The combination ' Synkavit-proteins ' cannot be effected instantaneously and depends on the local concentration of Synkavit. When the latter is introduced into the muscular tissues, it remains there longer and at a higher concentration than when it is introduced in the blood-stream and therefore cannot reach distant organs in sufficient concentration. B. JoLLES : Having used Synkavit in over two hundred patients with advanced malignant disease undergoing radiotherapy, and noted the beneficial effects of the administration of this compound as a coadjuvant of radiotherapy, I should like to draw attention to the formula of Karnofsky mentioned by Mitchell in view of the fact that often, particularly in the case of patients with carcinoma of the lung, the results assessed on the basis of survival times do not give the true picture of the efficacy of the treatment. This is often better assessed instead on the basis of criteria concerning comfort and relief of symptoms which accrue from a particular method of treatment. J. O. Laws and B. Jolles : Amongst the attempts to improve the results of the radiotherapy of some types of cancer, especially those in which results are unsatis- factory, by the ancillary use of chemical agents designed to act as radio-sensitizers, the work of MiTCHELLf (1948) has been most consistently and thoroughly pursued. After many clinical trials Mitchell J (1952) has shown that the administration of tetrasodium 2-methyl-l : 4-naphthohydroquinone diphosphate (synthetic vitamin K derivative, Synkavit, Roche) to patients with advanced malignant disease improves * Richard, M., Peguiron, L. and Neukomm, S. Arch, internal. Pharmacodyn. TMrapie, in press. t Mitchell, J. S. Brit. J. Cancer, 1948. 2 351-358. + Mitchell, J. S. Brit. Empire Cancer Camp. 30th Ann. Rep. London, 1952, p. 239. 190 DISCUSSION significantly the survival results of treatment. Laboratory tests with tissue cultures and animal experiments with a large series of chemical compounds (Mitchell and Simon-Reuss*, 1952), have shown the superiority of this temporarily solubilized quinol. The work here described is concerned with the animal experiments only as the clinical material available would not allow as yet any data regarding the effectiveness of Synkavit as a coadjuvant of radiotherapy to be presented. Thirty-five rabbits and 37 rats were given subcutaneous injections of Synkavit, the amounts given to rabbits ranging from 10-50mg daily, but constantly lOmg in the case of rats. The drug was given in some cases for a few days before the delivery of the X-ray dose and continued for a time varying from 10-17 days. The dose of X-rays given in the case of rabbits ranged from 600-1,500 r. There were three groups of rats which received respectively 480 r, 585 r and 650 r ; the first two groups contained 25 rats and the last 26. All the rabbits and all the rats were exposed to a beam of radiation covering the whole body. The physical factors were 180 kV, 10 mA, 50 cm F.S.D. with a H.V.L. varying according to the filtration, which was either the inherent tube shield filtration alone or 0-5mm Cu (H.V.L. 0-85 mm Cu). The irradiation was carried out with the tube head and the applicator inverted, with the box containing the animals standing on top of the applicator end. RESULTS Rabbits The difference in survi\al rates between the Synkavit plus X-rays series of rabbits with the series of rabbits which received X-rays alone was not significant when the assessment was made on the whole series of 35 rabbits which received Synkavit injections. In the Synkavit group the majority maintained their weight fairly well throughout the observation period or gained ground slightly. This was particularly so in the case of the rabbits which received Synkavit injections for a few days prior to exposure to X-rays. In the series of rabbits exposed to X-rays without Synkavit there was a consistent drop in the weight curve, with the exception of one surviving rabbit which gained some 10 per cent in weight over a period of 30 days. The Synkavit rabbits were on the whole more lively and taking food more liberally than the animals without Synkavit. Rats The 76 rats used in these experiments were dealt with in three separate series. Group 7—13 rats received lOmg of Synkavit injections daily (7 rats for four days and 6 rats for five days) prior to exposure to a dose of 580 r and subsequently daily until their death, and a group of 13 rats received a similar dose of X-rays to the whole body without any injections. In the Synkavit group there was 1 survivor and in the non-Synkavit series there were no survivors at 30 days. Group //—Of 25 rats in this group 24 were exposed to a dose of 650 r to the whole body. Of these, 11 were given injections of lOmg of Synkavit daily, and 13 were given daily injections of 1 c.c. of sterile normal solution of saline. With the exception of the control rat which survived the 30 days' period there were no 30-day survivals in the whole series. Group III^A group of 25 rats were exposed to a dose of 480 r to the whole body. Of these 9 received seven and 4 received five injections, 10 mg daily, of Synkavit prior to exposure to X-rays and eight to eleven daily injections after exposure. 8 rats received six pre-X-ray treatment and 10 post-treatment injections of 1 c.c. of * Mitchell, J. S. and Simon-Reuss, I. Brit. J. Cancer, 1952. 6 305, 317. 191 LABORATORY STUDIES AND CLINICAL TRIALS OF CHEMICAL RADIO-SENSITIZERS Sterile normal solution of saline and 4 received no injections whatsoever. In the group of rats which received saline injections there were no deaths at 31 days after irradiation. In the group of rats which received Synkavit injections there was only 1 survival at 31 days, the remainder dying within 12-16 days. The 4 control (X-rays only) rats survived 31 days. MICROSCOPICAL The spleen and adrenals of both rats and rabbits were examined in these experiments. The adrenal has been comparatively little studied, and yet is considered in the recent literature to play some part in the syndrome of irradiation sickness (Porter*, 1952), and in the repair of tissue damage following irradiation (CRAVERf, 1948). The importance of adrenal hormones in maintaining many vital functions also suggest a potential connection between this organ and the widespread changes present after irradiation. Spleen The changes in the spleen followed the same pattern in both species and were of the type previously described in the mouse by Jacobson et al% (1950). The lymphoid follicles disappeared rapidly in the first three weeks. In many of the animals dying in the earlier period there were further changes of an atrophic nature, in some cases little except blood vessels and fibrous tissue being seen. Iron containing pigment was often prominent in those specimens. There was in general no difference between animals given Synkavit and the controls dying after the same period. Rabbits In general the findings in these species are similar to those of Engelstad and Tor- GERSEN§. The changes in the cytoplasm consist of loss of lipoid at an early stage and in some cases a marked basophilia. This latter tends to disappear but lipoid is slow to reappear in comparison with what is usually seen in secondary adrenal involvement, often being small in amount at the end of 31 days. The nuclear changes affect particularly the zona fasciculata extending up to the medulla (the zona reticularis is hardly distinguishable in the rabbit). These come on after 3-5 days and appear to persist for weeks. The nuclei may undergo any of the commonly seen forms of degeneration, pyknosis, fragmentation or loss of basophilia followed by dissolution. Apart from occasional initial hyperaemia there is no inflammatory reaction and one has the impression that some of the degenerated cells remain in situ almost indefinitely. How much function may remain in spite of the nuclear changes is an interesting point for further study. In contrast to the constant degenerative changes in the inner zones of the cortex, the zona glomerulosa in the rabbit shows comparatively slight damage. Both cytoplasm and nuclei appear relatively normal. No mitosis was seen in the rabbit material, but in the presence of some of the relatively normal cells its occurrence cannot be excluded on the present evidence. Rats In the irradiated animals it was found that, unlike what had been seen in the rabbit, all zones seemed equally affected, showing cytoplasmic and nuclear changes. The cytoplasmic damage was similar to that seen in the rabbit. The nuclear alterations * Porter, E. C. Radiol. 1952, 58 246-257. t Graver, B. N. Amer. J. Roentgenol. 1948, 59 404-407. X Jacobson, L. O., Simmons, E. L., Marks, E. K., Robson, M. J., Bethard, W. F. and Gaston, E. O. J. Lab. Clin. Med. 1950, 35 746-770. § Engelstadt, R. B. and Torgersen, O. Acta Radiol. 1937, 18 671-687. 192 DISCUSSION varied according to the normal patterns seen in the various eones. In the reticularis, pyknosis was the rule ; in the fasciculata, either pyknosis or a loss of basophilia and 'fading' ; in the reticularis, a breaking up of the nucleus, the stroma disappearing and leaving the chromatin particles scattered. In animals dying in the first few days after irradiation, hyperaemia in the zona reticularis was common. As in the I'abbit no evidence of an inflammatory reaction was seen at any time. Mitosis was in general absent but a few mitotic figures were seen in two rats dying on the 29th and 31st days. They were all in the outer part of the zona fasciculata. In neither species was there any unequivocal evidence of the eflFect of Synkavit. In the rat, however, the general impression was one of greater damage in those receiving the compound than in the controls. SUMMARY (7) In rabbits the administration of Synkavit prior and after irradiation has shown slight increase in tolerance of irradiation, and although the difference in the mortality rate of rabbits and rats is not significant, the weight curve and the well-being of the animals suggested an increased tolerance of radiation in this species. In rats an opposite effect was found (Mitchell*, 1951 ; JoLLEsf, 1952). The histological changes in the adrenals stained only with haematoxylin and eosin can clearly give no more than a hint of the changes which take place in the irradiated animals. Nevertheless, enough has been found to suggest that a more detailed study of adrenal pathology and function might be fruitful in elucidating the mechanism of radiation effects. (2) The histological findings of the effects of Synkavit were disappointing, but as the main demonstrable effect of this compound is mitotic inhibition (Mitchell J, 1949), it is perhaps not surprising that the histological appearance of tissue already showing nuclear damage and mitotic arrest from the effect of the X-rays, should be little altered by the chemical. Nevertheless the suggestion of greater damage in the adrenals of rats receiving Synkavit makes it possible that this may at least have contri- buted to the deleterious effect of this compound in this species. Although it is hazardous to generalize from the described experiments the fact that patients suffering from malignant tumours who receive daily injections of Synkavit while undergoing radiotherapy stand treatment better, induces one to venture an opinion that the administration of this compound in humans produces a response similar to that found in rabbits rather than that in rats. In this context however it has to be borne in mind that a selective concentration of the drug in tumours has been shown by Mitchell by means of ultra-violet microphotography and that the use of Synkavit in normal individuals probably produces effects different from those to be expected in tumour-bearing patients receiving treatment to a part of the body only. * Mitchell, J. S. Brit. Empire Cancer Camp. 29th Ann. Rep. London, 1951, p. 192. I JoLLES, B. Brit. Empire Cancer Camp. 30th Arm. Rep. London, 1952, p. 325. X Mitchell, J. S. Brit. Empire Cancer Camp. 27th Ann. Rep. London, 1949, p. 214. 193 AUGMENTATION DE LA SYNTHESE DE L'HEMOGLOBINE IN VITRO PAR LES RETICULOCYTES APRES IRRADIATION A. NizET et A. Herve Laboratoires de Clinique Medicale B (Prof. L. Brull), de Pathologic Generale (Prof. Z. M. Bacq) et de Radiotherapie (Prof. P. Desaive) de I'Universite de Liege Les experiences que nous allons vous decrire ont pour but de mettre en evidence I'influence de I'irradiation, soit de Torganisme tout entier, soit du sang ou du plasma in vitro, sur la synthese de I'hemoglobine. Gette synthese est mesuree a partir de I'incorporation du carbone radioactif de la glycine- 2-i''C et de la phenylalanine-2-^*C a rhemine et a la globine par les hematics jeunes in vitro. L'un de nous (A. N.) a demontre, avec Robscheit-Robbins, que les hematics jeunes anucleees ou reticulocytes ont besoin d'une serie d'acides amines pour murir in vitro^^. Par ailleurs, une serie de travaux, bases sur la methode des indicateurs isotopiques, ont demontre I'utilisation par ces cellules de la glycine pour la synthese de I'hemine (London, Shemin, Neuberger et coll.'' ^'^), de la glycine, de la leucine, de la lysine et de I'histidine pour la synthese des proteines reticulocytaires (Borsook et coll.^), et de la glycine et de la phenylalanine pour la synthese de la globine (Nizet et Lambert^^"^''). La mesure comparative de I'incorporation d'anabolites radioactifs in vitro permet une etude precise de la vitesse de synthese de I'hemoglobine dans differentes conditions. techniques utilisees (a) Preparation des chiens Les animaux, adultes des deux sexes, sont soumis a deux saignees par semaine (environ 20 cm^ par kg de poids), par ponction de la veine jugulaire externe, jusqu'a ce que le taux d'hemoglobine soit abaisse jusqu'a 7 a 10 grammes pour cent. A ce moment, le taux de reticulocytes dans le sang peripherique atteint 40 a 200 p. 1000. Les chiens regoivent une alimenta- tion complete avec un supplement de fer et de vitamines. [b) Incubation du sang avec les amino-acides marques Les manipulations se font par voie aseptique. Le sang est additionne lors du prelevement, et a raison de 1/10 de son volume, de la solution suivante : heparine en poudre (Liquemine Roche) 20cg-glucose 2g-chlorure sodique 90cg-eau lOOcm^. Les amino-acides marques sont ajoutes au sang sous forme de solution dans le chlorure sodique a 9 p. 1000. Les echantillons de sang sont incubes pendant 5 a 10 heures a 37' C dans un bain thermostatique. Apres incubation, le plasma est elimine et les hematics sont lavees 4 fois au chlorure sodique 9 p. 1000, par centrifugations et decantations successives. 194 A. NIZET ET A. HERVE {c) Preparation de Vhemine et de la globine Hernine—\2 a 15 cm-'^ de puree globulaire est additionnee d'un volume egal de solution a 1 p. 1000 de glycine ou de DL-phenylalanine dans le chlorure sodique 9 p. 1 000 et la suspension globulaire versee dans 5 volumes d'acide acetique sature de chlorure sodique et chauffe a 95' C (Nencki et ZaleskiII). La temperature est ramenee a 95° G et maintenue a ce niveau pendant 5 minutes. Le liquide est filtre rapidement sur ouate et abandonne a la temperature du laboratoire. Apres 24 heures, les cristaux d'hemine sont separes par centrifugation, laves successivement une fois a I'eau, une fois dans une solution aqueuse a 1 p. 1000 de phenylalanine ou de glycine, 2 fois a I'eau, 1 fois a I'alcool ethylique a 95° C, 1 fois a I'ether, et seches sur chlorure calcique dans le vide. (rf) Mesures de radioactivite Les mesures de radioactivite de I'hemine et de la globine se font en double, apres combustion par vole seche, sur sources epaisses de BaCOg, au compteur de Geiger-Miiller a courant d'helium-isobutane. On trouvera dans les publications anterieures de I'un de nous (^2, le-is^ des details complementaires sur la preparation, I'hygiene et la dietetique des chiens, ainsi que sur les techniques d'etude de la synthese de I'hemo- globine in vitro. [e) Conduite des experiences Du sang heparine et glucose est preleve aux chiens soumis a I'anemie hemorragique et partage en plusieurs parties, de 50cm=^ chacune lorsqu'il s'agit de preparer I'hemine, de 5 cm^ dans le cas de la globine. La premiere partie, non irradiee, sert de reference ; la deuxieme est irradiee in vitro (20.000 a 1 00.000 r). D'autre part, I'animal est irradie in toto a la dose de 500 r et du sang est preleve |h a 1 h apres I'irradiation. Tons les echan- tillons de sang sont soumis a la centrifugation et le sang preleve apres irradia- tion (eventuellement dilue par rapport au temoin par suite de la soustraction sanguine precedente) est ramene au meme volume globulaire par sous- traction de plasma. Dans certaines experiences, le culot de globules rouges preleves avant irradiation du chien et debarrasses du plasma est additionne de plasma de I'animal irradie. Dans d'autres experiences enfin, des globules intacts sont respectivement mis, dans les memes conditions, en presence de plasma intact et de plasma irradie in vitro a 20.000 r. Les hematics sont remises en suspension, saturees d'oxygene par agitation prudente, et les difTerentes fractions sont additionnees de glycine-2-"C ou de DL-3-pheny- lalanine-2-i^C* aux memes concentrations dans tous les echantillons. La radioactivite est mesuree, apres incubation, dans la globine et dans I'hemine. {f) Donnees sur les conditions d'' irradiation A titre d'exemple, nous donnons ici un protocole : Exp. No. it'^— Irradiation du chien in toto (500 r) : 200kV— 18nx.A— filtre de cuivre de 0,5 mm — distance focale 180 cm — champ 50x30 cm — duree 15 minutes. * Glycine fournle par le Radiochemical Centre, Amersham, Bucks., Angleterre ; activite specifique 46,7 microcuries par mg. Phenylalanine fournie par Tracerlab, Boston, Mass., U.S.A. ; activite specifique 0,31 millicurie/millimole. 195 AUGMENTATION DE LA SYNTHESE DE l'hEMOGLOBINE in Vltro Irradiation du sang : a 500 r : memes constantes. a 1 00.000 r : 50kV— 2 mA— pas de filtre— distance focale 2 cm — champ 25 mm de diametre — duree 15 minutes. RESULTATS EX PE RIME NT AU X Les resultats sont transcrits dans les Tableaux I-III. Tableau I. — Irradiation in vivo et in vitro et Synthese de rhemine No. des experiences 61 87 102 108 No. des chiens BN49 N56 B58 N61 Taux d'hematies {par mm* de sang) 2.860.000 4.200.000 2.700.000 3.200.000 Taux de reticulocytes {par mm* de sang) 147.000 55.000 111.000 162.000 Taux de glycine-2-^^C (d.p.m. par cm* de sang) 222.000 222.000 74.000 92,500 Taux de glycine-2-^* C(y par cm* de sang) 2,36 2,36 0,79 0,98 Duree d'incubation du sang (heures) 7 5 6 7 Taux d'he'moglobine (g p. 1 00) 8,10 9,9 6,3 8,3 Activites {en disintegrations par minute et par eg d'hernine) Sang ternoin 14.200 1.092 11.793 9.151 Sang irradie in vitro (500 r) 15.700 1.554 27.035 — Sang irradie in vitro (20.000 r) — — — 10.507 Sang irradie' in vitro (1 00.000 r) 30.400 1.237 19.884 — Sang du chien irradie in toto (500r) . . 28.000 1.788 47.623 11.095 Hematies non irradiees {prelevees avant irradiation du chien) + plasma du chien irradie ' 19.180 13.088 Tableau II. — Irradiation et Synthese de la Globine No. de V experience 121 130 139 No. du chien N62 R65 B66 Taux d'hemoglobine (g p. cent) 7,15 7,45 8,7 Taux d'hematies par mm* de sang 3.000.000 3.100.000 3.500,000 Taux de reticulocytes par mm* de sang 210.000 295.000 210.000 Taux de Di.-3-phenylalanine-2-^*C, en disintegrations bar minute et par cm* de sang . . 277.500 294.500 590.000 Duree d'incubation du sang (heures) . . 5 5 7 Activitis {en disintegrations par minute et par eg de globine) Sang ternoin {avant irradiation) 851 999 703 Sang irradie in vitro (500 r) 1.244 — — Sang irradie in vitro (20.000 r) 1.019 1.234 722 Globules prilevis avant irradiation plus plasma prileve apres irradiation 929 1.097 — Sang d'animal irradii in toto 1.302 2.116 908 196 A. NIZET ET A. HERVE Tableau III. — Irradiation du Plasma et Synthese de I'hemine No. de rexperience No. du chien Taux d' hemoglobine (g p. cent) Taux d'he'maties par mm^ de sang Taux de reticulocytes (p. 1000 hematies) Taux de glycine-2-^*C {desintegralions par minute de sang) Volume de puree globulaire (cm^) Volume de plasma (cm*) Dure'e d'incubation du sang (heures) 128 P63 5,15 3.000.000 51,5 49.400 10,5 29,5 7 Activites [en desintegralions par minute et par eg d'he'mine) Hematies non irradiees plus plasma non irradie Hematies non irradiees plus plasma irradie (20.000r) 134 P63 5,75 3.500.000 87 17.000 30 40 5 4.350 5.200 DISCUSSION DES RESULTATS Les donnees experimentales autorisent les conclusions suivantes : {1) L'incorporation du carbone radioactif /« vitro a rhemine et a la globine des hematies jeunes de chien irradie est augmentee par rapport au sang preleve avant I'irradiation ; elle pent etre quadruplee. Cette donnee est en accord avec les resultats de Richmond et coll.^^. (2) L'irradiation du sang in vitro entraine, elle aussi, une augmentation de l'incorporation de ^^C a I'hemine et a la globine. {3) Cette incorporation est egalement acceleree dans les globules rouges preleves avant irradiation du chien et mis en presence de plasma preleve 1/2 heure apres irradiation de I'animal. {4) L'incorporation du ^*C par les hematies jeunes intactes est plus importante en presence de plasma irradie qu'en presence de ce meme plasma, non irradie. REMARqUES [a) II est souvent difficile de conclure avec certitude a une synthese proteique en se basant sur sur l'incorporation de metabolites radioactifs in vitro. On pent se demander s'il ne s'agit pas de phenomenes d'adsorption ou de liaisons chimiques anormales. A ce point de vue, I'hemoglobine nous donne de serieuses garanties, grace au controle que permet son groupement pros- thetique dont la structure est bien connue. Une augumentation de la radioactivite de la globine peut etre avec certitude attribute a un accroisse- ment de la synthese si elle s'accompagne d'une augmentation concomitante de la radioactivite de I'hemine^^. Au reste, le phenomene ne s'observe qvi'en presence d' hematies jeunes et il est bloque par des poisons metabo- liques ou respiratoires^''. D'autre part, I'activite specifique des amino-acides utilises est suffisamment elevee pour que leur introduction dans le sang ne modifie pas de fagon anormale le taux de I'amino-acidemie. {b) II n'est pas a priori certain que les syntheses de I'hemine et de la globine marchent de pair. Dans les presentes experiences I'effet excitant 197 AUGMENTATION DE LA SYNTHESE DE l'hEMOGLOBINE in Vt'tw a ete observe dans le cas de rhemine et de la globine ; nous sommes done autorises a parler d'une synthese de I'henioglobine, interessant a la fois la fraction proteique et son groupement prosthetique. (c) II n'y a pas lieu d'envisager I'influence possible d'une variation du volume globulaire consecutive au prelevement de sang effectue avant I'irradiation du chien. Le volume globulaire de tous les echantillons est ramene a celui du temoin et tous les echantillons subissent des manipulations identiques. II n'y a pas non plus de variation de taux des reticulocytes ; une crise reticulocytaire apres saignee ne survient qu'apres plusieurs heures. D'autre part, il ne s'agit pas non plus d'une liberation de facteurs excitants sous I'influence des saignees. La mise en evidence d'une stimulation de la synthese de I'hemoglobine dans le sang irradie in vitro et en presence de plasma irradie in vitro sufBt a refuter ces objections. II est hautement significatif que la stimulation de la synthese soit observee apres irradiation de plasma seul, prive d'elements figures. (d) La purete radioactive de I'hemine et de la globine a ete verifiee par plusieurs cristallisations et precipitations successives^'. (e) II n'existe pas de relation simple de proportionnalite entre la dose de rayonnement ionisant et le degre de stimulation de la synthese. II est possible, et meme vraisemblable, que des facteurs limitants, d'importance variable d'une experience a I'autre, et non controles, interviennent. Cette meme absence de proportionnalite a ete observee dans le cas d'autres facteurs stimulant la synthese^' ^. (/) Nous pouvons confirmer notre observation anterieure^'* relative a I'absence de toute hemolyse dans le sang irradie a des doses atteignant 200.000 r, dans nos conditions experimentales. (g) Les resultats que nous venons d'exposer ont un caractere preliminaire : ils ne nous renseignent pas sur la chronologic des phenomenes observes. lis ne nous apprennent pas a quel moment les proprietes stimulantes atteignent un maximum eventuel dans le sang de I'animal irradie, ni combien de temps elles persistent apres I'irradiation. Ces points restent a eclaircir. La stimulation de la synthese de I'hemoglobine, observee en premier lieu par Richmond et ses collaborateurs, et rapportee par nous a un phenomene humoral, n'est pas le seul cas d'acceleration d'un processus metabolique que Ton ait observe sous I'influence des radiations ionisantes. C'est ainsi que Back et Bloch-Frankenthal- ont constate une augmentation temporaire de la respiration de noyaux d'erythrocytes d'oiseau soumis a une irradiation de 500.000 a 1.500. 000 r. De meme, Forssberg et Hevesy^ ont montre que la fixation de phosphate marque au ^-P par le foie de jeunes souris irradiees (2000 r) etait considerablement augmentee par rapport aux temoins non irradies. L'irradiation augmente la teneur en fer du foie, de la rate, des reins et du serum du rat (10 a 1500r)^- ^. En ce qui concerne nos experiences, on pent se demander s'il ne s'agit pas d'une liberation ou d'une decharge sanguine brutale d'un facteur intervenant normalement dans I'anabolisme de I'hemoglobine (et peut- etre dans d'autres "anabolismes). Cette decharge pourrait etre suivie de troubles par deficit secondaire. Si cette hypothese se verifiait, I'identi- fication du facteur plasmatique pourrait presenter un interet pratique d'ordre therapeutique. 198 DISCUSSION CONCLUSIONS ET RESUME (1) La synthese de riiemoglobine in vitro par les reticulocytes de chien irradie est augmentee par rapport au sang preleve avant I'irradiation de ranimal. {2) La synthese de rhemoglobine est egalement augmentee dans les reticulocytes normaux mis en presence de plasma d'animal irradie. {3) Cette meme synthese est acceleree dans le sang irradie in vitro. (4) Le plasma irradie seul, in vitro, jouit egalement de proprietes stimula- trices de la synthese. (5) L'intervention d"un facteur humoral accelerant la synthese de rhemoglobine apres irradiation est de la sorte demontree. REFERENCES 1 Anson, M. L. et Mirsky, A. E. J. Gen. Physiol. 1930, 13 469. 2 Back, A. et Bloch-Frankenthal, L. Proc. Sac. Exper. Biol. a. Med. 1947, 66 366. 3 Borsook, H., Deasy, C. L., Haagen-Smit, A. S., Keighley, G. et Lowy, P. H. J. Biol. Chem. 1952, 196 669. * Chanutin, a. et Ludewig, G. Amer. J. Physiol. 1951, 166 381. 5 Forssberg, a. et Hevesy, G. Arkiv for Kemi, 1952, 5 93. 6 Lambert, S. Arch. Intern. Physiol. 1953, 61 462. ■^ London, L M., Shemin, D., West, R. et Rittenberg, D. J. Biol. Chem. 1948, 173 797 ; 1949, 179 463 ; 1950, 183 749. * Ludewtg, S. et Chanutin, A. Amer. J. Physiol. 1951, 166 384. ^ Lybeck, H., Lambert, S., Nizet, A. et Bar.\c, G. Arch. Intern. Physiol. 1954. 62 304. i« Muir, H. M. et Neuberger, A. Biochem. J. 1949, 45 163 ; 1950, 47 97. " Nencki, M. et Z.\LESKi, J. Z- Physiol. Chem. 1900, 30 384. 12 Nizet, A. Recherches sur la Nutrition et la Regeneration des Hematies, Liege, Vaillant-Carmanne, 1951. 13 Nizet, A. Rev. Med. Liige, 1953, 8 506. 1* Nizet, A., Bacq, Z. M. et Herve, A. Arch. Intern. Physiol. 1952, 60 449. 15 Nizet, A. et Lambert, S. ibid, 1953, 61 120. 16 Nizet, A. et Lambert, S. Bull. Soc. Fr. Chim. Biol. 1953, 35 771. 1^ Nizet, A. et Lambert, S. ibid, 1954, 36 307. 18 Nizet, A. et Robscheit-Robbins, F. S. Blood, 1950, 5 648. 13 Richmond, J. E., Altman, K. L et Salomon, K. J. Biol. Chem. 1951, 190 817. DISCUSSION H. Suit : I would like to mention in connection with Nizet's report and the well- known depression of the marrow iron turnover rate following low dose (25 r) total body radiation in the rat, that we have examined the effect of 300-5,000 r in vitro on the ability of normoblasts of human bone marrow in culture to take up iron. For the culture of the human bone marrow we used the technique of Lajtha of our laboratory. Irradiation was given at the beginning of the culture period (0 hr.). ^®Fe-globulin was added at the third hour of culture in the amount of 1 jjLc/ml culture medium. After 17-20 hours the culture was opened and autoradiographs prepared, which were developed and stained after a period of 4 weeks' exposure. This per- mitted a differential count on the erythroid series, a count of grains over individual cells, and the determination of the percentage of the cells of any group that showed a positive autoradiograph. 199 AUGMENTATION DE LA SYNTHESE DE l'hEMOGLOBINE Ul VltVO Figure 1 shows the result of such an experiment. Here a dose of 1,000 r was given in 3 mins. and the culture continued for 17 hours. The only change observed is that of a marked shift in the differential count of the normoblasts. There are 72 per cent early normoblasts (E.N. — pro, baso, and early polychromatic normoblasts) and 28 per cent late normoblasts (L.N. — late polychromatic and orthochromatic normoblasts) in the control culture while the irradiated specimen contained only 10 per cent E.N. but had 90 per cent L.N. The upper average grain count and the per cent positivity did not alter appreciably. These changes were not considered significant in any of the 12 marrows examined with doses varying from 300-5,000 r. 100- 90- 80 70 \ 60 I 50 t •^ H-0 30 20 10 EN m LN ■■2M:-:10y. LN FN y-30-: ■■'■10 Control Figure 1 Irradiated These data have suggested to us that those cells which had not received damage due to the in vitro radiation which was lethal by the time we observed them, had been able to take up iron at an essentially normal rate. We did not evaluate changes in reticulocytes properly and therefore cannot comment on them. However, we found nothing to indicate that iron uptake in vitro by human normoblasts was either accelerated or depressed by the rather large doses of radiation used. It seems, therefore, that the observed depression of marrow turnover rate in vivo following total body radiation may result from a mitotic inhibition or delayed maturation or some other factor than an interference with the actual uptake of iron. 200 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION D. VV. VAN Bekkum Medical Biological Laboratory of the National Defence Research Council, Rijswijk, Netherlands The interference with mitosis and the death of living cells after exposure to ionizing radiation are commonly assumed to be the outcome of some primary damage to the cell nucleus. Most morphological and biochemical changes so far described after irradiation have been localized in the nucleus. Pycnosis and karyorrhexis, structural damage to chromosomes and inhibition of DNA synthesis are among the most widely recognized cellular effects of irradiation. There are however reasons to question the assumption that radiation damage is primarily to the nucleus (Trowell^ and Mole-). Therefore some years ago studies were initiated in this laboratory with the object of collecting information on the effect of ionizing radiation on metabolic processes in the cytoplasma. It was postulated that the interference with nuclear function, e.g. cell division and DNA synthesis, which follows exposure to irradiation, might well be the consequence of damage to biochemical reaction systems outside the nucleus. Since synthetic processes in the nuclei are generally supposed to be dependent on energy-generating reactions which occur in the cytoplasma, the oxidative phosphorylation of mitochondria seemed to present the most obvious subject for investigation. In preliminary experiments with mitochondrial preparations from various tissues, it was found that spleen mitochondria showed a decreased phosphate uptake shortly after total body irradiation. Most of our subsequent work has been carried out with rat spleen tissue because of its radiosensitivity and its relative abundance per animal. A decrease of oxidative phosphoryla- tion of isolated spleen mitochondria following total body exposure to X-rays has since been reported by several investigators. In 1952 Potter and Bethel^ described a decrease of phosphate uptake by mitochondria, isolated from rat spleen after total body irradiation with 800 r. Similar observations were reported shortly afterwards by ourselves (van Bekkum et al^). In these experiments also rather large doses of X-radiation, namely 800 r and l,100r, were administered to rats and spleen mitochondria were isolated 2, 4 and 24 hours afterwards. In addition to a diminished phosphate uptake, some decrease of oxygen consumption was observed in most experiments, although the latter effect has been generally of lower magnitude. Thus a decrease of P/O ratios has been consistently found with several substrates. Maxwell and Ashvvell-^ have published comparable results obtained with mitochondrial preparations from mouse spleen at 1-7 days after a lethal dose of total body X-irradiation. It should be pointed out that in general the study of biochemical reactions in highly radio-sensitive tissues 201 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION can only be expected to throw some light on the mechanism of initial radia- tion injury, if performed within a few hours after exposure. After a larger interval, especially when relatively large doses of radiation are being employed, the structural changes in the tissue are so radical that a change in tissue composition rather than in cellular function is most likely to be reflected in the results. Therefore our experiments are usually performed at 4 hours or less after total body irradiation, when changes in organ weight are not yet apparent. INFLUENCE OF DOSE After the effect of large doses of total body irradiation had been established, we have attempted to assess the sensitivity of oxidative phosphorylation to this type of injury. Furthermore our studies have been extended to mito- chondria isolated from rat thymus. The methods employed with spleen 60 J .50 w so- so 10 c g ^ f- c ^ <=> Qi C3 Ci cs, "a ''^ tx Oi c f- f- (- o s> Ci ^ ^ ?> C^) TJ t\ Figure 1. Oxidative phosphorylation of rat spleen mitochondria at 4 hours after various doses of total body irradiation. Black bars : phosphate uptake in [j.moI/mgN ; white bars : oxygen uptake in [i,atoms/mgN. Each experiment consists of 4 groups including a control. mitochondria have been described previously (van Bekkum et al^) and were applied with minor modifications to the study of mitochondria from thymus. Mitochondria were prepared from the pooled tissues of at least 2 rats in case of spleen and of 4 rats in case of thymus. Oxidative phos- phorylation of these preparations was estimated in duplicate. Variation of activity between and to a less extent within batches of normal rats necessitated the inclusion of control animals in every experiment. Some of the results are depicted in Figures 1-3. It is apparent that in the case of spleen mitochondria, oxygen uptake is usually much less affected than phosphorylation, while in the case of mito- chondria from thymus this difference is not so outspoken, although P/O ratios are depressed after irradiation in most experiments. The minimal dose of radiation which was capable of inducing a decrease of mitochondrial phosphorylation was found to be between 50 r and lOOr for both tissues. Microscopical examination of the tissues used in these experiments revealed extensive cellular destruction at 4 hours after doses of 300 r and more. In 202 D. W. VAN BEKKUM the spleen after a dose of 50r, some cell debris is clearly recognizable in most of the lymph follicles, while in an occasional one very little if any sign of damage is to be found. After lOOr, a greater number of nuclear fragments c6 "o E ^60 ■50 -30 20 - 10 I Jl c t- c f- C (- Ci Ci S> 0 ■o <>> ie C Figure 2. Oxidative phosphorylation of rat spleen mitochondria at 4 hours after various doses of total body irradiation. Black bars : phosphate uptake in (jimol/mgN ; white bars : oxygen uptake in [xatoms/mgN and pycnotic nuclei have been observed, but the damage is markedly less than after the larger doses of radiation. In thymus slices only a small amount of nuclear fragmentation could be observed at 4 hours after 50 r and mitotic figures were still present. After ^60 "a so "I liO ri- 30 20 10 __ 1 n C i- c ^ c ^ Ci O Q> cs Ci C> ^ Co ^ m C f- c 1 u (7 f- C f- Figure 3. Oxidative phosplwrylation of rat thymus mitochondria at 4 hours after various doses of total body irradiation. Black bars : phosphate uptake in [xmol/mgN ; white bars : oxygen uptake in [xatoms/mgN. lOOr the signs of destruction were more extensive, although many apparently normal nuclei and a few mitotic figures remained. Although no accurate estimation of the amount of visible damage has been attempted, it appears that the minimal doses of total body irradiation required to produce morpho- logical evidence of nuclear damage in a sizable number of cells and those 203 1 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION necessary for the biochemical change under discussion to become measur- able do not differ widely if at all. TIME-EFFECT Since the process of oxidativ^e phosphorylation has thus been found to be highly radiosensitive, it seemed of interest to determine more accurately the earliest time after irradiation at which this lesion becomes discernable. These experiments were performed with rat spleen mitochondria at various times up to 4 hours after total body irradiation with a dose of 700 r. The time-effect curve shown in Figure 4 reveals a latent period of more than 1 hour after the irradiation before the oxidative phosphorylation becomes clearly impaired. From most of the tissues used in this series, samples were taken for histological study with the object to investigate whether the dis- turbance of oxidative phosphorylation precedes the morphological changes in the nuclei or otherwise. 100 50 Figure 4. Phosphate uptake of rat spleen mitochondria at various periods after total body irradiation with a dose of 700 r. Abscissae: hours after irradiation, ordinates: percentage of corresponding control value ; white circles with attached vertical lines : mean ± S.E. ; n : number of experiments V¥ V2 7 8 V- I I I 1 L 5 3 V^ 5 S -"K -Tl The first definite signs of damage, which consisted mainly of nuclear vacuolization, were found at 1 hour after irradiation, but the number of nuclei affected was very small at this time. At 2 hours after 700 r the degeneration was more pronounced with some pycnotic nuclei in most of the follicles. At this time the picture resembled that found at 4 hours after 50-lOOr in the previous series. The slices obtained at 4 hours after 700 r showed a domination of pycnosis and fragmentation in all of the follicles and a number of cells in the pulpa was involved as well. After that period degeneration progresses still further and removal of dead material by macrophages begins. In general this pattern closely resembles the observa- tions made by Trowell^ on lymph nodes after in vivo irradiation. The limited number of our results does not allow a definite conclusion as to the sequence in which the two effects develop after irradiation. It is therefore at present not possible to decide if a causal relationship between the two phenomena exists but the approximately simultaneous appearance of both the cytoplasmic and the nuclear changes seems to warrant further investigation. 204 D. W. VAN BEKKUM IRRADIATION OF SPECIFIC ORGANS In this connection it is perhaps of interest to mention that we have not been able to find any interference with the oxidative phosphorylation of mitochon- dria isolated under similar conditions from the livers of irradiated rats. Even a dose of 5,000 r administered to the liver region failed to produce an effect as observed in the case of spleen and thymus mitochondria. Since the liver is generally held to be a radio-resistant organ, these observations suggest a relation between the disturbance of oxidative phosphorylation and the radiosensitivity of the cells from which the mitochondria are derived. Because of the well known sensitivity of the spleen to various forms of stress, it was considered essential to investigate the effect of radiation on the zn E 700 Figure 5. Effect of intermittent hypoxia on oxidative phos- phorylation of spleen mitochondria. Black bars : phosphate uptake in [xmol/mgN ; white bars : oxygen uptake in [I atoms/mg N ; C ; controls ; IH : intermittent hypoxia 75 SO 25 C IH I IH spleen only. To this end the spleen was exteriorized during irradiation while the rest of the animal was being shielded. In this series the controls were subjected to a sham-procedure. The depression of oxidative phos- phorylation of the mitochondria isolated from the irradiated spleens was found to be comparable to that observed after total body irradiation. The interference with phosphorylating activity after total body exposure is therefore at least for the greater part the result of the action of X-rays on the spleen tissue itself. In vitro irradiation of isolated spleen mitochondria has been performed both in the inactive state at 0° C and during incubation in the presence of substrates and oxygen at 38° C. Following the irradiation the mitochondria were kept at 0° C in the presence of versene for 4 hours and reincubated 205 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION for the estimation of oxidative phosphorylation. No effect of irradiation even with doses of 20,000 r has been found, which is in accordance with results of Potter et aP. From this it should not be concluded that the mitochondrial defect is necessarily secondary to some radiation-induced aberration in other parts of the cell. For one thing, it has not been possible to keep the mitochondria in vitro in a state of active metabolism for a period of 2 hours, and this might well be essential for the lesion to develop, as has been observed with other forms of radiation-induced damage. The results obtained with rat spleen do not allow an evaluation of the radio-sensitivity of the mitochondria in the various haematopoietic cells, since this organ contains beside lymphoid elements, also variable quantities of erythropoietic and myelopoietic cells. Our results with thymus mitochondria indicate that the mitochondria of lymphoid cells are affected by total body irradiation. In order to assess the sensitivity of mitochondria from erythropoietic cells in 80 CO -^so o E W 20 J I Figure 6. Oxidative phos- phorylation of spleen mitochon- dria at 4 hours after various doses of total body irradiation of rats subjected to intermittent hypoxia for a period of 3-4 days. Black bars : phosphate uptake jmgN ; white bars : oxygen uptake jmgl^i 0 ^ ^ ^ C f- f- S ^ this respect, an increase of the red cell forming elements has been induced by exposure of the animals to intermittent hypoxia for a period of 3 to 4 days. Mitochondrial preparations from the spleens of these pretreated rats were found to exhibit an increased rate of oxidative phosphorylation, with a con- comitant increase of P/O ratios to nearly double the control values (Figure 5). This activity is also severely depressed after total body irradiation (Figure 6), which indicates that the activity of mitochondria from erythropoietic cells is also susceptible to irradiation. INTERPRETATION The work so far summarized has been mainly of a descriptive kind. The significance of the disturbance of oxidative phosphorylation with regard to the mechanism of radiation injury to the cell is not known. Also the nature of the derangement of oxidative phosphorylation is still obscure. There have been suggestions that it might be secondary to the increased ATP breakdown which occurs in spleen homogenates following total body 206 D. \V. VAN' BEKKUM irradiation. In 1952 Ashvvell and Hickman' reported a threefold increase of ATP dephosphorylating activity (to be denominated ATP-ase activity hereafter) of mouse spleen homogenates at 1 to 1 1 days after total body irradiation with lethal doses of X-rays and in 1953 more details on this subject were published (Ashwell and Hickman^). The authors conclude that this rise could be best explained by the premise that a large amount of inert cellular material had been destroyed after irradiation, while the par- ticular enzyme systems remained unaffected. More recently Dubois et aP showed that the increase of ATP-ase activity could be observed in rat spleen and thymus homogenates even after total body irradiation with doses as small as 25-50 r and they suggested the possible significance of this effect with regard to the maintenance of energy requiring reactions after irradia- tion. Previously it had been pointed out by Maxwell et al'^ that the increase Figure 7. ATP-ase activity of rat spleen homogenates at various periods after total body irradiation with a dose o/ 1 , 1 00 r . Values are expressed as a percentage of corre- sponding controls % 250 200 150 100 SO - 12 Zf 2¥K of ATP-ase activity cannot be the cause of the depressed phosphorylation of spleen mitochondria, because ATP-ase activity had been blocked by NaF in the system used for the measurement of oxidative phosphorylation. However in our hands the amount of NaF employed by Maxwell et al left a small part of the ATP-ase activity uninhibited and we found this remaining activity in homogenates to be proportional to the values obtained in the absence of NaF. Therefore a more extensive investigation of the possible role of ATP-ase in the disturbance of oxidative phosphorylation has been carried out, using mouse and rat spleen preparations. The results confirm the conclusion of Maxwell et al and may be summarized as follows : (7) The ATP-ase activity of rat spleen homogenates has been estimated at various intervals after irradiation and the results have been compared with the time-effect curve obtained in the experiments on oxidative phos- phorylation. It has been found that the increase of ATP-ase activity of the homogenates is not apparent within a few hours after irradiation, when the disturbance of oxidative phosphorylation is already well developed {Figure 7). 207 PHOSPHORYLATING ACTIVITY OF MITOCHONDRIA AFTER TOTAL BODY IRRADIATION {2) The ATP-ase activity of isolated spleen mitochondria after total body irradiation has been found to be normal even at 24 hours after irradiation, in the presence of a severely impaired phosphorylating capacity which was observed in samples of the same preparation {Figure 8). We have also investigated if the decrease of anaerobic glycolysis which has been described in mouse spleen homogenates at one day and longer ^ I/O o E 20 W $ J C ^'K C 2'/\ Figure 8. Phosphate uptake and ATP-ase activity of mouse spleen mitochondria at various periods after total body irradiation with a dose of I,100r. Black bars: phosphate uptake in pimol/mgN; hatched bars : ATP- ase activity in (jimoI/mgN of phosphate formed after irradiation (Hickman and Ashwell^*'), has any relation to the disturb- ance of oxidative phosphorylation. The fact that a normal rate of glycolysis was observed in the presence of a decreased oxidative phosphorylation {Figure 9) indicates that different mechanisms are probably involved in the development of these two types of radiation damage. In conclusion there is as yet no evidence of the depression of oxidative phosphorylation being secondary to some other known radiation-induced Figure 9. Oxidative phosphorylation of spleen mitochondria and anaerobic gly- colysis of spleen homogenates after total body irradiation with a dose of 700 r. Black bars : phosphate uptake in [jimol/ mgN [left ordinate) ; white bars : oxygen uptake in [j,atoms/mgN {left ordinate) ; hatched bars : lactate formation in [jimol/ ingN [right ordinate) > 60 3-50 o E 30 20 10 L CD r2i o W E 8 ^ 6 if - 2 C 2K U\ biochemical alteration. Therefore the effect must be ascribed to some block in the oxidation-coupled phosphorylating reactions, the exact nature of which is at present little understood. SUMMARY The depression of oxidative phosphorylation of mitochondria isolated from spleen or thymus has been found to occur after doses as low as 50 r. This biochemical change develops within a few hours after irradiation and a close parallelism with the appearance of morphological changes in the nuclei 208 DISCUSSION has been observed. The effect has also been found after irradiation of the exteriorized spleen only, but could not be produced by in vitro irradiation of mitochondria. The decrease of phosphorylation is not secondary to the increased ATP-ase activity and develops prior to the depression of anaerobic glycolysis, which are observed in spleen homogenates after total body irradiation. The sensitivity of oxidative phosphorylation as well as its relatively rapid impairment after irradiation suggest that this cytoplasmic lesion may be intimately connected with the primary radiation injury. REFERENCES 1 Trowell, O. a. J. Path. Bad. 1952, 64 687. 2 Mole, R. H. Brit. J. Radiol. 1953, 26 234. 3 Potter, R. L. and Bethel, F. H. Federation Proc. 1952, 11 270. * VAN Bekkum, D. W., Jongepier, H. J., Nieuwerkerk, H. T. M. and Cohen, J. A. Trans. Faraday Soc. 1953, 49 329. '" Maxwell, E. and Ashwell, G. Arch. Biochem. Biophys. 1953, 43 389. " VAN Bekkum, D. W., Jongepier, H. J., Nieuwerkerk, H. T. M. and Cohen, J. A. Brit. J. Radiol. 1954,27 127. ^ Ashwell, G. and Hickman, J. Proc. Soc. exp. Biol. Med. 1952, 80 407. 8 Ashwell, G. and Hickman, J. J. Biol. Chem. 1953, 201 651. 9 Dubois, K. P. and Petersen, D. F. Amer. J. Physiol. 1954, 176 282. 10 Hickman, J. and Ashwell, G. J. Biol. Chem. 1953, 205 65 1 . DISCUSSION R. H. Mole : I am sure van Bekkum is right in deciding to look for biochemical changes in the first few hours after irradiation. As he points out, at a time when there is visible cell death in the tissue examined, biochemical changes must be occur- ring from the fact of cell death alone. But in the light of this I wonder why he chose as long an interval as 4 hours since by that time cell death in the spleen is quite evident. I would also like to suggest that changes in oxidative phosphorylation by mito- chondria may be analogous to the morphological changes in cell nuclei which follow irradiation, and may be a consequence of lethal damage to the cell rather than the cause of it. Just as nuclear pycnosis occurs in radio-sensitive organs and not radio- resistant organs like muscle, so oxidative phosphorylation is altered in the spleen, but not in the muscle (as Laser mentioned). If there is one thing clear about the effects of whole body irradiation it is that some organs are radiosensitive and some are resistant. If there is a biochemical lesson due to irradiation — and a biochemical lesson there surely must be — then it seems to me it must be sought in the specific biochemical activities which distinguish radio-sensitive from radio-resistant tissues, not in the general metabolic properties which are common to all cells. 209 ACTION D'UNE DOSE UNIQUE DE RAYONS X SUR OUELOUES FRACTIONS DE PHOSPHORE ACIDO-SOLUBLE ET SUR LA RESPIRATION DE LA PEAU CHEZ LE RAT* p. Mandel, Ch. Gros et J. Rodesch Institut de Chimie Biologique, Faculte de Medecine, Strasbourg L 'importance du metabolisme energetique dans les syntheses cellulaires et dans le renouvellement des constituants tissulaires, a incite divers auteurs a rechercher les modifications des composes phosphores acido-solubles de la rate^' ^\ de la moelle osseuse^ ou de bacteries entieres^ sous I'effet des rayons X. II nous a paru interessant d'examiner Taction de ces rayons sur le phosphore acido-soluble de la peau et ceci pour des multiples raisons. Tout d'abord il est aise de comparer I'effet d'une irradiation de I'animal entier et d'une irradiation locale directe au niveau de la peau. D'autre part, tout en etant sensible aux rayons X, du fait des proliferations de la couche basale, la peau subit cependant sous I'effet des irradiations moins de destructions cellulaires que la rate, la moelle osseuse ou les cultures bacteriennes. On ne sera done pas astreint a tenir compte des disparitions de cellules pour I'interpretation des resultats dans le cas de la peau dans la meme mesure que lorsqu'il s'agit de la rate ou de la moelle osseuse. Enfin il est a peine necessaire de rappeler I'importance des lesions cutanees sous I'effet d'irradiations, objet de preoccupations de nombreux radiobiologistes. Nous rapportons ici nos resultats concernant Taction d'une irradiation totale sur diverses fractions de phosphore acido-soluble de la peau en meme temps que les repercussions sur la consommation d'oxygene de ce tissu. Rappelons que Tetude des composes phosphores acido-solubles tels TA.T.P., le phosphagene et les esters phosphoriques fournissent des indications a la fois sur les disponibilites en energie et sur le catabolisme des glucides. II ne saurait etre question, dans le cadre de cet expose de rappeler la biblio- graphic concernant les effets metaboliques des radiations et en particulier leur action sur le metabolisme des glucides. On trouvera des indications dans de nombreux articles, monographies et mises au point^' ^^•^**^. Nos essais ont porte sur un total de 120 rats Wistar, d'un poids variant de 150 a 250 g. Soixante de ces rats ont servi de temoins, les 60 autres ont subi une irradiation corporelle totale. (Dose : 700 r ; tension : 180kv ; Filtre : 1 Cu ; Distance foyer-rat : 50 cm ; Debit : 25r/mn.) Les animaux groupes en lots homogenes, du meme sexe, issus d'une meme portee et descendant de croisements repetes entre freres et soeurs a travers plusieurs generations, ont ete sacrifies par saignee 24 heures, 48 heures, 4 jours, 7 jours, 10 jours, 14 jours et 21 jours apres Tirradiation. En vue * Travail effectue avec le concours de I'lnstitut National d'Hygiene. 210 p. MANDEL, CH. GROS ET J. RODESCH des dosages de phosphore acido-soluble, la peau prelevee rapidement dans la region comprise entre les omoplates en haut, les cretes iliaques en has et limitee lateralement par la ligne axillaire, a ete congelee dans un melange d'acetone et de neige carbonique. Un jour avant le sacrifice la peau qui devait etre prelevee, a ete rasee. Le tissu cutane a ete ensuite extrait par de I'acide trichloracetique a lO^V) en chambre froide a 0°. Sur I'extrait ainsi obtenu nous avons dose le P total selon Briggs, le P mineral a I'etat de phosphate ammoniaco-magnesien et le P du phosphagene selon Lohmann'. p. 100 i-50 + W + 30 +Z0 + 10 0 -10 -20 -30 -50 Action des Rayons X sur fa Consommation d' 0 de la Peau {Resultafs rapporfes a 100 ^ de poids frais) Le reste de I'extrait a ete firactionne par precipitation a la baryte conforme- ment a la technique decrite par Sachs^^, que nous avons modifiee^'' compte tenu du procede de Lepage^. Nous avons ainsi determine dans la fraction des composes baryum-insolubles : le P labile de I'A.T.P. et les esters phos- phoriques ; dans la fi-action baryum-soluble, alcool insoluble : les esters glucose- 1-phosphoriques, glucose-6-phosphoriques et les trioses-phosphates. p. 100 fW +Z0 0 -20 -to Action des Rajons X sur dii^erses fractions de P acido-soluble de la Peau P. Mai p.100 +W +20 0 -20 -f-0 P. Mineral 21 Jours Dans le residu, apres delipidation, nous avons determine le P de I'acide desoxyribonucleique (A.D.N.) apres separation selon la technique de Schmidt et Tannhauser^^ legerement modifiee, suivie de dosages de phosphore et de desoxypentoses. La consommation d'oxygene a ete mesuree a I'appareil de Warburg selon la technique classique en milieu de Krebs glucose et a Zl". La peau a ete soigneusement rasee sous anesthesie generale 3 heures avant le sacrifice des animaux et a ete rapidement prelevee a I'aide d'un dermatome dans la meme region que les echantillons utilises pour les dosages de P acido-soluble. Des examens histologiques ont montre que la partie prelevee comprenait 211 ACTION d'uNE dose UNIQUE DE RAYONS X I'epiderme et le derme, a I'exclusion de toute formation musculaire. resultats de nos essais consignes dans les graphiques ci-contre : Les i Graphiques Nous y avons represente les variations des valeurs en p. 100 par rapport aux temoins non irradies. Chaqiie point de la courbe represente une experience comportant un minimum de 4 temoins et de 4 animaux irradies. P. Glucose-1- phosphates P. Triases -phosphates p.100 i-80 ii.- solubles) p. 100 f80 En ce qui concerne le P acido-soluble total qui est de 29,3 mg chez le rat normal on remarque apres un accroissement leger dans les premieres 48 heures, une baisse de sa valeur qui atteint 23 p. 100 aux environs du 7eme jour. Le P mineral (valeur normale 14,2mg en moyenne) accuse egalement un accroissement au bout de 24 heures approximativement du meme ordre de grandeur que celui du P total. On note par la suite une reduction qui se maintient plus longtemps que celle du P total, puisque le retour a la normale survient seulement apres 14 jours. P/ab/7e de I'ATP Le phosphagene dont le taux normal est de 3,33 mg environ accuse une baisse notable dans la periode qui s'etale entre le ler et le 13eme jour apres I'irradiation ; la valeur la plus basse ( — 40 p. 100) est enregistree entre le 6eme et le 7eme jour. II convient d'insister sur I'augmentation du phos- phagene apres le 14eme jour qui s'accentue encore jusqu'au 21eme jour. L'A.T.P. dont la valeur normale est de 3,75 mg, montre une oscillation tantot dans le sens negatif, tantot dans le sens positif au cours des premieres 48 heures. Par la suite on releve une reduction considerable ; les valeurs les plus basses se situent aux environs du 7eme jour. Apres le lOeme jour on assiste a une augmentation notable de I'A.T.P. 212 p. MANDEL, CH. GROS ET J. RODESCH Parmi les composes phosphores intermediaires du catabolisme des glucides, nous retiendrons revolution des esters phosphoriques baryum-insoluble et du glucose- 1 -phosphate de la fraction baryum-soluble. Les esters phosphoriques baryum-insolubles presentent une augmentation jusqu'au 14eme jour alors que Ton note une reduction apres ces delais. Les glucose-\-phosphates par centre se trouvent regulierement abaisses a partir du 2eme et ceci jusqu'au lOeme jour. Notons que les trioses-phosphates montrent egalement une baisse reguliere a partir du 4eme jour : (45 p. 100 le 4eme jour, 30 p. 100 le lOeme jour). Pour ce qui est de la consommation d'oxygene on releve un accroissement de celle-ci dans les premieres 24 heures suivie d'une baisse avec retour a la normale aux environs du lOemejour. Discussion des Resultats L'accroissement du P mineral comme celui du P total dans les premieres 24 heures est a rapprocher de I'augmentation de la consommation d'oxygene de la peau a la meme epoque. Une telle augmentation de I'oxygene consomme ete signalee en ce qui concerne la moelle osseuse^^. Nous ne P. Esters phosphori(jues (^B^-insofubles) saurions expliquer d'une fagon valable ce phenomene avec les donnees dont nous disposons actuellement. La reduction sensiblement parallele du P mineral et du P total jusqu'au 7eme jour, montre que la teneur en P organique qui englobe a la fois les composes intermediaires du metabolisme des glucides et les constituants riches en energie, n'a pas varie d'une fa^on sensible. Apres le 7eme jour le maintien d'un phosphore mineral abaisse avec un phosphore total qui revient a la normale, traduit un accroissement global des composes phos- phores organiques acido-solubles. Comme par ailleurs nous constatons durant la meme periode qui s'etale du 2eme au lOemejour une baisse tres accentuee des composes riches en energie, A.T.P. et phosphagene, on pent admettre une accumulation des composes phosphores intermediaires du metabolisme a la suite des blocages enzymatiques. Les blocages portent d'une fagon inegale sur les divers enzymes. Ceci nous explique I'augmenta- tion des esters phosphoriques de la fraction baryum-insoluble et la diminu- tion du glucose- 1 -phosphate a partir du 2eme jour de meme que la baisse reguliere des trioses-phosphates a partir du 4eme jour. II convient d'insister sur l'accroissement des esters phosphoriques de la fraction Ba-insoluble durant la periode de baisse de 1' A.T.P. Elle pent s'interpreter comme le reflet d'un blocage du catabolisme au stade de ces esters phosphoriques, dont la consequence evidente est une reduction de la formation de I'A.T.P. 213 ACTION D UNE DOSE UNIQUE DE RAYONS X Au moment ou I'A.T.P. revient a la normale et attaint meme des valeurs superieures a celles-ci, le blocage se trouve leve et le taux des esters phos- phoriques Ba-insolubles se trouve naturellement abaisse. Pour ce qui est du glucose- 1 -phosphate, stade initial de la degradation du glycogene, on constate une reduction apres 48 heures c(ui parle en faveur d'un blocage de la phosphorylase. On pourrait se demander si les modifications observees en rapportant les resultats au poids frais, ne sont pas en relation avec des modifications eventuelles de I'etat d'hydratation. Nous avons de ce fait determine la teneur en proteines des echantillons de peau analyses et recalcule les valeurs en les rapportant a ces proteines. Les conclusions qui se degagent se trou- vent en parfait accord avec ce qui vient d'etre dit plus haut. II en est de meme dans les grandes lignes si Ton considere les valeurs rapportees a 1 'A.D.N. Cependant, dans ce dernier cas, a la suite de la destruction d'un certain nombre de noyaux, que reflete la diminution de I'A.D.N., les reductions de I'A.T.P. et du phosphagene sont plus attenuees. II n'en reste pas moins vrai que la chute des composes phosphores riches en energie ne peut etre mise sur le compte de la disparition d'un certain nombre de cellules. La reduction de I'A.T.P. et du phosphagene que Ton retrouve en rapportant les valeurs a I'A.D.N., traduit un appauvrissement des cellules en ces composes. Addendum Nous avons etudie comparativement les effets d'une irradiation de I'animal entier et d'une irradiation locale superficielle sur le Phosphore acido-soluble de la peau. Dans les deux cas la meme dose de 700 r a ete administree. Si Ton compare les resultats que nous venons de discuter avec ce que nous avons observe a la suite d'une irradiation locale, superficielle, il apparait que dans ce dernier cas les reactions sont bien plus attenuees. En effet, apres I'irradiation locale, les reductions du P total, du P mineral, de I'A.T.P, et du Phosphagene de meme que I'accroissement des esters phosphoriques baryum-insolubles sont nettement plus faibles que dans le cas de I'irradiation Action des rayons X sur racide desoxypentose nucleique de la Peau ■p.ioo ^30 -W -20 -30 A Dose ■■ 700 r (irradiation corporelle fota/e) _ 1 i\ ij- 1 6 '7 70 lf ^'■^■^^^'^' J"*^^^ C de I'organisme entier. De plus I'accroissement reactionnel de I'A.T.P. et du phosphagene au 21emejour analogue a celui des acides nucleiques de la moelle osseuse et de la rate rapportes anterieurement est pratiquement inexistant apres une irradiation locale. 214 p. MANDEL, CH. GROS ET J. RODESCH Conclusion De I'ensemble de ces resultats se degage la notion d'une chute importante des composes phosphores riches en energie dans les premiers jours qui suivent I'irradiation. Ce fait est sans doute la consequence d'un trouble du cata- bolisme des glucides dont un autre aspect apparait a I'examen de revolution des esters hexoses-phosphoriques. Dans ce cadre il convient de noter le blocage des diverses activites enzymatiques provoquant la baisse de certains constituants (glucose- 1 -phosphates, trioses-phosphates, glucose-6-phosphates) et I'accroissement de certains autres (esters phosphoriques Ba-insolubles). A partir du 7eme jour on assiste a un retour a la normale des taux de I'A.T.P. d'abord, du phosphagene ensuite. II est interessant de noter qu'au 21eme jour on enregistre des valeurs d'A.T.P. nettement superieures a la normale. Pareil phenomene a deja ete observe pour I'acide ribonucleique et de I'acide desoxyribonucleique de la rate^ et la moelle osseuse* apres irradiation. II semble ainsi que dans la periode eloignee de I'irradiation on observe a la fois un effet stimulant sur la synthese des acides nucleiques et le metabolisme glucidique. II importe encore de souligner que les modifications rapportees ne sont pas dues seulement a une destruction de tissus avec disparition d'un certain nombre de cellules mais a des variations au niveau des cellules restantes. Si Ton oppose aux consequences de I'irradiation totale ceux observees a la suite d'une irradiation locale superficielle on constate que dans ce dernier cas les troubles sont beaucoup plus attenues. REFERENCES 1 BAcq, Z. M., Lecomte, J. et Herve, A. Arch. int. Physiol. 1949, 57 142. '^ BiLLEN, D., Strehler, B. L., Stapleton, G. E. et Brigham, E. Arch. Biochem. Biophys. 1953,43 1. 3 Gros, Ch. M. et Mandel, P. J. beige Radiol. 1952, 35 357. * Gros, Ch. M., Mandel, P., Metais, P. et Voegtlin, R. C.R. Acad. Set., Paris, 1951.233 1685. 5 Krebs, H. a. Biochim. biophys. Acta, 1950, 4 1249. ^ Lepage, G. A. In Manometric Techniques and Tissue Metabolism, Burgess Publishing Co., Minneapolis, 1949, 1 227. ■> LoHMANN, K. Biochem. Z- 1928, 194 306. * LouREAU, M. et LARTiquE. Experientia, 1950,6 25 ; Arch. Sci. physiol. 1950, 4 197 ; Arch. Sci. physiol. 1951, 5 83 ; J. Physiol. 1951, 43 593 ; C.R. Acad. Sci., Paris, 1952, 234 2022. 9 LuTw^AK, — , M.\NN, C. Biochem. J. 1951. 49 300 ; ibid, 1952. 52 356. 1" Mandel, P., Bieth, R. et Weill, J. D. Bull. Soc. Chun. bioL. Paris, 1953, 35 973. ^1 Maxwell, E. et Ashwell, G. .Arch. Biochem. 1953, 43 389. 12 Ord, M. G. et Stocken, L. A. Physiol. Rev. 1953,33 357. 1^ Richmond, J. E., Altmann, K. I. et Salomon, K. Science, 1951, 113 404. 1^ Sacks, J. J. biol. Chem. 1949, 181 655. 1^ Schmidt, G. et Th.\nnh.\user, J. S. ibid, 1945, 161 83. 1® Symposium of Radiology, \'o\. 1, p. 465, John VV'iley, New York, Chapman & Hall, London. 215 DNA SYNTHESIS IN BONE MARROW STUDIED BY AUTORADIOGRAPHY L. G. Lajtha, R. Oliver and F. Ellis Department of Radiotherapy, Oxford The purpose of this paper is to report on experiments on the incorporation of ^-P orthophosphate, adenine ^^C, and formate ^*C into deoxyribo- nucleic acid (DNA) by human bone marrow cells in vitro, and on the effect of X-irradiation and aminopterin on the synthesis of deoxyribonucleic acid. METHODS Cell suspensions from human bone marrows were cultured in a medium containing 80 per cent fresh human serum and 20 per cent balanced salt solution. The method of culture has been described in detail elsewhere (Lajtha^). The labelled compounds were added to the culture medium : 5[j.c/ml medium ^-P orthophosphate (with less than 0-1 [i,g inactive PO4 Figure 1. Localization of formate i*C in human bone marrow cells in vitro. Stained autoradiographs. X 2000. buffer) : 0-5[j.c/ml medium adenine i-^C (specific activity 10[xc/mg) : 1 [j.c/ml medium formate ^^C (specific activity 15[xc/mg). The cultures were incubated at 37° C for 2-48 hours, then smears were made, fixed in 95 per cent methylalcohol and hydrolysed in N HCl at 60° C for 6 minutes. 216 L. G. LAJTHA, R. OLIV'ER AND F. ELLIS This h)drohsis was found to remove all non DNA phosphorus or adenine from the cells while leaving DNA phosphorus and adenine behind (Lajtha^). After hydrolysis autoradiographs were prepared using the stripping film technique, and were exposed in light-tight boxes in a refrigerator. After processing, the slides were stained without removing the photographic emulsion (Lajtha^' *). The stained autoradiographs thus obtained allow differential and grain counting (see Figure 1) . The number of cells showing positive autoradiographs can be expressed as a percentage of the total counted. Grain counting over individual nuclei is also carried out and the maximum activity is expressed as 'upper average' grain count (the average grain count of 10-15 cells judged visually to show maximum activity). RESULTS [a] The cell cycle — If the cells synthesized DNA throughout the entire intermitotic period, then, after a few hours of culture in the presence of the isotope all the cells should contain labelled DNA. This, however, was found not to be the case. Experiments with ^^P or adenine ^'*C indicated that when cells were cultured for progressively greater lengths of time, a gradually increasing percentage of the cells showed DNA autoradiographs (per cent positive nuclei). The grain counting (upper average grain count per nucleus) demonstrated that the amount of labelled DNA per nucleus also increased with time, until a maximum was reached in about 15-18 hours. These findings indicate that DNA synthesis takes place during a limited period in the cell cycle. Lajtha, Oliver and Ellis^ showed that labelling of the DNA indicated a length of period of DNA synthesis (S period) of the order of 12-15 hours, and a total cell cycle time (intermitotic period) of about 40-45 hours. When only the mitotic figures were counted on the smears (metaphase and ana- phase) it appeared that, while in a 2-4 hours' culture with '^-P the majority of the mitotic figures did not show autoradiographs, in a 6 hours' culture most of the mitoses already contained labelled DNA. This observation suggests that immediately prior to mitosis there is a 3-4 hours' stage of the cell cycle (Gg period) during which no DNA synthesis takes place. Prior to this second gap is the period of DNA synthesis (S), which is preceded by the long first gap (G^) during which again no DNA synthesis takes place. The cell cycle related to DNA synthesis measured bv the incorporation of 32p or adenine i^C into DNA : ' 25-30 h 12-1 5 h 3-4 h (,i S G. This timing, in principle, agrees well with that found in the bean root by Howard and Pelc^. Ejfect of X-irradiation — Large doses of X-rays (5,000 r in 15 minutes, 140kV 1mm Al filter) exerted a marked inhibition on DNA synthesis. When in a 0-6 hour culture in isotope, radiation was delivered in the third hour (0-3x-6) then both the number of cells showing DNA auto- radiographs and the grain count over the nuclei give similar counts to those in a 3-hour culture (0-3). This suggests that cells in the period of DNA synthesis are immediately and completely prevented from synthesizing DNA. 217 DNA SYNTHESIS IN BONE MARROW STUDIED BY AUTORADIOGRAPHY Radiation delivered at the beginning of the cukure period in isotope (Ox-6 : Ox-24) also markedly inhibited DNA synthesis. Since the grain counts indicated that subsequent to irradiation no cells, even with long culture times, synthesized more DNA than a 2-3 hours' non-irradiated culture wovdd, it was suspected that the cells in the G^ period suffered a latent damage. If the isotope was added to the cultures three or more hours after irradiation (x-3-24) hardly any DNA synthesis was observed, as opposed to those cultures in which the isotope was added immedi- ately following the irradiation (Ox 24). These observations suggest that cells in the G^ period, immediately before the S period receive a latent damage from a dose of 5,000 r. During the first 3 hours following radiation these cells may enter the S period ; after about 3 hours, however, even these cells will be incapable of DNA synthesis. All G^ cells are damaged by a dose of 5,000 r. The grain counts indicate that the difference between 6 and 24 hour cultures after radiation is pro- duced by some G^ cells entering the S period for a limited time. The radiation damage in these cells becomes apparent in that although they may enter the S period, they cannot synthesize more than 2-3 hours' worth of DNA as compared with the normal 12-15 hours' worth. The effect of irradiation on the G^ period was indicated by the fact that no mitoses were observed in any of the irradiated cultures. Formate ^'*C uptake — The incorporation of adenine or phosphorus is not necessarily the true picture of DNA synthesis, therefore in a series of experi- ments the uptake of formate ^*C as a purine and pyrimidine precursor was investigated. Formate ^^C as a one-carbon compound takes part in many of the reactions in the cytoplasm of the cell, and therefore it is surprising that most of the labelled carbon taken up by the cells was localized in the nucleus, even on the non-hydrolysed autoradiographs. This suggests a preferential viptake of formate into DNA and RNA and also the central role of the nucleus (? nucleolus) in RNA synthesis. On the acid hydrolysed preparations a quantitative assessment of the formate ^*C into DNA was possible. The maximum uptake per nucleus was of the order of 1 -2 X 10'' atoms DNA ^^C (50 grains in 10 days' expos- ure, 2 grains/electron) which is only slightly greater than that found by previous adenine ^*C experiments. (The concentration of adenine ^*C in the cultures was 0-5[j.c/ml (specific activity 1 •56mc/mM) i.e. 0-3 milli- molar. The concentration of the formate ^'*C : 2-0[jLc/ml (specific activity l-04mc/ml) i.e. 2-0 millimolar.) The adenine stock was labelled in the ratio of 1 : 45 and the formate stock in the ratio of 1 : 75. Therefore, if a cell took up 1 ,000 molecules of adenine or formate they would include 22 molecules adenine ^^C or 13 molecules formate ^*C. Further, since corre- sponding cultures contained 1,000 adenine molecules or 6,000 formate molecules in equal volumes of the medium, if the uptake was parallel to the concentration, then for each 22 labelled adenine molecules 78 labelled formate molecules (i.e. over 3x) should have been taken up. This in fact was not the case. It was considered that the concentrations used were in excess of those permitting maximum uptake. Subsequent experiments have confirmed this. Effect of Aminopterin — As can be seen from Table I, aminopterin, in a con- centration of 0-5[j.g/ml medium markedly inhibited the incorporation of 218 DISCUSSION formate ^'*C into DNA, while showing only slight inhibition on the uptake of adenine ^'*C into DNA, It appears therefore that folic acid antagonists, unlike X-rays, do not affect significantly the assembly of the DNA molecule Table I. Effect of Aminopterin on uptake of ^^C into DNA on formate ^^C uptake into DNA Culture time Control 0-5!JLg/ml aminopterin Exp. No. % + nuclei Upper av. grain % + nuclei Upper av. grain count count Formate ^^C 200 18hr 74 80 48 30 205 18hr 33 30 11 5 207 18 hr 62 45 14 5 210 20 hr 55 40 12 10 217 20 hr 65 50 34. 30 224* 21 hr 74 50 60 50 Adenine ^^C 205 18hr 35 25 27 20 207 18 hr 58 25 39 20 210 20 hr 64 50 47 40 • 217 20 hr 52 40 48 40 224* 21 hr 50 50 50 50 * Old aminopterin solution from its constituents (purines and pyrimidines, desoxyribose, phosphate) but inhibit the synthesis of pyrimidines, and, probably to a lesser extent, that of the purines. Citrovorum factor, in a concentration of 75 [xg/ml medium reverses the inhibiting effect of aminopterin. This work is part of a project supported by the British Empire Cancer Campaign. ^ Lajtha, L. G. 2 Lajtha, L. G. 3 Lajtha, L. G. * Lajtha, L. G. ^ Lajtha, L. G., REFERENCES J. Clin. Path. 1952, 5 67. Nature, Lond. 1954, 174 587. Exp. Cell. Res. 1952,3 696. J. Photogr. Sci. 1954, in press. Oliver, R. and Ellis, F. Brit. J. Cancer, 1954, in press. * Howard, A. and Pelc, S. R. London. Isotopes in Biochem. Ciba Conf. 1951, Churchill, DLSCUSSION ' R. Oliver : It has been shown that radiation prevents the incorporation into DNA of adenine added to the culture. However, it is not at present clear whether this is due to interference with the synthesis of the other necessary purines or with the assembly of the DNA molecule. As an extension of the work reported here it is suggested that ^*C labelled adenine be added to the irradiated culture together with the other three purines required. If then it is only the purine synthesis which is inhibited DN.-\. synthesis should be able to proceed as indicated by incorporation of the labelled adenine. The radiation effect on each purine synthesis could be investigated in this way. 219 THE INHIBITION OF DNA SYNTHESIS BY IRRADIATION WITH SPECIAL PREFERENCE TO IRRADIATION IN THE EARLY STAGES OF LIVER REGENERATION Barbara E. Holmes and Lorna K. Mee Department of Radiotherapy, University of Cambridge It has been noticed by Hevesy\ by Vermund et al", by Skipper and Mitchell^ and by ourselves^ and various other authors that irradiation with a considerable range of X-ray doses will reduce the rate of deoxyribo- nucleic acid formation in growing tissues to about 50 per cent of normal. Provided that the technique of irradiation is so arranged that the animal suffers no undue shock from pain or fear or constricting strapping, the dose may be as high^ as 9,000 r and still not reduce the DNA forma- tion below this level. This fact may be of interest in a general con- sideration of mitotic process, since it has been suggested by Von Euler and Hevesy^ and later by other authors, that the reconstitution of the DNA already present, as well as the formation of new DNA takes place in dividing- cells and that the new formation is sensitive to irradiation whereas the reconstitution is not. These views receive support from the work of Hevesy^ and of Stevens, Dauost and Leblond^ who show that the rate of DNA formation, as indicated by isotope uptake, is double the amount required for the formation of new cells actually appearing. The number of new cells appearing is judged sometimes by the increase in weight of an organ or tumour and sometimes by mitotic counts after colchicine. Most of these estimations of rate of DNA synthesis were based on ^-P uptake and it was therefore possible that only the phosphate of the resting DNA was being replaced. We have, however, carried out some double labelling experiments with regenerating liver (using glycine or adenine containing ^^C and injecting it at the same time into the same animal as the ^^P), which suggest that the new formation of the basic part of the nucleic acid is proceeding at the same rate as the new formation of the phosphate. These experiments are still incomplete, but as far as we can see, if the DNA of the cell is indeed broken down and rebuilt during the mitotic cycle, it is the whole molecule which must be broken down and rebuilt. This may be difficult to reconcile with a template theory of DNA synthesis. Since the work of Howard and Pelc^ it has been realized, as Lajtha has pointed out in this symposium, that synthesis of DNA is complete some hours before mitosis and we cannot, therefore, suppose that the breakdown of the original DNA is some part of the chromosome division and general rearrangement of mitosis. In our material, Howard has been able to show that the synthesis, as measured by adenine incorporation, is complete about 220 BARBARA E. HOLMES AND LORXA K. MEE 7 hours before mitosis. In the very early stages of rat Hver regeneration, just before the first mitoses take place, enlarged cells with nuclei carrying their double loads of DNA are found. Since the total uptake of isotopes into the DNA up to this point can be measured and the number of enlarged cells counted, it should be possible to decide here also whether double the amount of isotope needed for new synthesis has been taken up. It is possible that this apparent doubling is due to an occurrence at actual mitosis. Various authors have been led to believe that there is a doubling of DNA content at mitosis or directly after. It can also be pointed out, though this is not meant as a serious suggestion, that a throwing out of old, unlabelled DNA at mitosis might occur and would reduce the actual amount of DNA present while causing a sudden rise in the specific activity of the DNA now extractable from the cells. In our curves w^e find a sudden rise in the specific activity of DNA phosphorus just before the first burst of mitoses in the tissue, though no new ^^p is now entering the DNA. c < Q WOO VJ 500 100 0 Mitotic rotes shown by isolated points • Control V Afier 150 T G After li 50 r -30% 50 40 20 70 t n 76 24 30 Hours after liepatectomy Figure 1. Effect o/ 1 50r and 450r X-ray doses on rate of formation of DNA (^'P used) Another possible way, and quite a different one, of accounting for the X-ray resistant 50 per cent of DNA synthesis w^as brought out by the work of Pelc and Howard^. They showed that DNA synthesis could be inhibited for some hours b)' very small doses of X-rays given shortly before the s\n- thesis began. During the period of actual DNA synthesis even considerably larger doses had no effect on it. It therefore seemed possible that, when a growing tissue containing cells in every stage of the mitotic cycle was irradiated, a proportion of them would be in an insensitive stage and a proportion in a sensitive stage as regards DNA synthesis. Thus it might happen that only part of the synthesis would be inhibited. The early stages of rat liver regeneration provide very easy and straight- forward material for investigating this possibility. In fact Kelly^ has already shown that the formation of DNA can be inhibited by total body irradiation in the pre-synthesis period much more easily than by irradiation in the synthesizing period in this material also. It can be seen from Figure 1 that no synthesis of DNA takes place in the remaining lobe during the first 12 or 15 hours after partial hepatectomy. Synthesis then begins and the 221 THE INHIBITION OF DNA SYNTHESIS BY IRRADIATION rate of synthesis increases rapidly up to about 24 or 27 hours after hepa- tectomy. A fast rate is maintained for some hours ; at about 42 hours, however, the rate of synthesis and the rate of mitosis are lower than before, but remain at a fairly constant level. During this later period, the tissue can probably be regarded as an ordinary growing tissue containing all stages of the mitotic cycle in an established proportion. In the following experiments rate of synthesis has usually been measured by uptake of ^^P, but many confirmatory experiments have been done with glycine ^^C as marker. If irradiation is given at 12 hours, no cells will yet be in the synthetic stage. It is easy to show the sensitivity of DNA synthesis to irradiation at 12 hours, since 450 r will delay synthesis for many hours and some delaying action can be seen with as small a dose as 150r. This small dose will also cause a delay of a few hours in the appearance of the first mitoses and 450 r will cause a longer delay (9 hours). This early-stage irradiation with small doses can also cause chromosome breakage ; Roller has seen about 10 per cent of breaks about 15 hours after irradiation with 150r, in the first mitoses to appear ; about 20 hours after 450 r he has found chromosome breakage in up to 100 per cent of the first dividing cells. Twelve or fifteen hours after irradiation (that is, 24-27 hours after the partial hepatectomy) all these effects are very apparent. Mitosis is sup- pressed and the DNx\ synthesis, which is very rapid in the controls, is scarcely beginning. The period from about 15 to about 24 hours after the hepatectomy is one of DNA synthesis, but still without mitosis ; this begins at about 24 hours but is much more active at 27 or 30 hours, when from 3 per cent to 6 or even 10 per cent of cells may be found to be in mitosis. It is once more very easy to show, thus confirming other authors, that irradiation with doses of 450 r has no immediate effect on DNA synthesis if delivered during this period of active synthesis. It must be emphasized, however, that mitosis can still be greatly delayed by this irradiation and it seems that we must believe that inhibition of some other mechanism than DNA synthesis must here be the cause of the temporary inhibition of mitosis. If we consider the case of a growing tissue, containing cells in all stages of mitosis, we can see that the 50 per cent inhibition of deoxynucleic acid synthesis produced by fairly large doses of X-rays could not be produced if only the cells in the pre-synthetic stage were aflfected. The 50 per cent inhibition is an immediate effect, whereas some hours must elapse to allow the pre-synthesis inhibition to demonstrate itself at all strikingly. An immediately visible inhibition must be due to an immediate inhibition in the synthetic period. Lajtha has described experiments in which this inhibition can be brought about by 5,000 r. In a confirmatory experiment (only one so far) we have found that 2,200 r will cause an immediate 50 per cent inhibition of DNA synthesis during the synthetic period ; this is a dose sufficient to cause regression in at least 50 per cent of various implanted tumours and would probably cause the death of the liver lobe. The effect of 450 r on DNA synthesis is temporary only ; the effect of 2,200 r is probably irreversible {see Table I). When X-radiation is acting on a dividing tissue with cells in all stages (46 hours), 450 r is not sufficient to produce an immedi- ate reduction in rate of DNA synthesis, whereas 2,200 r is sufficient. If on the other hand, the period from 9-12 hours after the irradiation is chosen 222 BARBARA E. HOLMES AND LORNA K. MEE (48-60 hours, ^-P injected at 57 hours) the effect of the low dose is obvious. Several other examples of the lack of immediate effect and the marked delayed effect of the low dose are shown in Table I. It seems that the immediate 50 per cent reduction in synthesis seen after clinical doses of X-rays is not accounted for by the action on cells in the sensitive state only. There is still a possibility that the new synthesis of DNA is stopped but that some sort of renewal of old DNA is taking place Table I. — Immediate and delayed effects q/"450r and 2,200 r Rate of Dj\A Synthesis Expressed as Percentage of Controls ^-P used and injected 3 hours before killing Time after liepatectomy Mitotic counts per mi lie Irradiation Killing Rate of DNA Synthesis Control Irradiated 150r 12 hours 24 hours 30 0-75* 0 450 r 12 hours 24 hours 20 10-0* 0 450 r 12 hours 27 hours 30 64 0-3 450 r 1 7 hours 29 hours 30 23-4 0-5 450r 19 hours 22 hours 100 0 0 450r 19 hours 23 hours 96 4-8* 0 2,000 r 19 hours 23 hours 50 0 450 r 24 hours 27 hours 95 26-8 0 2,200 r 24 hours 27 hours 37 0 450 r 21 hours 27 hours 100 46 6 450 r 24 hours 36 hours 29 15 6 450 r 24 hours 36 hours 30 16 8-7 450 r 36 hours 48 hours 26 6 0-5 450 r 36 hours 39 hours 100 4 0 450 r 46 hours 49 hours 92 19-3 0-2 2,200 r 46 hours 49 hours 50 0 450 r 48 hours 60 hours 20 2-7 1 2,200 r 48 hours 60 hours 20 0 * Mitotic counts irregular from l';i-26 hours, but always high from 27-33 hours at the same time and is not inhibited. If the renewal of the old DNA is taking place without much expenditure of energy, by exchanges of base and phosphate with free base and phosphate in the medium for instance, this could well be true. To reconcile this idea with the template theory (and particularly if we find that the synthesis of the new and the renewal of the old takes place at the same time) we have to suppose that the components of the old DNA become more mobile at the moment when the new DNA is being formed, or has just been formed, upon it. REFERENCES ^ Hevesy, G. Radioactive Indicators. Interscience Publishers, New York. 1948. 2 Vermund, H., Barnum, C. P., Huseby, R. A. and Stenstrom, K. W. Cancer Res. 1953, 13 633. 223 THE INHIBITION OF DNA SYNTHESIS BY IRRADIATION 3 Skipper, H. E. and Mitchell, J. H. Cancer, 1951, 4 363. * Holmes, B. E. and Mee, L. K. Brit. J. Radiol. 1952, 25 273. ^ Von Euler, H. and Hevesy, G. Arkiv. Kemi, Mineral, geol. 1944, A17, No. 30. « Stevens, C. E., Dagust, R. and Leblond, C. P. J. Biol. Chem. 1953, 202 177. ' Howard, A. and Pelc, S. R. Heredity, 1953, 6 261. ® Pelc, S. R. and Howard, A. Aarhus Conference of Radiobiology, 1953. ^ Kelly, L. S. Proc. Amer. Ass. Cancer Res. 1954, 1 24. DISCUSSION Z. M. Bacq : If I have clearly understood, Mrs. Holmes has observed several times after irradiation that DNA synthesis may be normal when there is no mitotic activity. Is it possible that mitosis and DNA synthesis are unrelated events ? A. Howard : It is obvious that a cell must synthesize DNA if it is to divide, but DNA synthesis does not always lead to division. The existence of many cells having tetraploid or higher amounts of DNA in tissues with low rates of division indicates this clearly, and is one of the grounds for criticizing Leblond's conclusion that the high rate of incorporation of ^^P relative to mitotic rate means that existing DNA is being broken down. In view of the clear separation in time of the two events, it would, I think, be surprising if DNA synthesis were inevitably followed by mitosis. 224 INCORPORATION DU ^^G DANS LE GLYCOGENE DU FOIE APRES IRRADIATION* Mme Lourau-Pitres Institut de Biologic Physico-chimique, Paris UN grand nombre d'observations^ montrent qu'une irradiation generale par les rayons X provoque un trouble du metabolisme des glucides. On sait par exemple qu'au cours du jeune on trouve toujours plus de glycogene hepatique chez les animaux qui viennent d'etre exposes aux rayons que chez des temoins jeunant depuis le meme temps-. C'est la signification de ce fait qui a ete examinee dans ce travail : I'irradiation augmente-t-elle la synthese du glycogene— et dans ce cas est-ce a partir de molecules privi- legiees — ou bien ralentit-elle sa vitesse de renouvellement ? La methode utiHsee est celle des indicateurs radioactifs. L'animal d'ex- perience, qui est la souris, regoit, en injections, des metabolites, marques par du i-^C, qui sont incorpores dans le glycogene du foie : glucose, bicarbon- ate. Au bout de temps variables, les animaux sont sacrifies, le glycogene isole et purifie et sa radioactivite mesuree sur un compteur de Geiger- Muller a fenetre mince. Dans ces experiences preliminaires les doses de rayons X etaient largement lethales (2.000 et 2.500 r) et la periode d'observa- tion limitee aux 24 premieres heures qui suivent I'irradiation. Les experiences realisees jusqu'a ce jour peuvent etre reparties en 3 groupes, 2 d'entre eux destines a suivre la synthese du glycogene, le troisieme plus specialement congu pour determiner sa vitesse de renouvellement. I. INCORPORATION DANS LE GLYCOGENE DU ^"^C CONTENU DANS LE GLUCOSE CIRCULANT Ces experiences^ montrent que I'irradiation modifie Tincorporation, dans le glycogene, du ^^C fourni par le glucose circulant. Lorsque le glucose marque est administre en une seule injection, I'activite specifique du glyco- gene atteint tres rapidement son maximum, au bout de 40 a 50 minutes, puis decroit. L'irradiation ne deplace pas la position de ce maximum mais elle augmente tres fortement sa valeur, d'au moins trois fois ; aussi les courbes representant les variations de I'activite specifique en fonction du temps ont-elles une pente beaucoup plus forte chez les irradies que chez les temoins. Des controles experimentaux ont montre que cette augmentation d'activite est reelle, en particulier qu'elle n'est due ni a une impurete radioactive entrainee avec le glycogene, ni a une acceleration de la resorption du glucose marque {cf.'^), ni a une baisse de la glycemie. Toutefois une hypothese meritait d'etre envisagee et discutee, c'est que I'irradiation accelere le * (Travail du Laboratoire Pasteur a I'lnstitut du Radium, realise avec une subvention du Commissariat a I'Energie Atomique.) 225 Q INCORPORATION DU ^*C DANS LE GLYCOGENE DU FOIE APRES IRRADIATION catabolisme des glucides. On sait en effet* que des accelerateurs du meta- bolisme modifient le profil des courbes : activites specifiques/temps dans le sens que nous avons constate, en augmentant leur pente. Mais on sait aussi que cet effet persiste dans la periode d'activite decroissante de sorte que les courbes donnees par des animaux a metabolisme accelere coupent toujours celles des temoins dans leur branche descendante. Or les experi- ences de ce premier groupe ne permettaient pas d'envdsager cet aspect du probleme car les activites specifiques developpees par une seule injection de glucose marque sont trop faibles pour qu'il soit possible de suivre leur decroissance et d'y relever des differences significatives, Un plan different a done ete recherche pour les experiences du deuxieme groupe. II. VITESSE DE RENOUVELLEMENT DU GLYCOGENE Le plan adopte est base sur les observations resumees dans la Figure 1. Le metabolite porteur du ^^G est du bicarbonate ; on favorise son incorpora- tion dans le glycogene en faisant disparaitre le glycogene inactif par un jeune Figure 1. Activites specifiques du glycogene extrait du foie de souris nor males ay ant regu temps 0, une injection de glucose inactif et une injection de bicarbonate de sodium marque par du ^*C. 5 10 15 ZO 25 JO 35 V-O V5 50 55 heures Injections de quelques heures, puis en reconstituant les reserves au moyen de glucose inactif injecte, en petites quantites, quelques milligrammes, en meme temps que le bicarbonate. (Activite injectee environ 1,7 microcurie.) On voit dans la figure que le ^*C du bicarbonate est rapidement incorpore et utilise. Mais le phenomene qui a attire notre attention est I'apparition, 28 a 30 heures apres les injections, d'un deuxieme maximum aussi eleve que le premier, excellent point de depart pour etablir une courbe de decroissance. Ce deuxieme maximum est done choisi comme origine des experiences ; I'irradiation est faite a ce moment la, les deux groupes d'animaux, temoins et irradies, ayant a leur disposition un materiel marque identique. La Figure 2 donne un exemple des resultats, qui sont extremement nets : il n'y a aucune difference dans la decroissance de I'activite specifique des temoins et des irradies, la premiere observation etant faite 5 heures apres I'irradiation et la derniere 17 heures apres. Avec une echelle semi-logarith- mique les valeurs experimentales se disposent suivant une droite et celle-ci a la meme pente. L'irradiation ne modifie pas la vitesse de renouvellement du glycogene du foie et les resultats donnes par les experiences exposees dans le premier 226 MME LOURAU-PITRES paragraphe semblent iiidiquer que I'irradiation accelere la synthese du glycogene a partir du glucose circulant. Figure 2. Activites specifiques du glycogene extrait du foie de souris normales et de souris irradiees, preparees au temps 0 comme dans la Figure 1. U irradiation a ete faite au moment indique par lafieche, 29 hemes apres les injections de glucose et de bicarbonate. J c 100 -X ■^80 CO* -\:V. o 5 W .s~ ?:; ^»V v§ ^» §- i'' — X ■^ t -^ 10 1 1 1 1 1 23 33 37 11 115 Heures apres 1 'injection III. SYNTHESE DU GLYCOGENE A PARTIR DE MOLECULES 14 C DU BICARBONATE MARQUEES PAR LE Le dernier dispositif experimental a ete repris pour etudier la synthese du glycogene a partir de molecules marquees par le ^^G du bicarbonate que I'organisme a synthetisees avant d'etre irradie. Ge probleme est different de celui qui a ete traite dans le premier paragraphe. En effet le glycogene n'est pas seulement forme a partir du glucose circulant, mais aussi, avec une egale importance, a partir de molecules plus petites formees au depend des protides et des lipides aussi bien que des glucides. La radioactivite qui apparait dans le glycogene 30 heures apres I'injection de bicarbonate marque est due, selon toute vraisemblance, a I'incorporation de ces petites molecules, et il etait interessant de savoir si I'irradiation perturbe aussi cette synthese. Les animaux sont prepares comme en II, mais I'irradiation a ete faite avant le deuxieme maximum, dans la deuxieme branche ascendante et meme dans la premiere branche descendante de la courbe n° 1 et les examens ont ete faits de 1 a 10 heures apres I'irradiation. Qiiatre experiences de ce genre ont ete faites. Les figures obtenues sont assez differentes, plus complexes que les precedentes. Toutefois elles ont 100 . 80 o w- 20- 10 - ' \ J • / \ \ / \ t 1 — 1 1 1 1__ 1 Figure 3. Activity spicifiques du glycogene extrait du foie de souris normales et de souris irradiies preparies au temps 0 comme dans la Figure 1. L" irradiation a ete faite au monunt indique par lafieche, 24 heures apres les injections de glucose et de bicarbonate . Z¥ 26 28 30 _ 32 SV- Heures apres I'injection en commun une perturbation tres nette de fincorporation dans le glycogene des metabolites marques. Ghez les temoins, la courbe representative est toujours une droite, et quand il y a un point d'inflexion celui-ci est toujours unique. Au contraire chez les irradies il y a toujours plusieurs points 227 INCORPORATION DU ^*C DANS LE GLYCOGENE DU FOIE APRES IRRADIATION d'inflexion et les activites specifiques sont generalement superieures a celles des temoins {Figure 3). RESUME ET CONCLUSIONS {1) Une irradiation generale par des doses de rayons X largement lethales augmente I'incorporation dans le glycogene du foie du ^*C contenu dans le glucose circulant et dans divers metabolites qui ont fixe le ^*G fourni a Forffanisme sous forme de bicarbonate. (2) En revanche Tirradiation ne modifie pas I'utilisation du glycogene dont la vitesse de renouvellement n'est pas modifiee dans les 24 heures qui suivent I'irradiation. REFERENCES 1 Ord, M. G. et Stocken, L. A. Physiol. Rev. 1953, 33 357. 2 North, N. et Nims, L. F. Fed. Proc. 1949, 8 119. NiMs, L. F. J. chim. Phys. 1951, 48 280. Ross, M. H. et Ely, J. 6. J. cell. comp. Physiol. 1951, 37 163. Stocken, L. A. Chem. Ind. 1951, 32 116. MacKee, K. W. Fed. Proc. 1952, 11 256. LouRAu, M. C.R. Acad. Sci. 1953, 236 422. Nims, L. F. et Sutton, E. Amer. J. Physiol. 1954, 177 51. 3 LouRAU, M. Arkivf. Kemi, 1954, 7 211. 4 Beeckmans, M. L., Caiser, H. et Hevesy, G. Arch. Int. Phartnacodyn, 1951, 186 33. ^ Hevesy, G. et Forssberg, A. Ce symposium. DISCUSSION P. Mandel : Je pense que des experiences negatives concernant le renouvellement du glycogene ne permettent pas a eux seules de conclure a I'absence d'un trouble du metabolisme des glucides apres une irradiation aux rayons X. E. H. Betz : Les recherches que Lourau-Pitres vient de nous exposer montrent I'existence d'une augmentation precoce du glycogene hepatique chez I'animal irradie. Une augmentation similaire s'observe au cours des jours qui suivent I'irradiation. Le taux du glycogene hepatique semble se modifier parallelement a revolution de I'activite surrenalienne telle que Patt I'a decrite. Les modifications du glycogene hepatique semblent bien etre le reflet de I'activite cortico-surrenalienne puisqu'elles sont inhibees par la surrenalectomie, ainsi que I'a montre Fischer. Mme Lourau-Pitres : Les experiences de Fischer et celles de Nims et Sutton sont en effet favorables a I'interpretation de Betz. On devait s'attendre a ce que I'irradia- tion diminuat la vitesse de renouvellement du glycogene, puisque, selon I'opinion admise, les hormones corticales preservent le glycogene du foie en diminuant les oxydations peripheriques. Or ce n'est pas ce que j'ai observe. Mais peut etre les hormones corticales agissent-elles par un autre mecanisme ? II faut aussi noter qu'aux doses de rayons que j'ai utilisees (2.000 r), Hevesy et Forssberg observent des effets qui s'expliquent mieux par une deficience d'hormone surrenale que par un exces. 228 OBSERVATIONS MADE ON THE HUMAN RESPONSE TO A SINGLE DOSE OF X-RAYS— THE LATENT PERIOD W, M. Court Brown and John D. Abbatt Medical Research Council, London It is clearly desirable that human material should be used for radiobio- logical investigations. This can only be done if a reproducible response to irradiation occurs, of which at least some of the governing factors are known. The length of time which elapses from the commencement of irradiation to the onset of the symptoms of radiation sickness, the latent period, seems such a response. Experience with more than 150 patients has shown that provided a large enough single dose of irradiation is given to a large enough. Irradiation of whole length of spine (Ankylosing spondylitis) «^ 250 ^ 200 ^ SO J L J \ \ L 7 2 3 ¥ 5 6 7 Latent pen/od •Figure 1 8 9 10 volume of tissue in the trunk, or to the whole body, a symptomatic dis- turbance occurs which is sudden in onset, and that the time required to initiate this disturbance can be measured with reasonable accuracy. Experi- ence with control sham irradiations has indicated that the symptoms which terminate this latent period are the genuine symptoms of radiation sickness ; these symptoms are acute and sudden nausea, and the development of uncontrollable and often persistent vomiting. Observations on the latent period have been made in two classes of patients : those irradiated with medium kilovoltage X-rays and those given large doses of ^^^I. This period and the ensuing symptomatic disturbance, together with such physio- logical changes that occur, appears to correspond to the initial phase of the General Radiation Syndrome in man and may well be the himian equivalent of the first phase of response in the rat as described by Lamerton and his co-workers^. 229 THE HUMAN RESPONSE TO A SINGLE DOSE OF X-RAYS THE LATENT PERIOD Three known factors influence the length of the latent period, the dose of radiation, the body size and the particular anatomical site irradiated. It has been found that for a sjiven anatomical site of irradiation, i.e. the whole 300 I I 800 Irradiation of wtiole length of spine (Anl(yiosing spondylitis) \ Figure 2 ioo\_ 3 15 Latent period 10 "h. length of the spine, or the whole abdomen, or the whole pelvis, a distinct relationship exists between the dose of radiation expressed in terms of body size and the length of the latent period, and that this relationship is a curvi- linear one {Figure 1). If the log dose per unit of body size is plotted against the latent period, the relationship is linear provided only that a restricted range of time is used [Figure 2). By plotting the dose against the reciprocal 7cV Figure 3 % i 20 30 SO 7-5 Irradiation of tyhoie iengiii of spme ( An ls. All X 3,000. (a) Chromatid interchange between long and short chromosomes. Note the short achromatic regions at points of exchange, {b) E.xamples of anomalous changes which are difficult to distinguish from chromatid breaks. Probable chromatid break at top-left. (c) and {d) The four types of proposed intrachange, with explanatory diagram. S. H. REVELL which should be occurring with a frequency equal to the duplication- deletion (type 1). However, the resolution of this problem could depend on one assumption : that if the loops of these /«/rachanges were always small enough in Vicia then, as the whole configuration shortened to metaphase, the original chromatid relations would be lost. Type 2 would then yield the bent chromosome with minute juxtaposed ; type 3 would probably be difficult to see at all since it is merely a small inversion ; and type 4 would appear as an isochromatid break with sister union {Figure Id). Carried a stage further, this interpretation could account for a proportion of chromatid breaks : if, like interchanges, these m/rachanges may also be incomplete — that is, with one of the two pairs of chromatid ends not joined up in the exchanged relation {Figure 2a ; see Catcheside et al^), then types 1 to 3 could yield ' chromatid breaks ' (compare Figures 2b and 2c) and type 4 could give failed 'sister union'. This interpretation is supported by three observations. (7) If the aberrations in question are really collapsed m/rachanges — that is to say, chromatid exchanges like the interc\\a.ngc?, — then some of the small unstained regions already mentioned which were evidently associated with these aberrations may now be rationally explained (no matter what they actually are) as marking the points of exchange, just as they are seen to do in the case of m^^rchanges {Figure la). For example, if 'sister unions' are really collapsed mifrachanges then the points of chromatid exchange must be to one side of the ' end ' of each fragment. It is frequently possible to confirm this by observation {Figure Id, type 4 photomicrograph). Such achromatic points may also be observed on the other presumed ?«/rachanges {e.g. Figure lb). Previous to the development of this hypothesis, the author had inter- preted many of these points as chromatid breaks. (2) In contrast to Trillium and Tradescantia, no clear intra-arm mimchanges are observed at metaphase in Vicia. However, such /«/rachanges are demon- strable at prophase although they are always relatively much smaller than those in the former species {Figure 2d). Therefore these m/rachanges must reach metaphase in a disguised form, and it seems that the most rational explanation available is that they are represented by the aberrations already described* {Figure Id). {3) As was observed by Lewitsky and Araratian^^^ the related parts of affected chromosomes are frequently closely juxtaposed at metaphase. Thus the fragments from an isochromatid break are often very close together and minutes are usually close to the chromosomes from which they are presum- ably deleted {Figure Id). (A similar phenomenon can be observed in the case of ' triradials ' and certain other types of interchange — see below.) This observation is not readily accounted for by the orthodox hypothesis since breakage and reunion are assumed to have occurred at the time of treat- ment, there being no subsequent relation between the separated chromosome parts. It can, however, be plausibly explained by the hypothesis now sug- gested, which postulates that the chromatids of the minute loops which * The same explanation could be advanced for these aberrations in Trillium and Trades- cantia, although in these species the largest zViirachanges retain their form even at metaphase and are therefore recognized as such. 247 a M # M i Figure 2. Photomicrographs of Vicia faba chromosomes in Feulgen squashes of root tip mitoses {all colchicine treated), after treatment with X-rays. All X 3,000. {a) Incomplete interchange between long and short chromosomes. (b) Complete intrachange in long chromosome. (c) Proposed incomplete intrachange in long chromosome {^: chromatid ' break '). {d) Intrachange at prophase. (e) Interchange with isochromatid break in one arm [i^' near triradial'). (/) and (g) Triradial {^chromatid-isochromatid interchange) with diagram of suggested interpretation. S. H. REVELL undergo rearrangement only lose their paired relation during prophase. If this were correct then the two fragments from an ' isochromatid break ' might still remain close together at metaphase because the association between them had only just lapsed (compare with Figure 17b of Catcheside et al^). In addition to these three items of supporting evidence, it should also be pointed out that the new interpretation gives a satisfactory explanation of the original difficulty encountered in scoring chromatid breaks. All the aberrations which previously were difficult to distinguish would now be assumed to be equivalent : all are either complete or incomplete small m/rochanges. But only one out of the four types — that which gives an 'isochromatid break' (type 4) — would be observed and discriminated effici- ently. The others, owing to their small size, would frequently be missed or else confused with one another, and it would moreover be difficult in some cases to decide whether they were incomplete (that is, whether or not they constituted ' chromatid breaks ') . The hypothesis also accounts for the occur- rence of chromatid and isochromatid breaks in the same nucleus. A large proportion of the whole class of 'chromatid ' aberrations as defined in the orthodox theory — both chromatid and isochromatid breaks, chromatid zn/^rchanges and ?Vz/rachanges — may thus be interpreted in terms of one unit of change : a chromatid exchange. But it will be noted that there is still an important group of m/^rchanges which remains unaccounted for ; namely those which are conventionally interpreted as involving reunion of one or more isochromatid breaks. It is not obvious how this type of {nterch.a.nge could arise by chromatid exchange. Nevertheless, certain facts suggest that they might be formed in essentially the same way. The most frequent of this last group of aberrations is the chromatid- isochromatid /«/^rchange ( = triradial) which, according to the orthodox interpretation, results from reunion between one isochromatid fragment and a single chromatid break, the other isochromatid fragment commonly undergoing sister union. It is often the case (a) that this latter fragment remains close to its related configuration at metaphase (see paragraph (3) and Figure 2g) ; and (b) that the short achromatic region is eccentrically placed as it is in sister unions of non-/«/frchanged chromosomes (see para- graph (/) and Figure 2g). These two facts strongly suggest that the sister union event, whatever it is, which occurs in a triradial is the same as that which results in ordinary isochromatid fragments. Now it is occasionally evident that a sister union event can occur close to a chromatid interchange {Figure 2e). In view of this, the items of evidence (a) and (b) above suggest that a triradial could consist of two chromatid exchanges very close together — one interchange and one intrachange (Figure 2f) : the chromatid relations would be obscured during contraction, the fragment thus separating from the main configin-ation by the time of meta- phase {Figure 2g). Similar interpretations in terms of two or more exchanges near together may be given of other less common types of 'interchange'. Thus it is possible, at least qualitatively, to interpret all recognized ' chro- matid ' aberrations in terms of one or more chromatid exchanges. It is evident that since incomplete m/erchanges, failed ' sister unions ' and chro- matid ' breaks ' are all assumed to be incomplete exchanges their numbers should be positively correlated. There is, of course, some evidence that 249 A NEW HYPOTHESIS FOR 'CHROMATID' CHANGES this is so (Catcheside et al^), the conventional explanation being that there is a certain likelihood that any break may remain open until metaphase. The quantitative aspects of the exchange hypothesis given above, and especially the question of whether it can account for all changes convention- ally scored as chromatid breaks, will be discussed in detail in another paper. SURVEY It must be borne in mind very clearly that the hypothesis which has been proposed above does not rest on any completely critical evidence. It is, like the breakage-and-reunion hypothesis, a scheme for the interpretation of visible changes in metaphase chromosome structure, and through which they may be related to the quantitative data. Although these changes are observed at metaphase they are, of course, only the consequence of much earlier alterations in chromosome development. Obviously, a knowledge of this development is essential for a detailed understanding of the nature of its aberrations. At present this knowledge is almost entirely lacking, and it therefore seems unlikely that the examination of the proposed exchanges at the time they reach metaphase could alone throw much light on the process of their formation (Loveless^). It is for this reason that the new hypothesis, as it now stands, only attempts to account for the observed aberrations in terms of a chromatid exchange, as being the least biological entity resolvable by the technique employed— that of metaphase analysis. Now if the exchange and the breakage-and-reunion hypotheses alone are considered, then it seems to the author that three main possibilities may be distinguished. Thus, the exchange hypothesis might be totally incorrect. Or it might be only partially correct : this could mean that the breakage- and-reunion interpretation was not essentially incorrect, but had been inaccurate insofar as it had failed to recognize that certain ' breaks ' were actually intrachsinges ; or it could mean that aberrations happening to resemble one another in some respects were arising by the two quite different mechanisms. Or, lastly, the new hypothesis might be entirely correct : in this case all the ' chromatid ' group of aberrations would be correctly inter- preted as exchanges (complete or incomplete) which occurred either between points on different chromosomes or else between points close together on the same chromosome. In the author's opinion the items of evidence numbered 7 to 5 on pages i 247-249 suggest rather strongly that the new hypothesis is at least partially I correct. However, it seems impossible at present to be more definite than | this, although it should be pointed out that the observations as a whole provide no critical objections to its being wholly correct. It must therefore be emphasized that the object of the ensuing discussion is to consider briefly what would follow if the new hypothesis were correct, and not what neces- sarily follows from its having been proved correct. If all defined ' chromatid ' aberrations were chromatid exchanges then would it be possible to say anything at all about the exchange process ? The conventional explanation would be that each exchange is due to two chromatid breaks and reunions. The complete acceptance of the new hypothesis would implicitly question this view because all the ' breaks ' seen 250 S. H. REVELL at metaphase would themselves be the result of exchange : hence the ob- served ' breaks ' would not represent the residue of primary aberrations, the rest of which had either reunited in pairs as exchanges or else restituted. Inter- preted in this way, the visible changes thus provide no evidence of ' breakage first ', or indeed of breakage at all. Of course, it might still be postulated that both intra- and /«^«7-changes arise by breakage and reunion, but this would now be an entirely separate question connected with the general problem of whether resting-stage ' chromosomes ' are actually permanent structures. If the new hypothesis were correct, then, for the reason already given, it could hardly be expected at present that much could be deduced about the exchange process, except in the most general terms. But it is clear that, for exchange to occur, the loci involved must come close together at some stage ; also, it seems difficult to conceive of such an exchange as other than an organized process. At least for the author, it is difficult to see how ex- change could be the result of any random damage and repair, even if such events were guided by a proximity of 1 [ji. Nor does it seem to the author that the incomplete exchanges themselves constitute evidence that a breakage process is involved : these should be regarded without prejudice simply as imperfect exchanges. The case for considering the exchange as a develop- mental entity, until more is known about it, is greatly strengthened by the fact that exchange does actually occur as a physiologically determined process at meiosis, where it seems to be connected essentially with chromo- some doubling. It would obviously be tempting to regard the aberrations as induced heterologous exchanges of the same developmental type : at present, however, such a comparison must only be considered with caution. Thus the question which was posed earlier in this paper in connection with the homologous exchanges alone now arises again with a much wider application. Are there inter- and /«/rfl-chromosomal associations in somatic cells which may be defined by their capacity to exchange in response to stimuli such as ionizing radiations ? It seems to the author that, whatever may be the general view of this hypothesis, there need be no essential conflict between it and the observed dose-/n/frchange relation for X-rays, although at first sight this might appear to be so. It is agreed that all types of ' chromatid ' aberration increase linearly with doses of the densely ionizing radiations (Giles^^, Thoday^*' ^^ ; KoTVAL and Gray^^), and because of this and for other reasons it has been deduced that chromosome loci have to be very close together [ca. 1 [x, Lea ^) before exchange is possible between them, and that this is so even with X-rays where two electrons are necessary. This last fact, however, does not by itself appear at all to exclude the possibility that the chromosomes may be in ' contact ' — if this means their being functionally associated so that they can exchange — unless ' contact ' is also specifically defined as capacity to respond to a single X-ray hit. Now this is, of course, exactly the definition which has been adopted, and it is possible that it might be a misleading one since it has always been so closely bound up with the assumption that it is two breaks which are the two primary events associated with the two ionization paths. It is just this assumption which is now in question. So far as the author can judge, it may equally well be proposed that an exchange process is initiated at the chromosome association, but that there 251 A NEW HYPOTHESIS FOR 'CHROMATID' CHANGES is not always a one-to-one correspondence between ionization paths and exchanges. The properties of the associations where intra- and /«/f?-changes may occur differ in some way which happens to be critical for the different radiations. Thus, in the case of small w^rachanges, either the two loci involved are always close enough together, or other conditions at the critical region of proximity are always such that one ionizing particle is enough to involve both chromosomes in the exchange. But the properties of inter- chromosomal associations cause them to react differently to the different radiations : the amount of ionization — or the distribution of its effects, or the precise chemical changes induced — along the tracks of recoil protons and a-particles is such that they are singly effective in causing inter- as well as /w^rfl-changes. In the case of X-rays, the passage of a mean of two primary electrons is required for /w/^rchanges, and these passages must occur within a very short time to effect an exchange. If this were correct, then there would be in this respect a primary event — an unstable effect of some kind — corresponding to what is conventionally visualized as breakage, its decay being ' restitution '. A successful exchange initiation, on the other hand, would correspond to ' reunion '. It should be stressed that whatever actual processes it is they represent, these tw^o stages need not be the only ones connecting the release of the dose energy with the final metaphase exchange : it could merely happen that they are the only two stages which are separable by the experimental method of varying the amount of the X-ray dose or its intensity. In similar neutron or a-ray experiments they are not separable at all. In distinguishing the new hypothesis from the breakage-and-reunion hypothesis, the present problem is that the data do not reveal anything about the nature of the ' association ', as it has been conveniently called in this paper. Thus, although it seems certain that the two chromosomes are close together (and that they are sometimes even homologously close to- gether), it is not known whether it is this closeness itself which alone confers sensitivity or whether there are specific physiological conditions necessary for exchange induction which exist only at these associations. Now whether this hypothesis is really a ' contact ' one seems to depend definitively on the answer to the question of whether or not the primary event — whatever it is, which corresponds to ' breakage ' — can be induced at loci other than those in associations. The orthodox theory necessarily assumes that this may happen because the premise is that chromatid breaks are the primary events induced and that two go to make an exchange : therefore it must follow that all single breaks which are actually observed as such are primary events which have not participated in mf^rchanges and hence were not in ' contact ' by definition. This is an assumption which in no way depends on the statistical relation between interchanges and X-ray dose. It also follows that, if all parts of the chromosomes are equally liable to breakage, there is about ten times the observed number of breaks which disappear without trace, due to restitution. Both these postulates appear to depend on the initial assumption that the observed discontinuities are breaks and that they are visible examples of the primary units of change. On the other hand, in terms of the interpretation presented in this paper, there is no visible evidence that the primary events occur anywhere 252 S. H. REVELL Other than at the associations, although of course this is by itself quite incon- clusive. If the exchange interpretation were correct then the nature of the primary events would have to be known, and a method would have to be devised which could detect them wherever they occurred, before the ' con- tact ' question could be decided conclusively. It will be clear that the present exchange hypothesis is much less unlike the breakage-and-reunion hypothesis of Sax, Catcheside and Lea than the somewhat different version of this hypothesis proposed by Darlington and others. It may be pointed out that the exchange hypothesis disposes of those arguments for delaved reunion which are based on the observation of chromatid and chromosome ' breaks ' in the same cell, and on the obser- vation of certain rearrangements such as triradials. In conclusion, the reservation made in the introduction to this paper must be repeated : the present hypothesis is only suggested as an explanation of the aberrations which characteristically occur in cells about to undergo mitosis, namely ' chromatid ' changes. Such a restriction may seem arbi- trary, for it has been customary to consider observations on all types of chromosome change together, and in general to seek a common explana- tion for them all. If all this information were to be considered now, much of it could certainly not be reconciled with the present scheme. In spite of these discrepancies, the author considers that there is enough evidence in favour of an exchange hypothesis for ' chromatid ' changes to justify its being regarded as a possible alternative to the orthodox hypothesis. ACKNOWLEDGEMENTS The author is greatly indebted to Dr. L. H. Gray, to Dr. L. F. Lamerton, and to Dr. A. Loveless for their advice and for criticism of the ideas presented in this paper. This investigation has been supported by grants to the Royal Cancer Hospital and Chester Beatty Research Institute from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the Xational Cancer Institute of the National Institutes of Health, U.S. Public Health Services. REFERENCES ^ Catcheside, D. G., Lea, D. E. and Thoday, J. M. J. Genet. 1946,47 113. 2 Sax, K. Genetics, 1938, 23 494. 3 Sax, K. ibid. 1940,25 41. * Catcheside. D. C, Lea, D. E. and Thoday, J. M. J. Genet. 1946, 47 137. 5 Ford, C. E. Proc. 8th Intern. Cong. Genet. Suppl. to Hereditas, 1948, 570. « MuLLER, H. J. J. Genet. 1940, 40 1. ■^ Loveless, A. Suppl. to Heredity, 1953. 6 293. 8 Catcheside, D. G. Advanc. Genet. 1948, 2 271. ^ Lea, D. E. Actions of radiations on living cells. Cambridge University Press, 1946. 10 Morrison, J. W. Suppl. to Heredity, 1953. 6 83. " Darlington, C. D. and La Cour, L. F. J. Genet. 1945. 46 180. 1^ Lewitsky, G. a. and Araratian, A. G. Bull. appl. Bot. Genet. PL-Breed. 1931, 27 part 1 (U.S.S.R.), 265. 13 Giles, N. H. Proc. nat. Acad Sci. Wash. 1940, 26 567. 1" Thoday, J. M. J. Genet. 1942, 43 189. i» Thod.\y, J. M. Brit. J. Radiol. 1951, 24 572. 622. i« KoTVAL, J. P. and Gray, L. H. J. Genet. 1947, 48 135. 253 EFFECT OF OXYGEN TENSION ON THE PRODUCTION OF CHROMOSOME BREAKAGE BY IONIZING RADIATIONS : AN INTERPRETATION* C. p. SWANSON The Johns Hopkins University, BaUimore, Maryland, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee It was first demonstrated by Thoday and Read^ (1947) that anoxia reduces the eflfectiveness of any given dose of X-rays. This is strikingly illustrated when the frequency of chromosome aberrations is used as the criterion of effect, but it should be recognized that the protective action of anoxia extends to other phenomena as well : survival of organisms, delay in cell division, and the stickiness of chromosomes. The time is not yet ripe, however, for an integrated interpretation of these multiple effects even if it is presumed that they are related to a common mechanism. It has been recognized that the sensitivity of cells to X-rays, as regards breakage, stickiness, and delay in cell division, is greatest at late prophase and very early metaphase, which suggests possible interrelations. This discussion will, however, be confined to chromosome breakage in the microspores of Tradescantia, and a more extensive interpretation will be attempted in the light of new evidence. Firstly, it will be well to recount those circumstances which have been established with some certainty, and then to reinforce and extend them with additional observations made recently. All other things being equal, it is the oxygen tension of the cell which determines, in part, the frequency of detectable chromosome aberrations. The oxygen-dependent portion of the total frequency will vary in magnitude with different ionizing radiations, but that oxygen is indeed part of a reactive system which influences breakage can be considered established by the studies of Giles and his co-workers. They have irradiated cells in hydrogen, helium, and in vacuum, and the results are essentially similar. Clearly, then, the oxygen tension of the cell is an important factor in the breakage of chromosomes, a finding reinforced by the observation that it is the oxygen tension during irradiation that is important, post-treatment changes in oxygen tension being without detectable effect. Gray- ( 1 953) has adequately discussed these aspects of chromosome breakage in terms of radiochemistry, and it is apparent that a more meaningful picture is beginning to emerge. Secondly, it is now quite clear that the degree of reduction resulting from anoxic irradiation is not the same for all types of aberrations even when the quality of radiation is the same. This was first demonstrated by Riley, Giles and Beatty^^ (1952), who showed that chromatid deletions were less * Work performed under Contracts No. W-7405-eng-26 and No. AT-fSO-D-BSl, U.S. Atomic Energy Commission. 254 C. p. SVVANSON affected by the absence of oxygen than isochromatid deletions and chromatid exchanges. If the reduction in frequency of aberrations is expressed as an air/nitrogen ratio, then that for chromatid deletions was about 1 -4 as com- pared to 2-6 for isochromatid deletions and exchanges. These observ'ations have been confirmed (Svvanson and Schwartz"*, 1953), and in addition it was possible to show that the air/nitrogen ratios varied with the stage of cell division. Dominant lethals and translocations in Drosophila also exhibit different air/nitrogen ratios with the same quality of radiation (Baker and Edington^, 1952 ; Baker and Von Halle^, 1953), therefore any hypo- thesis dealing with the relation of oxygen tension to radiation effect must be flexible enough to account for differential reductions among the possible aberration types. Table I. Chromatid Aberrations Produced in Tradescantia by Four Qiialities of Radiation in Air and in Nitrogen. 150 r a< 8-9 r/minute Aberrations per 1 00 cells Radiation Atmosphere Chromatid Isochromatid Exchanges Air deletiotu deletiotu X-rays-50 kvp 49-3 \ 84-6 35-3 unfiltered N^ 98-2 15-0 9-5 Air/Ng ratio 0-50 5-6 1-9 X-rays- 100 kvp Air 66-0 62-5 30-5 1 mm of Al N. 79-3 14-0 7-3 Air/Nj ratio 0-83 3-9 2-0 X-rays-250kvp Air 78-0 44-0 28-0 4 mm of Cu N^ 63-5 16-6 6-0 Air/Ng ratio 1-25 2-7 2-3 Gamma rays Air 96-2 32-5 26-0 M-l-3MeV N. 50-5 14-0 5-0 Air/Nj ratio 1-9 2-3 2-3 Thirdly, it must also be recognized that the influence of oxygen tension on the frequency of breakage is closely linked to the type of radiation em- ployed. Thoday and Read'' (1949) have demonstrated that the oxygen effect is much less with alpha rays than with X-rays, and that neutrons appear to occupy an intermediate position (Giles, Beatty and Riley^, 1952). The ion density of the radiation will therefore be a determining factor in governing the magnitude of the oxygen effect. Additional infor- mation has now been obtained from Tradescantia studies in which the effects from three qualities of X-rays, together with 1-1- to 1 -S-MeV gamma rays from a cobalt-60 facility, have been compared (Sw^anson^). The results of one of four similar experiments are given in Table I. If we first consider the frequencies of aberrations obtained in air, it is apparent that the chromatid deletions increase in frequency as the ion density of the radiation decreases but that the reverse holds true for isochro- matid deletions. In fact, this shift in frequency of the two types of deletions 255 THE PRODUCTION OF CHROMOSOME BREAKAGE BY IONIZING RADIATIONS is a compensatory one, for the total frequency of deletions does not vary appreciably with quality of radiation. In view of the results of Kirby- Smith and Daniels^" (1953), who have demonstrated a decrease in total frequency with decreasing ion density of the radiation, these findings are somewhat surprising. However, since four rather extensive and essentially identical experiments have yielded almost identical results, it appears that the compensatory shift in type of deletion is a real one. The relation of chromatid exchanges to quality of radiation in air is similar to that found for isochromatid deletions, and thus agrees with the earlier data of Kirby-Smith and Daniels. Irradiation vmder anoxia yielded isochromatid deletions which, although greatly reduced in frequency, appeared to show no relation to quality of radiation. This, of course, leads to air/nitrogen ratios which are large with 50-kvp X-rays, and which become progressively smaller as the ion density of the radiation decreases. The chromatid deletions, on the other hand, now exhibit an opposite trend to that found after irradiation in air. They increase rather than deciease as the ion density of the radiation increases, and this leads to air /nitrogen ratios which are largest in value with gamma rays and smallest with 50-kvp X-rays. With 50- and 100-kvp X-rays, there is, in fact, an actual increase in the absolute frequency of chromatid deletions obtained in nitrogen as compared to air, and the air/nitrogen ratios, as a consequence, have values less than 1 . The chromatid exchanges, like the isochromatid deletions, are much reduced in nitrogen, but the air/ nitrogen ratios follow a trend comparable to that for chromatid deletions, as might be expected. As pointed out in an earlier account of this work (Swanson^), it is believed that the differential air /nitrogen ratios for the several types of chromatid aberrations can be accounted for by assuming that there is a shift, in nitrogen, of one type of aberration into another. Thus, if we consider that the forma- tion of isochromatid deletions and exchanges involves two broken chro- matids, the repair or restitution of one of these but not of the other would essentially transform potential isochromatid deletions and exchanges into chromatid deletions. It is diflficult to account in any other way for the increases in chromatid deletions at 50 and lOOkvp in nitrogen. If we now convert the aberrations obtained in air and in nitrogen into terms of total breakage, i.e. by scoring chromatid and isochromatid deletions as single events, and exchanges as resulting from two independent breaks — • it can be demonstrated that the inverse relation of ion density of the radia- tion to magnitude of the oxygen effect holds very well {Table II), as Thoday and Read' (1949) and Giles, Beatty and Riley^ (1952) pointed out earlier. An interpretation Two hypotheses have been advanced to explain the reduction in frequency of aberrations obtained when irradiation is carried out under conditions of anoxia. Giles and his co-workers^ proposed a hypothesis in which the principal effect of oxygen during irradiation is considered to be on the breakage mechanism, but they do not specify in detail how this is accom- plished. Schwartz ^^ (1952) considers that oxygen affects the processes 256 C. p. SWANSON Table II. Percentage Reduction in Total Breakage in Tradescantia as the Result of Exposure in the Absence of Oxygen Radiation Experiment C Miscellaneous* Gamma ravs M-l-3Mev 59 — X-rays-250 kvp 48 58 X-rays- 100 kvp 43 — X-rays-50 k\p 36 — Neutrons — 33 Alpha rays — 0 * Reduction of 58 per cent at 250-kvp X-radiation was extracted from Riley, Giles and Beatty (1952, Table 1 at 150 r). The neutron and alpha ray reductions were derived from data in Giles, Beatty and Riley {1952, Table 2 at 10 n). Thoday and Read (1949) indicate that some reduction is obtained with alpha rays when ex- posure is made in nitrogen. of restitution in such a manner that restitution, which follows the actual breakage of chromosomes, is favoured by anoxia. As will be pointed out, neither hypothesis is satisfactory in its present form. If we adopt the point of view of Gray- (1953) that a variety of primary events are produced in or near the chromosome by ionizing radiations and that the relative import- ance of these events, when translated in terms of breakage, is determined by the physiological condition of the cell during irradiation and up to the time of final rupture of the chromatin strands, it becomes clear that breakage and restitution can be effected in a variety of ways and at various levels. In what follows, a more detailed analysis of possible events will be made, and the two schemes to be presented {Figures 1 and 2), although obviously tentative, will at least serve a useful purpose as points of departure for dis- cussion and possibly for future studies. Before proceeding with an explanation of Figures 1 and 2, it will be well to point out why the two previous hypotheses are inadequate. Insofar as the breakage hypothesis is concerned, increases in any type of aberration — for example, the chromatid deletions induced by 50-kvp X-ra}s in nitrogen as compared with air ( Table 1) — cannot arise by a reduction in breakage alone. Partial repair or restitution, which converts one type of aberration into another, as explained earlier, must be postulated as an additional pro- cess. Also, if breakage alone is affected by oxygen tension, it becomes equally difficult to explain how aberrations such as chromatid and isochro- matid deletions, which have somewhat similar relations to dose, vary quite differently with stage of division (Swanson and Schwartz*, 1953). Similar awkwardness is encountered if an attempt is made to explain the data in Table 1 in terms of the restitution hypothesis. It can be seen that as the ion density of the radiation increases the chromatid deletion /isochro- matid ratio decreases. With the more densely ionizing particulate radia- tions it would be expected, on the restitution hypothesis, that this ratio would be even lower in value than that obtained with 50-kvp X-rays, and that the values for the air/nitrogen ratios would be lower for chromatid deletions and higher for isochromatid deletions than those obtained with 50-kvp X-rays. That this expectation has not been realized has been 257 s THE PRODUCTION OF CHROMOSOME BREAKAGE BY IONIZING RADIATIONS demonstrated by Thoday and Read' (1949) for alpha rays and by Giles, Beatty and Riley« (1952) for neutrons. It must therefore be considered that restitution as defined by Schwartz ^^ (1952) cannot be the process principally affected by oxygen tension in Tradescantia. Figure 1 is an operational scheme adopted, in modified form, from Thoday12 (1953), and applicable only to the data obtained from the study of chromatid aberrations in Tradescantia. In agreement with Gray^ (1953) that a variety of initial events can take place in the chromosome as the result of exposure to ionizing radiations, it is suggested that both potential (latent) PHASE I IRRADIATION r-». STRUCTURALLY INTACT CHROMOSOME POTENTIAL BREAKS } PRIMARY BREAKS Og INDEPENDENT- RELATIVE FREQUENCIES OF POTENTIALS TO PRIMARIES DEPENDENT ON ION DENSITY REPAIR, FAVORED BY ANOXIA PHASED PHASE m SEPARATION OF BROKEN ENDS, FAVORED BY O2 NOT AFFECTED BY 02 PRIMARY BREAKS V FAILURE OF BROKEN ENDS TO ADHERE, (HEALING?) OBSERVED BREAKS (CHROMATID DELETIONS) r OLD WAY i RESTITUTION (INTACT CHROMO- SOME) ADHESION OF BROKEN ENDS 1 NEW WAY \ RECOMBINATION (ISOCHROMATIDS IF SISTER REUNION, EXCHANGES IF NON- SISTER REUNION) FATE OF POTENTIALS DEPENDENT ON O2 TENSION O2 INDEPENDENT Figure 1. A schematic diagram representing in brief the major events taking place in the chromosomes of Tradescantia during and after radiation, with suggestions as to the most likely manner by which oxygen affects the frequency of aberrations. and primary breaks are formed, and that in Phase I their formation is inde- pendent of oxygen tension. Valid objections to the terminology employed may be raised, but these are terms of convenience and may perhaps best be considered as the termini of a continuous spectrum of damage inflicted in the chromosome, the primary breaks being sites of irretrievable damage and of subsequent rupture, and the potential breaks, lesions or sites of lesser damage capable of repair or of complete disruption, as the cellular circum- stances might determine. Furthermore, it is proposed that the relative frequencies of the two types of breaks would be a function of ion density, for it is inconceivable that radiations of such quantitatively diflferent ioniza- tion characteristics as gamma and alpha rays could produce the same 258 C. P, SWANSON spectrum of initial events. Since there is little or no oxygen dependence with alpha rays, and since the probability of a single alpha particle produc- ing a break is close to unity (Gray 2, 1953), the initial events induced by alpha rays would probably lie far to the right of the spectrum, i.e. they are principally primary breaks. Their transformation into actual breaks with separated broken ends may be immediate or delayed— this we cannot establish, and the time factor may be different with different organisms — but complete rupture of the chromatids takes place regardless of the oxygen tension (Phase II). The less densely ionizing gamma and X-rays would produce changes more to the left of the spectrum — neutrons would be intermediate in this respect — although some primary breaks would still be produced. The fate of the potential breaks is determined -by the available oxygen in the cell in such a fashion as to favour their complete disruption in the presence or their repair in its absence. Also, the less the damage, the greater the dependence on oxygen for complete transformation into actual breaks. If these changes induced by radiation are in single-strand chromosomes, only a reduction in total frequency of aberrations can be expected if anoxia exists during irradiation, and rings and dicentrics, the customary types of aberrations scored, would be reduced to the same degree. Giles and his co-workers^ have shown that this expectation is realized. However, if double-strand chromosomes are irradiated in the absence of oxygen, repair of one or two damaged chromatids becomes possible, and a situation is therefore available for the transformation of latent isochromatid deletions and exchanges into actual chromatid deletions. Variable air/nitrogen ratios among chromatid aberrations are therefore possible, and the extent of variability will be determined not only by the ratio of potential to primary breaks in Phase I, but also by the ratio of single- to double-strand lesions. Since both ratios are dependent on the ion density of the radiation, a complex situation exists, but there can be no doubt that there is a correlation between oxygen dependence and ion density. The data obtained to date provide no reason for believing that oxvgen tension exerts an influence once the actual breaks are formed (Phase III). The scheme presented appears to be consistent with the biological data derived from the study of irradiated chromosomes of Tradescantia as w ell as with the physical facts of radiation. To what degree it corresponds to actual events taking place in irradiated chromosomes remains to be demonstrated for it is becoming increasingly evident that the final answers lie in the area of radiochemistry (Gray 2, 1953). The work of Lea and others has provided strong support for the belief that individual chromosome breaks are pro- duced by individual ionizing particles, but the role of oxygen suggests that both direct and indirect energy transfer to the molecular bonds within the chromosome can lead to breakage. Presumably, the indirect energy transfer is through the medium of reactive substance produced by the radiation in or near the chromosome. With this in mind, the scheme in Figure 2 provides a more general approach to the problem of how oxygen may aflfect breakage. Several possibilities exist, and for convenience they may be listed as follows : (i) Phase /—At this level the physiological conditions of the cell or the surrounding medium can have an influence on the amount of indirect 259 THE PRODUCTION OF CHROMOSOME BREAKAGE BY IONIZING RADIATIONS energy available for transfer to chromosomal substances. Thus bacteria irradiated in water and in broth have different survival rates even though the oxygen tension in the cells may be similar. So far as breakage in Tradescantia is concerned, it is likely that this is an important step because it is here that the relative amounts of direct versus indirect energy are determined. IRRADIATION PHASE I \ INDIRECT ENERGY TRANSFER DIRECT ENERGY TRANSFER I / / PHASE n SPECTRUM OF CHROMOSOMAL DAMAGE INCREASING DAMAGE Figure 2. A schematic representation in more general form of the possible roles of radiation and oxygen tension in inducing chromosomal aberrations. {Prepared in collaboration with Dr. K. G. Liining, Institute of Genetics, University of Stockholm, and published with his kind permission.) (2) Phase //^Several steps can be involved here, but at present there is no way of disdnguishing between them experimentally. Oxygen, through the medium of indirect energy transfer, can contribute to the spectrum of chromosomal damage, or it can enhance the damage already induced by direct ionization. Anoxia, therefore, would tend to repair damage created by direct energy transfer or to lessen the amount of damage created by 260 C. p. SWANSON indirect energy transfer. Phase II consequently corresponds to Phase II of Figure 1 where the fate of the potential breaks is considered to be deter- mined, and the influence of oxygen is greater at the left than at the right end of the spectrum of chromosomal damage, as demanded by the data in Table II. (3) Phase III — Primary breaks are distinguished from actual breaks only by a time factor, and the transformation may be immediate or only after an interval of time as indicated. The Tradescantia data suggest that oxygen is ineffective at this level, but we cannot be certain that this is true for breakage in other organisms. {4) Phase IV — The Tradescantia data again suggest that the fate of actual breaks is not influenced by oxygen, but the possibility is included since it may be important in other organisms. ACKNOWLEDGEMENTS This paper was uritten at the Institute of Genetics, University of Stockholm, where the author was in residefice as a Guggenheim Fellow. The author wishes to acknow- ledge his indebtedness to Professor Gert Bonnier for the facilities placed at his disposal, and for the many courtesies extended. Figure 2 and the discussions revolving around it are to be considered part of a collaboration with Dr. A . G. Liining, and they are included here with his kind permission. REFERENCES ' Thoday, J. M. and Read, J. .Vature, Lond. 1947, 160 608. 2 Gray, L. H. Brit. J. Radiol. 1953. 26 609. 3 Riley, H. P., Giles, N. H. and Beatty, A. V. Amer. J. Bat. 1952, 39 592. * SwANSON, C. p. and Schwartz, D. Proc. Nat. Acad. Sci. U.S. 1953, 39 1241 5 Baker, W. K. and Edington, C. W. Genetics, 1952, 37 665. « Baker, W. K. and Von Halle. E. S. Proc. Nat. Acad. Sci. U.S. 1953, 39 152. " Thoday, J. M. and Read, J. Nature, Lond. 1949, 163 133. 8 Giles, N. H., Beatty, A. V. and Riley. H. P. Genetics, 1952, 37 641. * SwANSON, C. P. ibid, in press. 10 Kirby-Smith. J. S. and Daniels, D. S. ibid, 1953, 38 375. 11 Schwartz, D. Proc. Nat. Acad. Sci. U.S. 1952, 38 490. 1^ Thoday, J. M. Heredity (Symp. on Chromosome Breakage, Springfield, 111., Thomas), 1953, 6 299. 261 THE RELATIVE EFFECTIVENESS OF VARIOUS IONIZING RADIATIONS ON CHROMOSOME BREAKAGE IN TRADESCANTIA J. S. Kirby-Smith, C. W. Sheppard and Doris L. Craig Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee A DETERMINATION of the relative biological effectiveness (RBE) of X-rays of mean energies approximately 60 and 200 kV respectively, mixed 1-17- and 1 -SS-MeV gamma rays from ^"Co and high-energy beta rays from ^-P on Tradescantia pollen has been reported recently by Kirby-Smith and Daniels^. These studies have shown medium-energy X-rays in the 60-kV range (0-20 A wavelength) to be twice as effective as 1-MeV gamma rays or beta rays of approximately 500-kV mean energy in producing chromo- some aberrations in this material. The effectiveness of 200-kV X-rays was found to be midway between that for 60-kV X-rays and 1-MeV gamma rays. This work now has been extended to cover the effects of fast neutrons on dry pollen and inflorescences of Tradescantia. In the course of these later investigations, the twofold increase in biological effectiveness in 60-kV X-rays over 1-MeV gamma rays has been confirmed for both of these biological materials. The source of fast neutrons in the present studies has been the Oak Ridge National Laboratory 86-inch, 22-MeV proton cyclotron with the reaction of protons on an internal beryllium target. Measurements of the fast neutron flux and dosage have been made by means of proportional counter dosimeters and tissue-equivalent ion chambers. A comparison of these two different methods, as well as an independent determination of the sf'ay gamma rays present with neutron insensitive ion chambers, has shown the gamma-ray contamination to be approximately 10-15 per cent. In order to reduce the gamma-ray contamination to this figure, a bio- logical exposure facility consisting of a lead box with 1 -inch-thick walls was constructed outside the dee chamber. The neutron energy spectrum within this facility from the proton reaction includes considerably lower energies than the well-known curve for deuterons on beryllium, and there is also the degradation in neutron energies and scattering produced by the lead and by the other metal structures of the cyclotron. The neutron energy spectrum within the exposure facility has been determined approximately by means of various threshold and fission detectors. As is to be expected, it is a much degraded spectrum, with a broad maximum in the 1-MeV range and an appreciable thermal component. Both Tradescantia dry pollen and inflorescences were exposed to fast neutrons in the lead-walled facility described. Following irradiation, the pollen was cultured after the methods of Bishop- and Conger^. Aberra- tions were scored at metaphase in the pollen tube division. In the inflor- escences, four-day chromosome aberrations were scored. Results of the 262 J. S. KIRBY-SMITH, C. W. SHEPPARD AND DORIS L. CRAIG pollen irradiations are shown in Table I together with aberration frequencies for X-ray and gamma ray treated samples of the same material for com- parison. Data for the neutron-irradiated inflorescences, both hydrated and Table I. — Effect of fast neutrons, medium-energy X-ray, and ^^Co gamma rays on Tradescantia pollen Treatment Dose No. of cells scored Percentage normal „\o. of Aberrations per cell Cd Iso Ex Fast neutrons X-rays (250 kVp 3-mm Al) Gamma rays (6OC0I 24 -7 rep 49 -3 rep 73 -8 rep 98 -8 rep 200 r 200 400 300 300 150 300 300 46 21 12 8 12 38 015 0-28 0-39 0-41 0-34 0-17 0-44 0-91 1-31 1-56 0-99 0-49 0-17 0-35 0-46 0-55 0-42 0-23 desiccated, as well as for an additional independent experiment in which normal buds were used, are summarized in Table II. These data show no differences in aberration frequencies for the wet and dry material and thus indicate that variations in the degree of hydration normally encountered in the flower buds do not affect their neutron sensitivity. Table II. — Effect of fast neutrons on hydrated and nonhydrated Tradescantia inflorescences No. of Dose (rep) No. of cells scored Percentage normal Aberrations per cell Exchanges Deletions Wet buds 24-7 200 60 0-23 0-28 49-4 200 44 0-44 0-44 73-8 150 27 0-74 0-61 98-3 100 14 1-02 0-89 Dry buds 24-7 49-4 140 350 63 39 0-22 0-52 0-20 0-45 Normal buds . . 24-7 200 63 0-24 0-22 49-3 200 42 0-44 0-45 73-8 150 26 0-65 0-69 The RBE of fast neutrons with respect to cobalt gamma rays and 60-kV X-rays is presented in Table III. The results of previous work^ on the relative effectiveness of X-, gamma and beta rays are also included in this table. Owing to the variation in biological effectiveness over the X-ray region, the effectiveness of 1-MeV gamma rays in producing the biological effects of chromosome breakage is assumed to be unity. This convention, which differs from previous usage in which X-rays are made the reference 263 CHROMOSOME BREAKAGE IN TRADESCANTIA Table III. — Relative biological effectiveness of various ionizing radiation': Type of Radiation Relative Biological Effectiveness Tradescantia pollen Tradescantia inflorescences fioCo gamma rays ( 1 • 1 7 and 1-33 MeV) ^2p beta rays (mean energy ~' 500 kV) . . 200-kV mean energy X-rays (0-06 A) . . 60-kV mean energy X-rays (0-20 A) Fast neutrons (1-MeV mean energy) 1 1 1-5 2-0 8-0 1 1 1-5 2-0 10-0 index, gives an unambiguous reference point for comparing the effects of the various radiations. These studies indicate the type of rough quantitative radiobiology possible with fast neutrons from a large internal target cyclotron source. Certain physical difficulties continue pending installation of a beam deflector and external target. Serious problems remain in precisely characterizing the neutron spectrum particularly at the very low energy or epithermal range. Owing to the very high values of the RBE for chromosomal breakage in the material used, the unavoidable presence of the contaminating gamma rays is not as serious an objection in the present work. The dose figures which have been given in all cases in rep are probably accurate to better than i 15-20 per cent. A more detailed discussion of the fast neutron dosimetry in the cyclotron facility is scheduled for future publication. REFERENCES 1 Kirby-Smith, J. S. and Daniels, D. S. 2 Bishop, C. J. Stain Technol. 1949, 24 9. 3 Conger, A. D. ibid, 1953, 28 289. Genetics, 1953, 38 375. 264 CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS G. Roy Lane Botany Department, Uni\ersity of Southampton The effects of X-rays and other ionizing radiations on the meristematic cells of the root of Vicia faba have been studied by a number of workers, e.g. Darlington and La Cour\ Ford"^, Thoday^-*, Gray and Scholes^, and Revell^' ^. The latter author has made a special study of the effects of di(2 : 3-epoxypropyl) ether and some other radiomimetic substances on Vicia root cells whilst McLeish^ has studied the effects of maleic hydrazide on the same material. This paper deals with experiments now being carried out with X-rays and radiomimetic substances on the root cells of Tradescantia. This material was chosen for the following reasons : (7) The effects of radiation on the haploid pollen cells of this plant have been extensively studied by many workers and of certain radiomimetic substances by Darlington and Roller'' and recently by Smith and Lofty^". Until now, no study has been made of the effects on the diploid root meristem cells of this plant, although it forms adventitious roots freely and the roots can be grown readily in culture under controlled conditions. (2) The chromosome breaks induced by diepoxide in the root cells of Vicia occur largely in the demonstrable heterochromatin (Revell^'^). So also do those induced by maleic hydrazide (McLeish^). The effects on Tradescantia chromosomes, which possess no demonstrable heterochromatin should make an interesting comparison. {3) Chromosome reunion (R") to form polycentrics and rings is relatively rare in Vicia following radiation treatment. This has been related to the presence of heterochromatin by Darlington^^. Chromosome reunion (R") probably does not occur at all in the first mitosis after treatment with diepoxide or maleic hydrazide, reunion being entirely between chromatids, i.e. of the SR and R' types. In Tradescantia pollen cells, chromosome reunion is extremely common and predominates if treatment is carried out during the first part of the resting phase. Here again the effects of diepoxide and maleic hydrazide on the root cells of Tradescantia should make an inter- esting comparison. {4) Revell^^' ^^ has postulated a new interpretation of the mode of origin of X-ray and radiomimetic chromosome structural changes, based on his work on Vicia. Results from Tradescantia in which the chromosomes differ in their chromatic make up and in their properties of reunion may be expected to assist in assessing the alternative interpretations. EXPERIMENTAL Details of the culture and technique vised in this work will be published elsewhere. The plants used are of a single clone of Tradescantia paludosa. 265 CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS Roots are induced by appropriate pruning and grown in culture at controlled temperature (22° C.) and illumination (16hr/day) in aerated culture vessels. Treatment with diepoxide solution is carried out at the controlled tempera- ture and for the last 5 hours before fixation the roots are grown in 0-05 per cent colchicine, also at the controlled temperature. Treatment with X-rays is carried out as nearly as possible at the temperature of culture. This material does not respond to the usual maceration methods used in making Feulgen squash preparations after aqueous fixation. New methods of maceration had to be worked out. The root tips are fixed in La Gour's 2 BD osmic fixative, bleached in hydrogen peroxide and placed in an extract of Bacterium aroideae for 3 to 5 days, a method suggested by the work of WooD^"*. This treatment greatly assists separation of the cells during squashing and does not interfere with subsequent staining. The root tips are stained by the Feulgen method, hydrolysis being carried on for 20 minutes and staining for one hour. The chromosomes of Tradescantia paludosa are so well known that no detailed description is necessary. There are six pairs of chromosomes in the somatic cells all with approximately median centromeres, consequently the individual chromosomes are not easy to distinguish from one another. One pair has arms of approximately equal lengths and is fairly easily picked out. In the other five pairs the length of the shorter arm is from about one-half to four-fifths of that of the longer arm {Figure 2). There is no demonstrable heterochromatin and no distinguishable nucleolar constriction. The material was given X-ray doses of 50r-100r (intensity 27r/min. approx.) and fixed at various intervals from 6 hours to 4 days after treatment. Diepoxide treatment was given with an M/2,000 solution for 10, 20, 30 and 40 minutes. Material was fixed at intervals from 12 to 78 hours after treatment. RESULTS X-rays— The effects of X-rays are similar to those shown in the pollen cells. There is, as is to be expected, a complete overlap of the different effects obtained by earlier or later treatment of the pollen grains. This is due to the lack of any synchronization in the development of the cells and to the varying rates of development. At 36 hours after 200 r (22° C.) most of the reunion is of the R' or SR type but dicentrics and centric rings (R") and minutes (m) also appear in considerable numbers together with Xg cells showing breaks and micro- nuclei. Dicentrics and rings survive in Xg mitoses and at 3 days paired cells showing equal-sized dicentrics or rings are quite frequent. Diepoxide— MicY a treatment of 20 minutes with M/2,000 diepoxide the breakage is quantitatively approximately the same as that obtained by Revell*'- ' with Vicia using the same dose {Table I and Figure 1). Reunion is entirely between chromatids, either chromatid reunion (B') or sister reunion (SR), at all the time intervals {Figure 2). No dicentrics or rings (R") like those observed after X-ray treatment were observed even at the longest time interval of 60 hours. Approximately half of the changes are simple chromosome breaks, the majority of which show sister reunion in both centric and acentric fragments. 266 G. ROY LANE Table I. — Frequency of breaks and interchanges after treatment with di-{2 : 3-epoxypropyl) ether, MI2,000 for 20 minutes, and Mj2,000 for 30 minutes Chromatid Time interval in hours :3 Damaged cells B" 03 1 Co interchanges Total interchanges 03 Total B 5 03 Treatment Between chromosomes Within chromosomes -a Ratio of in changes to *M/2,000 18 100 34 20 5 3 4 0 1 19 39 0-35 20 min M/2,000. 22 100 40 28 9 9 5 0 14 37 71 0-50 20 min M/2,000, 36 100 54 34 12 18 5 1 24 60 94 0-71 20 min M/2,000 48 100 65 68 7 14 7 2 23 53 121 0-34 20 min Totals 400 193 150 33 44 21 13 3 68 169 325 0-47 M/2,000, 31 100 52 30 11 27 4 44 99 128 1-47 30 min M/2.000. 52 100 85 74 20 65 33 6 104 228 302 1-41 30 min Totals 200 137 104 31 92 46 10 148 327 430 1-42 * 50 cells from each of 2 slides 30 m\n. treatment 20 mm. treatment Time m tergal h. Figure 1. Frequency of breaks and interchanges after treatment with di{2 : 3- epoxypropyl) ether M {2, 000 for 20 minutes and MI2,000for 30 minutes 267 CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS A few single chromatid breaks occur, but most of the remaining structural changes are chromatid interchanges, either symmetrical or asymmetrical (in about equal quantity) . A few triradial figures appear with accompanying centric or acentric fragments which may or may not show sister reunion [Figure 3). I i/\ Figure 2 Approximately one-third of the interchanges are within the same chromo- some, frequently between the two arms [Figure 2). With treatment of 30 minutes the proportion of interchanges appears to be considerably higher, the ratio of chromatid interchanges to chromosome breaks rises to 1 -4 as compared with 0-47 for the lower dose [Table I and Figure 1). This is at variance with the results with Vicia where Revell^''^ 268 ROY LANE found that the ratio of interchanges to B" remained at between 0-41 and 0-52 at all doses tried. This high ratio of 1 -4 is consistent in the two time intervals analysed (31 and 52 hours), but in view of the relatively small f Figure 3 sample (200 cells) needs confirmation by further experiment and analysis. Another difference in the interchanges induced by diepoxide in Trades- cantia roots is that there appears to be no tendency towards interchange 269 CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS between homologous chromosomes at apparently homologous points, as found by Revell^- "^ in Vicia. As regards localization of breaks in general, this is difficult to estimate in Tradescantia owing to the close similarity in form between the different chromosomes. Further analysis should enable me to make a decision on this important point. A few minute chromosome fragments (m) are found after all treatments, often associated with chromatids of unequal length or chromatid breaks. These are probably mainly minute interchanges of the type figured by Table II. — Frequency of mitoses and of micronuclei after treatment with M 12,000 di-{2 : 3-epoxypropyl) ether. Roots grown in 0-05 per cent colchicine for 5 hours before fixation. Two slides for each treatment Time interval in hours Percentage Micronuclei Treatment Total no. of cells Percentage mitoses ' resting cells with micronuclei per 100 resting cells None — 1,298 7-30 0-25 0-25 M/2,000, 10 mill 48 1,412 10-20 5-37 5-92 M/2,000, 20 mill 12 1,282 5-85 0-58 0-75 18 1,240 2-58 0-66 0-66 24 1,249 1-68 2-28 2-62 30 1,306 4-90 3-38 3-90 36 1,398 6-93 7-75 8-55 42 1,402 7-45 7-55 9-10 48 1,417 7-35 8-65 9-55 54 1,044 7-27 13-80 15-60 60 1,647 11-20 18-00 20-50 M/2,000, 30min 22 1.221 0-49 1-24 1-24 > J 31 1,250 2-16 1-88 2-13 52 1,735 4-85 16-70 25-40 J> 78 1,411 5-38 10-10 11-60 M/2,000, 40min 22 1,214 0-33 0-83 1-08 RevelP in relation to his explanation of the mode of origin of the chromosome breaks with sister reunion. An occasional dicentric chromosome appears in the 48-hr sample from 10-min treatment. At this time-interval after the smaller dose no primary breakage is seen. Most of the mitoses are evidently of the second cell generation after treatment (Tg). These dicentrics are accompanied by a micronucleus in the same or neighbouring mitosis, and clearly have their origin in asymmetric interchanges, the micronucleus representing the acentric fragment. Similarly, neighbouring cells in this sample are found with short chromosomes of equal size, the products of separation of centric fragments which have not undergone sister reunion. Sister reunion is frequently absent in the centric fragment when the break is close to the centromere. 270 G. ROY LANE An analysis was made of the frequency of mitosis (after the usual colchicine treatment) and of micronuclei in the resting cells at different time intervals after doses of diepoxide {Table II and Figure 4). This shows that mitosis is suppressed by the diepoxide, the duration of the effect being dependent upon the dose. It can be seen that the frequency of mitosis is reduced from that of untreated samples. Mitosis drops to a minimum at about 24 hours after 20 minutes' treatment and recovers by about 36 hours. It rises still higher right up to 60 hours. After 30 minutes' treatment the recovery is slower, whereas after 10 minutes' treatment the mitotic frequency has already climbed to a high level and the cells in mitosis appear to be already of the 30 mn. treatment 20 m\r\ treatment 12 78 2t- 30 se f-2 Time internal fS S¥ SO h. Figure 4. Frequency of mitoses and of micronuclei after treatment with M 1 2, 000 di{2 : 3-epoxypropyl) ether for 20 minutes and 30 minutes T2 cell generation. This indicates that after this dose, suppression is of still shorter duration. The different degree of mitotic suppression with different doses means that it is dangerous as with X-ray effects to draw comparisons between the effects of different doses at one given time period after treatment. The data obtained on the frequency of micronuclei after treatment also show the effect of dose in retarding mitosis. At 30 hours the frequency is less than after the larger dose. SURVEY / The data so far obtained on the effects of diepoxide on the meristematic root cells of Tradescantia raise many questions to which there is as yet no certain answer. It is useful, however, to consider these questions in order to clarify the direction which future investigation should take. 271 CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS One thing is clear, that in spite of the lack of demonstrable heterochro- matin, di(2 : 3-epoxypropyl) ether is as effective in causing structural change in the chromosomes of Tradescantia as it is in Vicia. The changes in Vicia, though not confined to the demonstrable heterochromatic segments, occur largely in them. It may be that changes outside these segments occur in small undetectable segments of heterochromatin or at points which have certain properties in common with heterochromatin. This of course raises the whole question of the definition of heterochromatin. The chromosomes of Tradescantia are broken at many scattered locations. Acentrics of certain lengths seem to predominate, but it has not yet been possible to determine with certainty whether any particular points on the chromosomes are sensitive to diepoxide. As Revell** has pointed out, there is considerable evidence that the hetero- chromatic regions of Vicia associate with one another in the chromocentres during the resting phase. These are just the regions in which, in the main, breakage by diepoxide occurs. It is probable that no such association between special regions sensitive to diepoxide occurs in Tradescantia where there is no demonstrable heterochromatin and no obvious chromocentres. Consequently it might be expected that interchange between chromosomes (reunion) would occur in a more random manner. My observations suggest that this is indeed the case. There is no such tendency towards homologous chromatid interchange. RevelP- 'lays stress on the tendency for apparently homologous interchange to occur in Vicia and compares this phenomenon with chiasma formation. Clearly, reunion or interchange can take place only at points of association. In meiosis, and to a certain extent in mitosis, in Vicia these points of associa- tion are not random. Consequently, interchange will also tend to occur between certain restricted regions of the chromosomes which are normally associated rather than between points of chance association. This might be held to account for the lack of an exponential relation between the frequency of interchanges and the dose. There are indications in my data that there may be such an exponential relationship in Trades- cantia. If both chromosome breaks (B") and interchanges show a linear relationship with dose, their ratio should remain constant at all doses provided that the threshold dose, the origin of the curves, is the same for both (and there is no reason to believe that it is not). This is the case in Vicia according to Revell*^- '. In Tradescantia the ratio rises from 0-5 at 20 minutes' exposure to approximately 1-4 at 30 minutes' exposure. The apparent great difference with a relatively small change of dose may not truly reflect the relationship. Firstly, the samples are relatively small and secondly, the actual dose may change to a much greater degree than is suggested by the time values of the treatment. I have now reason to believe that penetration into the root in Tradescantia may be much slower than in Vicia. Certainly fixatives penetrate more slowly. It is there- fore planned to carry out experiments with longer treatment at lower concentrations. Revelli2 has interpreted all types of aberration including all the chromo- some breaks with sister reunion (and presumably those without) as chro- matid interchanges. Certainly there is considerable evidence that some of 272 G. ROY LANE the chromosome breaks have their origin in interchange of the type he describes. This may well account for the greater than linear relation between chromosome breaks with sister reunion (so-called isochromatid breaks) and the dose, reported by some workers in irradiated material. I have observed an apparent chromosome break with sister reunion of Ijoth centric and acentric fragments with the two fragments interlocked by the reunion. This clearly cannot be the result of failure of reproduction of broken chromosome ends. However, other evidence suggests that only a fraction of the sister reunions are the result of minute interchange at loops. Firstly, if all B" are the result of interchange at loops, one would expect the frequency of the three alter- native types of detectable interchange to be approximately equal. In fact, the B" with sister reunion far outnumber the other types {i.e. minutes (m) with or without B' and unequal or looped chromatids). Secondly, after treatment with diepoxide, breaks cutting straight across both chromatids without sister reunion in the centric or acentric fragment occur fairly frequently. In addition to these clear breaks, constrictions or lesions occur either on both chromatids at apparently the same locus or on one chromatid opposite a chromatid break. These constrictions have been observed by McLeish^ in Vicia after treatment with maleic hydrazide and by many workers in irradiated material. They are usually interpreted as restitution and may be considered as evidence supporting the view that diepoxide and other radiomimetic substances produce direct chromosome breakage by local inhibition of chromosome synthesis, and that the aberration is not always an exchange as RevelP"^ has suggested. Finally, RevelFs suggestion that the diepoxide effects are to be considered as a kind of artificially induced chiasma formation is a tempting one to follow, at least in regard to the clear cases of interchange. The similarities between certain types of interchange and the chiasma are obvdous, and they both occur at a restricted period in the cell cycle, probably at or near the time of chromosome reduplication. However, it should not be forgotten that the differences are many, a most significant one being that the chiasma is never an asymmetric interchange. This suggests a different mechanism for the two types of chromatid exchange, the chiasma and the artificially induced interchange. On the other hand, the complete absence of chromo- some reunion (R"), even with treatment during early resting phase, suggests that breakage occurs near to the time of reduplication at whatever part of the resting phase the treatment is given. SUMMARY (/) Di(2 : 3-epoxypropyl) ether causes chromosome structural changes in the meristematic cells of the roots of Tradescantia paludosa. These changes are similar to those reported in Vicia faba root after similar treatment. This substance also has a delaying effect on mitosis, the effect increasing with dose. {2) The chromosome breaks and reunions do not appear to be localized as in Vicia nor is there the same tendency towards apparently homologous chromatid interchange betw^een homologous chromosomes. From the limited data so far obtained, interchange appears to be favoured by higher dose. 273 T CHROMOSOME BREAKAGE BY DIEPOXIDE AND BY X-RAYS {3) Some of the apparent chromosome breaks have their origin in chro- matid interchange. (4) Dicentrics and centric rings are frequent after X-ray treatment, but no evidence was found of chromosome reunion (R") after treatment with diepoxide even at the long time-intervals. ACKNOWLEDGEMENTS / am indebted to Professor P. C. Koller and to Dr. S. H. Revell for supplying me with samples of diepoxide, to Dr. J. R. Clarkson for irradiating material and to Mrs. J. Paton for invaluable assistance. This investigation is supported by grants from the British Empire Cancer Campaign. REFERENCES 1 Darlington, C. D. and La Cour, L. F. J. Genet. 1945, 46 180. 2 Ford, C. E. Proc. 8th Intern. Cong. Genet., Heriditas Suppl. 1949, 570. ^Thoday, J. M. Proc. 8th Intern. Cong. Genet., ibid, 1949, 672. * Thoday, J. M. Brit. J. Radiol. 1951, 24 572 and 622. 5 Gray, L. H. and Scholes, M. E. ibid, 1951, 24 82. « Revell, S. H. Brit. Ernp. Cancer Camp. Ann. Rep. 1953, 30 42. ^ Revell, S. H. Symposium on Chromosome Breakage, Heredity Suppl. 1953, 107. * McLeish, J. Symposium on Chromosome Breakage, ibid, 1953, 125. 9 Darlington, C. D. and Koller, P. C. Heredity, 1947, 1 187. " Smith, H. H. and Lofty, T. A. Amer. J. Bat. 1954, 41 589. " Darlington, C. D. John Innes Hart. Inst. Ann. Rep. 1952, 42 26. 12 Revell, S. H. Brit. Emp. Cancer Camp. Ann. Rep. 1954, 31 33. 1^ Revell, S. H. This Symposium. 1* Wood, R. K. S. Nature, Lond. 1951, 167 771. 274 THE RELATIONSHIP BETWEEN CHROMOSOME FRAGMENTATION AND REJOINING IN TRILLIUM ERECTUM FOLLOWING THERMAL NEUTRON AND X-IRRADIATION Eleanor E. Deschner* Brookhaven National Laboratory', Biology Dept., Upton, Long Island, New York An investigation into one of the factors affecting the degree of radio-sensi- tivity of the nuclear cycle in Trillium erectum was reported by Sparrow and Maldawer^ in 1950. Evidence was presented by them indicating that greater rejoining occurred at a relatively radio-resistant stage, interphase, accounting for a part of the difference in the total visible chromosome fragmentation while a small amount of rejoining occurred after X-irradia- tion at first meiotic metaphase, a highly sensitive stage. However, the increased number of reunions at the apparently resistant interphase stage was not sufficient to explain the decrease in total visible fragmentation. A further study along these lines has been carried out in an effort to determine if this inverse relationship between breakage and I'ejoining would hold true at other stages of meiosis and if, in general, the frequency of reunions be closely associated with the degree of sensitivity. In addition, a similar study was undertaken using thermal neutron irradiation in an effort to learn how the two radiations differ, if at all, in their effect on the processes of breakage and reunion^. Propiono-carmine smears of anthers irradiated at known stages of micro- sporogenesis were examined at first anaphase and microspore anaphase in order to pick up both immediately visible and delayed breakage. Frag- ments were scored as an index of the breakage sensitivity and dicentric and ring chromosomes were used as an index of the degree of reunion. Data collected at first anaphase as a result of 50r X-irradiation at zygotene, pachytene, and diplotene indicates that while no statistically significant difference in breakage sensitivity existed, the amount of recombination of broken ends was significantly higher at diplotene. Cells irradiated at diplo- tene displayed a higher breakage sensitivity at microspore anaphase than at first anaphase although the same degree of rejoining was found to occur at both stages. The combined data at first anaphase and microspore anaphase revealed that a significantly greater degree of fragmentation and reunion occurred after diplotene irradiation than after pachytene or zygotene irradiation. In comparing the relative sensitivity of diplotene and first metaphase, an analysis of variance showed that there was no significant difference in * Present address : Radiobiological Research Dept., Mount \'ernon Hospital. Northvvood, Middlesex, England. 275 CHROMOSOME FRAGMENTATION AND REJOINING IN TRILLIUM ERECTUM fragmentation occurring at microspore anaphase between stages nor was there a difference in the number of reunions taking place as a result of diplotene and first metaphase irradiation. However, when the total amount of reunion occurring as a result of diplotene irradiation was calcu- lated, that is, the combined rejoining at first anaphase and microspore anaphase, it was found that rejoining was significantly higher in cells irradi- ated at diplotene than in cells irradiated at first metaphase, although both stages were equally sensitive to breakage. The combined data of first anaphase and microspore anaphase also indicates that rejoining calculated as a function of fragmentation occurs 2-3 times more frequently after diplotene and interphase than after first metaphase X-irradiation. Ratios formed between the number of fragments and rejoins observed for each stage irradiated with thermal neutrons and X-rays indicate an over-all similarity in response to both types of radiation. Diplotene chromosomes are comparably sensitive and appear to have a greater ability to rejoin than chromosomes at other stages while first metaphase irradiated material consistently shows high sensitivity and low rejoining capacity. In conclusion, no consistent relationship has been found to exist between the rejoining capacity and degrees of sensitivity exhibited by chromosomes of Trillium erectum exposed to irradiation at various stages of microsporo- genesis. Consideration of the data seems to indicate that the processes of breakage and reunion may be controlled by two independent mechanisms or alternately that they are two processes whose relationship in time is obscured because while they are closely associated, both phenomena do not proceed at the same rate. In addition, it would seem that not only are there differences in radio-sensitivity of various stages to chromosome break- age, as for example, first metaphase and interphase, but also, stages similar in sensitivity to fragmentation, like first metaphase and diplotene, differ with respect to the degree of rejoining which follows. REFERENCES 1 Sparrow, A. H. and Maldawer, M. Proc. Nat. Acad. Sci. 1950, 36 636-643. 2 Deschner, Eleanor E. and Sparrow, A. H. Genetics, in press. 276 DISCUSSION ON PAPERS BY REVELL, SWANSON, KIRBY-SMITH, LANE AND DESCHNER L. H. Gray : ' Contact first ' hypothesis — I think it is extremely interesting tliat Reveli has feh compelled to return to the view that exchanges only occur between chro- matids at points at which they lie in contact, in order to account for his observations on the aberrations induced in Vicia chromosomes by diepoxide. Over twenty years ago this view was advanced in connection with the production of exchanges by ionizing radiation, and so far as I know, the alternative hypothesis that exchanges arise by union between chromosome ends which result from independently produced breaks was adopted in preference to the 'contact first' hypothesis when it was shown that exchanges resulting from the irradiation of Drosophila sperm increase as the 3/2 power of the dose. Subsequent studies with Tradescantia, Vicia and many other materials have, so far as I know, always yielded the result that exchanges induced by X- or gamma rays increase more rapidly than the first power of the dose, and often as the square of the dose. In one respect the two hypotheses do not differ very greatly, for it was found neces- sary by Lea and Catcheside, in developing quantitatively the generally accepted view of interchange formation, to assume that in Tradescantia exchanges rarely take place between breaks which are more than 1 micron apart at the time of their formation, and it might become a rather fine point to distinguish between this and actual contact. There is, however, one big and crucial difference, as Reveli has pointed out, namely, that while the usually accepted hypothesis supposes that a chromosome may be broken anywhere along its length, Reveli supposes that break- age is secondary to exchange and can only occur at places at which chromatids are already in contact. These are two hypotheses between which it should not be too difficult to decide by experiment. It is clear, in the case of both chemically induced breaks and those induced by ionizing agents, that many steps intervene between the acts of initiation and the definitive formation of the aberrations, and in my view we already know of a number of differences between aberrations induced by diepoxide and those induced by ionizing radiation which make it clear that the steps cannot be identically the same in the two cases. This is shown {a) by the selective localization of aberrations induced by diepoxide (as well as by some other chemical agents) within restricted regions of the chromosome, by contrast with the more or less random distribution of radiation-induced aberrations, and {b) by the fact that in the case of Vicia meristem cells studied by Reveli, the chemical agent is most effective when given during the first half of interphase, whereas ionizing radiation is most effective in the second half of interphase. I would suggest, therefore, that the hypothesis which we adopt as to the mode of action of each agent should be that which best accommodates the facts known to us about the aberrations produced by that particular agent, without any attempt to arrive at a single hypothesis which would be applicable to all agents. For myself, I find no compelling reason at the present time to abandon the accepted view of aberration production by ionizing radiations. In the case of diepoxide, Reveli has been led by the high proportion of aberrations which involve the hetero- chromatic regions of the long M chromosome, and from the approximate linear relation between exchanges and dose, to adopt the 'contact first' hypothesis for the chemical agent. By the same token, the random distribution of breaks and the square dose law for exchange production indicate a two-particle process for X- and gamma radiation. I agree with Reveli that the two electrons might not initiate breaks in two different chromosomes ; it might be that even when two chromatids are lying in contact an amount of energy requiring the co-operative action of at 277 CHROMOSOME ABERRATION least two electrons is necessary to provide a disturbance adequate to initiate an exchange. I prefer the alternative view, which I think is supported by the dose relations observed with neutrons and a-particles. At not too high doses, exchanges which follow a square-law dose with X-rays are linearly related to dose when produced by neutrons, but even so show a small square-law component at high neutron doses. Again, they are linearly related to dose with all doses of a-radiation so far studied. Having regard to the number of electrons, protons and a-particles by which a cell is traversed when exposed to a given dose of X-, neutron- or a- radiation, the dose relations observed with these three radiations are just those to be expected if exchanges result from two independently produced breaks. More- over, in the case of a-radiation, such low doses have been used that most of the aberrations observed have been produced in cells whose nuclei have been traversed by only one a-particle. It would seem difficult to account for the high efficiency with which a-particles produce aberrations if the induction of aberrations is restricted to occasions on which a particle passes through or in the immediate vicinity of a region in which two chromatid threads are lying in contact. We all exercise some degree of selection, conforming our hypothesis to fit the facts which impress us most, and it may be that I am placing too much emphasis on the quantitative aspects of the dose relations observed with different types of radiation. The fact that Revell has adopted the ' contact first ' hypothesis for chromosome breakage cannot but result in a critical re-examination of the ionizing radiation data, and will, we hope, lead to further experiments designed to decide between the alternative hypotheses. R.B.E. FOR X-RAYS OF DIFFERENT TUBE VOLTAGES In connection with the papers by Kirby-Smith and Daniels, and by Swanson, I would like to add a few remarks concerning the influence of kilovoltage and filtra- tion on the yield of aberrations produced per unit dose. On a number of occasions (Lea\ Gray"^, Spiers^) attention has been drawn to the fact that the mean energy of the secondary electrons generated in tissue remains almost constant over the entire range of photon energies from 25kV to 100 kV, and that on this account it would be surprising if biological efficiency showed any appreciable dependence on X-ray tube kilovoltage over the range 50-200 kV. As is well known, the constancy of the mean electron energy arises from a fortuitous balance between the varying pro- portions of the more energetic photoelectrons and the low energy recoil electrons, and I thought it would be of interest to see how far this balance might be disturbed in the case of a phenomenon such as the production of chromosome structural damage for which we have reason to believe that the contribution of the very slow electrons should be heavily weighted. From a consideration of the relative effective- ness with which X-rays, neutrons and a-particles produce chromosome aberrations in Tradescantia microspores, Lea concluded that in order to break a chromatid thread an electron must [a] have sufficient residual range (0 • 1 [x) to traverse the thread and {b) dissipate at least 0-5 keV of energy in crossing the thread. (Lea^, p. 276). In terms of the energy of the electron as it enters the chromatid thread, this limits chromatid breakage to electrons having energies between about 1 -6 keV and 2 • 8 keV. Having applications to radiation chemistry as well as to chromosome breakage in mind at the time I made my calculations, I have assigned unit efficiency either to (fl) electrons having energy of 0-5keV (Column I of the Table) ; or {b) electrons having energy between 0-5 keV and 3 keV. (Column II of the Table) and zero efficiency to all contributions to the total dose from electrons whose energies lie outside the assigned limits. 278 DISCUSSION It will be seen that the second postulate resembles that of Lea but is not identical with it. Some typical results are given in the table. Allowance has been made for : (/) The low energy electrons fS-rays) produced by the more energetic electrons. (2) The relative contributions of photoelectric absorption and Compton scattering to the total tissue dose, and the distribution of energy among the recoil electrons. (j) The spectral distribution of photon energy from tubes operated at particular kilovoltages and filtrations, where such data were available. Unfortunately no such data are available at present for the radiations used by Kirby-Smith and Daniels. The figures given in columns I and II for lightly and heavily filtered 250 kvp X-radiation therefore refer to the equivalent monochromatic X-radiation. On the basis of either postulate, a significant variation of biological efficiency with X-ray quality in the range 50-200 kV is theoretically to be expected. In the case of postulate (a) the biological efficiency falls steeply from unity at 0-5keV to a local minimum value of 0-135 at 29keV, rises to a local maximum of 0-183 at Table I. — Relative Effectiveness of X- and Gamma Radiation Calculated values of y) compared with experimental data of Kirby-Smith and Daniels. (Chromosome Structural Damage) Column I values of y; for Q_= 0 • 5 keV Column II values of/; for 0-5-2nd generation- t t later generations t Secondary processes Observed effects : Death Chromo- somal dis- turbances Growth inhibition Elimina- tion of genetic changes Sterility Mutations Evolutionary effects Figure 1. Development of a barley seed, irradiated in the dormant state. Observed effects and possible post-irradiation influences and, after further growth, to a mature plant. By sexual reproduction the latter gives rise to second generation seeds, which pass through the same ontogenic cycle, and an unlimited number of later generations then follow. In irradiation experiments designed to produce mutations^, any of these developmental stages can be irradiated. In the present study dormant seeds or seeds at a time just after the start of germination were irradiated. The (biological) observed effects registered at any moment after the irradiation are due to the chemical changes produced at the moment of irradiation (primary act) and to a series of secondary processes following the primary act. These may be studied {a) by a variation of the conditions under which the radiation is performed, and [b) by post-irradiation treatment at different times. 285 THE INFLUENCE OF POST-RADIATION FACTORS ON EFFECTS PRODUCED IN BARLEY When the irradiation conditions are changed {e.g. temperature, oxygen tension, presence of protectors), it is often difficult to prove if there is an influence at the moment of energy absorption or if the treatment affects the secondary reactions. To a certain extent the absence of an effect of such a change, when applied immediately after the irradiation, can prove that the factor has an influence on the primary act. The post-irradiation influences provide information about the secondary processes, which may be chemical, biochemical or biological and are referred to here as post-irradiation effects, or after-effects. The water content of the seeds at the moment of irradiation influences the radio-sensitivity markedly. For all observed effects^- ^ of sparsely ionizing radiations (e.g. X-rays) the damage (or change) is greater when the water content of the seeds is reduced, and smaller when the water content is increased. In the case of such a densely ionizing radiation as fast neutrons Figure 2. Mutation frequencies in per cent per spike progeny. Dormant seeds equilibrated with air oJO, 30 and 100 per cent relative humidity irradiated with different X-ray doses. Water content of the seeds 8-6, 10-5 and 18 ■ 1 per cent, respectively. (around 8 MeV), the water content of the seeds has no effect on the radiation sensitivity. The growth of seeds equilibrated with dry air is four times more sensitive to X-rays than for seeds equilibrated with air of 100 per cent relative humidity. Figure 2 illustrates the influence of the water content on the frequencies of chlorophyll-deficient mutations obtained after irradia- tion with X-rays (180 kV, unfiltered). At low doses the mutation fre- quencies are linear functions of dose, the greatest deviation being obtained for the lowest water content. For the production of mutations an increased water content protects. At higher doses the curves bend, because of the elimination of genetically changed cells which shows a different dependence on the water content of the seeds than does the mutation frequency. The influence of the water content on the radiation sensitivity of the seeds seems to be restricted to the primary reaction, since changes of the water content, immediately after the irradiation but still before the starting of the germina- tion, have no influence on the observed effect (measurements of seedling growth). Effect of germination temperature — Figure 3 shows the relative growth of seedlings, after irradiation of seeds of two different water contents with 286 L. EHRENBERG different doses of X-rays. The seeds were sown at two different tempera- tures, -|-25° and +12° C. The damage developed is greater at the lower than at the higher temperature. This is in agreement with Gelin's"* findings, that the frequency of chromosomal rearrangements in the root tips is about twice as high in the cold as in the warmth. Figure 3 demonstrates Figure 3. Relative growth of seedlings from seeds with 9-3 and 22-0 per cent water, respectively, treated with X-rays and grown at 25° C. ( X ) and 12° C. (o). 28-0 %K0 further what seems to be a general trend in experiments of this type, viz. that the temperature influence is smaller when seeds of a low water content are irradiated. In Figure 4 a similar experiment, comparing temperature 100 20 w eo so fVarmffy, °/cgrou'fh WO Figure 4. Dormant seeds irradiated with X-)ays and neutrons (■ — ' 8 MeV). Relative growth at 12° C. given as function of relative growth at 25° C. 100 80 \60 <3 20 Rfg. o y^ Germ. / ■/ ( Y •/ X / y^ /Doi ^m. ^ ^^^,00^ X 20 VO 60 80 Warmth. % growth 100 Figure 5. Germinating seeds [pre-soaked for 24 hours in water) and dormant seeds irradiated with different X-ray doses. Relative growth at 12° C. given as function of relative growth at 25° C. effects on seeds irradiated with X-rays and neutrons, is shown diagram- matically : the relative growth at the lower temperature ( + 12° C.) is given as a function of the relative growth at the higher temperature (+25° C). In the case of X-rays, a 25 per cent decrease of the growth in the warmth is found to correspond to as much as 70 per cent decrease in the cold (i.e. in accordance with the result given in Figure 3). For neutron irradiation, the influence of the germination temperature is appreciably smaller. In Figure 5 the influence of the germination temperature has been studied for the case of seeds irradiated with X-rays after the start of the germination 287 THE INFLUENCE OF POST-RADIATION FACTORS ON EFFECTS PRODUCED IN BARLEY process and there is no influence of the temperature, contrary to the effect obtained with dormant seeds irradiated simultaneously (see Figure 4). Effect of storage after irradiation — In Figure 6 the relative growth of seeds sown after storage for two weeks at room temperature is given as a function Figure 6. Dormant seeds irradiated with neutrons and X-rays. Relative growth after storage for 6 weeks at 20° C given as function of relative growth obtained after immediate sowing. 20 ¥-0 60 80 % Growth at zero time too of the relative growth of parts of the same irradiated samples sown immedi- ately after irradiation. The storage is found to lead to an increase in damage with X-rays, but not with fast neutrons. The storage effect is relatively greater when seeds of a high water content [i.e. equilibrated with moist air) irradiated and when the seeds are stored at about 25° C. than at about 12°C. When germinating seeds are irradiated and afterwards dried again and then stored, no change of the degree of radiation damage is observed. CONCLUSION Although the data presented (and summarized in Table I) do not suffice to explain the mechanism of radiational action, they provide a foundation for Table I State of barley seeds Dormant, equilibrated to ' dry air moist air Germinating Effect on radiation damage {growth inhibition) of : lowered growth temperature X-rays small increase large increase none neutrons small or none storage {esp. high temp. X-rays small increase large increase none neutrons small or none further work on the biochemical level. The relative absence of post-irradia- tion effects in the case of neutron irradiation indicates that the damage is fixed at the moment of energy absorption, and is in agreement with the finding^ that the neutron damage is confined chiefly to nuclear material. 288 L. EHRENBERG In the case of X-irradiation of dormant seeds, especially those irradiated at high humidities, the development of the lesion can be interfered with. The effects of germination temperature and storage are additive. The relative protection obtained at the higher germination temperature, and which can be measured as a lower frequency of chromosomal rearrangements* as well as a reduced growth inhibition, cannot be explained only as a repair of chromosome breaks. Earlier results^ had already indicated cytoplasmic damage by X-rays, as a cause of growth inhibition. The protective effect of an increase in water content seems to be related to the increased respiration rate. When germinating seeds are irradiated, the damage seems to be caused by quite a different mechanism, and the observed effect cannot be influenced by post-irradiation factors. Compared to the dormant seeds, with a relatively low water content (10-20 per cent) and a low metabolic rate, the germinating seeds contain more water (about 40 per cent) and show a much higher metabolic rate, different enzyme systems being activated. The difference in the action of neutrons and X-rays is well illustrated by barley seedlings ; these show a higher dry-weight after their growth has been inhibited by neutrons than by X-rays^. One cause for this difference might be that neutron damage is primarily located in the roots of the seedling and interferes with water uptake, while the X-rays cause a relatively greater inhibition of the shoot part of the plant. REFERENCES ^ GusTAFSsoN, A. This Symposium. 2 Ehrenberg, L. and Nybom, N. Acta Agr. Scand. 1954, 4 396. ^ Ehrenberg, L. and Andersson, G. Nature, Lond. 1954, 173 1086. * Gelin, O. Agri. Hort. Genet. 1953, 11 66. ^ Unpubl. investigation in co-operation with D. v. Wettstein. 289 u PATHOLOGY OF MICE IRRADIATED AFTER INJECTION OF CYSTEAMINE (P MERCAPTOETHYLAMINE) M. A. Gerebtzoff and Z. M. Bacq, Laboratory of Anatomy and Laboratory of Pathology of Liege University There is no doubt that cysteamine injected into mice before a lethal irradiation, confers protection. But the site of this action is unknown. In what organs does the protection appear ? And does it protect the cells themselves or a factor necessary to their regeneration ? In an attempt to obtain an answer to these questions, we have studied the lesions in three radio-sensitive organs (spleen, thymus and intestinal epithelium) and in the liver ; the importance of this organ in regeneration has been stressed by Maisin and his co-workers. We have compared C57 mice subjected to 700 r with or without an injection of 3mg of cysteamine just before the irradiation. The detailed results are published elsewhere^. Only the main observations will be described here. For every organ listed above, we have measured the degeneration due to the primary action of X-rays, as seen 6 hours after irradiation, and the regeneration observed 3 to 6 days later. {]) Spleen (a) Degeneration— In the lymph nodes of the spleen, the spread of nuclear pycnosis is smaller in mice treated with cysteamine than in control animals. The relation between the intact surface and the total surface of the nodes is, in the mean, 0-151 for untreated mice and 0-371 for treated mice. The difference is quite significative, {b) Regeneration — Four days after irradia- tion, pycnotic nuclei are very rare in treated animals, but still numerous in some nodes of controls. In these, elimination of degenerated cells and regeneration are slower. (2) Thymus (a) Degeneration— The difference in pycnotic areas is not significative. Pycnosis is massive in both groups of mice, {b) Regeneration — Count of mitosis for 10 microscopic fields gives 48 mitosis for controls and 60 for treated animals. But a statistic study of the results shows that this difference is not quite significant. We attribute the uncertain action of cysteamine on thymus to the strong radio-sensitivity of this organ. (3) Intestine {a) Degeneration— There is no difference between treated and untreated mice. {b) Regeneration— The number of mitotic nuclei is 61 for controls, 83 for treated animals. The difference is significant and regeneration is more intense after an injection of cysteamine, 290 M. A. GEREBTZOFF AND Z. M. BACQ^ {4) Liver In this organ, the lesions are predominantly cytoplasmic. Their intensity seems to be related to the mode of fixation of the tissue. When the liver is fixed in formalin, there is no difference between treated and untreated mice : 6 hours after irradiation, the cytoplasm of hepatic cells shows a few small vacuoles ; 4 days later, the vacuolar state is very pronounced. When it is fixed in formalin and picric acid, there are marked differences between treated animals and controls : in these, the vacuolar state is evident 6 hours after irradiation and very pronounced 4 days later ; in cysteamine injected mice, the hepatic cells seem to remain normal. The vacuolar state of the cytoplasm suggested lipidic degeneration. But a histochemical study showed that, if some lipids were present at the begin- ning of the degeneration, they disappeared later on. The lack of vacuolar degeneration in cysteamine-injected animals when the liver is fixed in a liquid containing picric acid may be related to the fact that this acid is an excellent fixation medium for glycogen. It is possible that the linkage between gly- cogen (and other polysaccharids) and proteins is fragile in the liver of irradiated animals. But this fragility is greater in controls than in cysteamine- treated mice : in these, picric fixation is sufficient to maintain the poly- saccharids in the cytoplasm. This observation connects our study with some biochemical researches, particularly those of Fischer. CONCLUSION The injection of cysteamine before irradiation eflfects a direct protection of . liver and spleen. It has a strong accelerating action on regeneration in spleen, intestine and probably thymus. Our observations favour the hypo- thesis that protection by cysteamine is eflfective at the level of some factors concerned with glycogen metabolism. REFERENCE 1 Gerebtzoff, M. a. and Bacq. Z. M. Experientia, 1954, 10 341. 291 SOME FACTORS CONTROLLING THE HAEMATOPOIETIC REGENERATION IN WHOLE BODY IRRADIATED RATS* E. H. Betz Department of Pathology, University of Liege, Belgium, National Foundation for Scientific Research Many papers in the field of radiobiology have shown that there is a close relationship between the survival rate of whole body irradiated animals and their ability to regenerate the destroyed haematopoietic tissues. During the last yeai^s, it has been shown that different chemicals are able to protect animals against a lethal dose of X-rays. Whereas animals submitted to a lethal whole body irradiation do not show any regeneration of their haemato- poietic tissues, there is an extensive regeneration of bone marrow, spleen, lymph nodes and thymus in the animals receiving a protective agent before the lethal irradiation. Qiiite similar results have been described in animals protected with potassium cyanide (Betz^ ; Betz and Fruhling^), gluta- thione (Cronkite et al^), thiourea (Mole^), cysteine (Lorenz^). An identical stimulation of haematopoiesis is observed in animals protected by spleen or bone marrow homogenates injected after X-irradiation (Jacob- son et al^, Lorenz et aP). In earlier experiments, we have shown that, in animals submitted to a lethal dose of X-rays, there is an inhibition of haematopoiesis which is independent of the tissular lesions themselves. It is possible, indeed, by grafting the spleen of an irradiated mouse to a normal one to induce an extensive regeneration of myeloid and lymphoid tissues within the graft. Such a regeneration would never have taken place if the splenic tissue had remained within the radiated body till death. From these observations, we may conclude that the regeneration of the haematopoietic tissues depends not only on tissular lesions, but also on the humoral conditions within the body where such tissues are living. These observations led us to investigate different factors which could possibly control the haemopoietic regeneration of irradiated rats (Betz^). The first factor we started to investigate was the influence of the adrenal cortex. It is well known that whole body irradiation stimulates the activity of the adrenal cortex (Patt et aP). The increased production of adreno- cortical hormones could influence the haematopoietic regeneration ; Baker and Ingle^" have observed indeed an atrophy of bone marrow in rats treated with large doses of cortisone and AGTH. Therefore, the hyper- corticism existing in whole body irradiated rats, could possibly explain the inhibition of the haematopoiesis observed in such animals. * A more detailed paper on this subject has been published in Revue d'Hemaiologie, 1953, 8 489. 292 E. H. BETZ (i) Influence of cortisone and desoxycorticosterone acetate [D.C.A.) on haematopoietic regeneration after a sub-lethal dose of X-rays The experiments are made in order to see whether adrenocortical hor- mones are able to inhibit the haematopoietic regeneration after a sublethal irradiation (500 r). Former experiments have shown that rats submitted to such a dose of X-rays regenerate very quickly the haematopoietic tissues destroyed by the irradiation. Methods : Six groups of 12 albino rats each were used. The animals of the first group are controls. They are irradiated with 500 r (Picker app., 250 kV. 18 mA, filter 0-25mm Cu, F.D. 50cm output 90r/min.) The rats of the three following groups are irradiated in the same way. After the irradiation, the animals are injected daily with cortisone. The doses used were respectively 2-5, 5 and lOmg daily. The rats of the two last groups are irradiated and injected daily with 3 or 5 mg of Figure 1. Femoral bone marrow of the rat, 15 days after 500 r ; extensive regeneration of the myeloid tissues {200 X ) desoxycorticosterone acetate (D.C.A.). In each group, two animals are killed on the sixth, eighth, tenth, twelfth and fifteenth days. Cervical lymph nodes, spleen, thymus, femoral bone marrow are fixed in Helly's fluid, and studied histologically. The control rats, which have been irradiated only, have an extensive and early regeneration of thymus, spleen, lymph nodes and bone marrow. The repair of the destroyed tissues starts on the fourth day after irradiation. The daily injection of 2 • 5 mg of cortisone does not modify in any way the histological picture of the haematopoietic tissues. Daily injection of 5mg of cortisone acetate to rats irradiated with 500 r does not influence the regeneration of spleen, lymph nodes, or bone mar- row ; the regeneration of the thymus only is inhibited by such a dose of cortisone. On the contrary, the injection of lOmg of cortisone daily does block the regeneration of spleen, lymph nodes and bone marrow as well as the repair of thymus. The histological picture of the haematopoietic 293 THE HAEMATOPOIETIC REGENERATION IN WHOLE BODY IRRADIATED RATS tissues of these rats is quite similar to that observed in rats receiving a lethal irradiation of 800 r. The daily injection of 3 or 5mg of D.C.A. does not modify the regeneration of haematopoietic tissues. {2) Influence of adrenalectomy on the haematopoietic regeneration Methods : four groups of rats were used. The animals of the first group are irradiated with a lethal dose of X-rays (800 r) . The rats of the second group are adrenalectomized and 4 days later submitted to 800 r. The animals of the third group and the fourth group are adrenalectomized and irradiated. In addition, they were supplemented with a dose of D.C.A. (3 mg daily) or cortisone (2-5mg daily) too small to influence the haematopoietic activity. The haematopoietic tissues are taken and studied histologically between the fourth day and the moment of death. 4^1^%... Figure 2. Femoral bone marrow of the rat, 15 days after 500 r and injection of cortisone acetate ( 1 0 mg daily) : inhibition of the myelopoiesis {200 X ) The control rats receiving 800 r died between the sixth and the tenth day. They never showed any repair of the haematopoietic tissues destroyed by the irradiation. The adrenalectomized rats were very sensitive to a lethal dose of X-rays (800 r) and died before any regeneration could possibly take place (fourth day). In the adrenalectomized group of rats supple- mented with a dose of D.C.A. (3 mg daily) or cortisone (2 • 5 mg) the resistance appeared to be normal. Some of them survived for 10 days. Although any possibility of hypercorticism was excluded, no haematopoietic regeneration has been observed in these rats. The results of these experiments are not conclusive. Should an increased adrenocortical secretion be able to inhibit the haematopoietic regeneration, there is no doubt that the hypercorticism following a lethal whole body irradiation is not the only factor involved in the inhibition of haematopoiesis. The adrenalectomy does not succeed, indeed, in stimulating the haemato- poietic regeneration of irradiated rats. Some other factors must be involved in the regulation of this phenomenon. 294 E. H. BETZ Recently Selye^i ^^s shown that somatotrophic hormone is able to counteract the catabolic effect of protein which normally occurs after a stress or an injection of cortisone. On the other hand, it is known that the production and differentiation of blood cells is closely related with the protein metabolism. Therefore, we studied the effect of somatotrophic hormone on the haematopoietic regeneration of irradiated rats. We com- pared the results with thoic obtained by using other substances such as testosterone propionate and vitamin Bj., whose effect on protein anabolism is also well known. (5) Influence of somatotrophic hormone on the haematopoietic regeneration Methods : Three groups of rats were studied, all irradiated with a lethal dose of X-rays (800 r) . The first group is used as control. The animals of the second group are irradiated and injected daily with 5I.U. of growth hormone. The rats of the Figure 3. Femoral bone marrow of tlie rat. 10 days after 800 r .• almost no regeneration of t/ie marrow has taken place {200 X ) third group are adrenalectomized before the irradiation. Afterwards, they are injected daily with 2-5mg of cortisone and 5I.U. of growth hormone. The haematopoietic tissues are studied histologically. In the control group, all the animals died within the eleven days following the irradiation, without showing any sign of haematopoietic regeneration. The rats irradiated and injected with growth hormone behaved like the controls ; they died between the fourth and the tenth day without regenera- tion of their haematopoietic tissues. The adrenalectomized rats injected with costerone and somatotrophic hormone proved more resistant to a lethal dose of X-rays ; about 25 per cent of the individuals of this group survived, showing an extensive regeneration of lymph nodes, spleen and bone marrow. In the bone marrow and spleen, the differences between control rats and treated rats are striking. In the control rats, the bone marrow is congested and oedematous; the myeloid cells have almost all disappeared ; the spleen is 295 THE HAEMATOPOIETIC REGENERATION IN WHOLE BODY IRRADIATED RATS small and lymphoid, or myeloid structures are no longer present. In the treated rats, the bone marrow is packed with myeloblasts and myelocytes and is even more cellular than the normal marrow. In the spleen the regeneration of lymphoid and myeloid cells is conspicuous ; numerous myeloid foci are to be seen in the red pulp. Influence of testosterone propionate and vitamin B^2 ^" ^^^ haematopoietic regeneration The experiments were made on the same scheme as the one used for the assay of growth hormone. The two substances used in this experiment have the same action as growth hormone. They are quite inactive when injected to normal irradiated rats, while they do, on the contrary, stimulate the haema- topoietic regeneration of adrenalectomized animals ; here again, about 20 per cent of the individuals survived a lethal whole body irradiation. Figure 4. Femoral bone marrow of the rat 10 days after adrenalectomy, 800 r and injection of growth hormone and cortisone acetate (2 • 5 mg daily) . Regeneration of myeloid tissue is conspicuous. {200 X ) . SURVEY From these experiments, we may conclude that substances stimulating the anabolism of proteins are active in stimulating the haematopoietic regenera- tion of irradiated rats provided they are given to adrenalectomized animals. They are quite inactive in normal rats. The results indicate that, as far as haematopoiesis is concerned, there is an antagonistic effect of adrenal hormones and substances like growth hormone, testosterone propionate and vitamin B^g- It is likely that such an antagonism is related to the action of these substances on the metabolism of proteins. In whole body irradiated rats, hypercorticism plays a role in the increased catabolism of proteins which is observed in animals. Betz and Jehotte^^ have shown that a lethal dose of X-rays produces in the rat a quick reduction of food intake, a reduction of nitrogen excretion, together with a negation 296 E. H. BETZ of the nitrogen balance. After five days, a secondary increase of the nitrogen excretion takes place with a still more marked negation of the nitrogen balance. Adrenalectomy prevents the secondary increase of nitrogen excre- tion although the nitrogen balance remains negative. Our results confirm the relationship between the regeneration of blood cells and the metabolism of proteins. The disturbances of this metabolism which follows a whole body irradiation are poorly understood. Besides the hyperactivity of the adrenal cortex, there are other unknown factors interfering with the nitrogen metabolism. A better knowledge of these factors would probably be a great help in the discovery of the factors which control the haematopoiesis of the irradiated body. REFERENCES 1 Betz, E. H. C.R. Soc. Biol. 1950, 144 1439. 2 Betz. E. H. and Fruhling, L. C.R. Soc. Biol. 1940, 144 1013. 3 Cronkite. E. P.,Brecher, G.and Chapman, W. H. Proc.Soc.exp. hiol. 1951.76396. * Mole. R. H. Colloquium centenaire de la decouverte du Radium, Paris, 1950. 5 Lorenz, W. Strahlentherapie, 1952, 88 190. sJacobson, L. O., Simmons, E. L., Marks, E. R., Robson, M. J., Bethard, W. F. and Gaston, E. O. J. Lab. Clin. Med. 1950. 35 746. Jacobson, E. O., Simmons, E. L., Marks, E. K., Gaston, E. O., Robson, M. J. and Eldredge, J. H. J. Lab. Clin. Med. 1951, 37 683. ' Lorenz. E.. Uphoff, D., Reid, T. R. and Shelton, E. J. Nat. Cancer Inst. 1951, 12 197. 8 Betz, E. H. Ann. Endocr. 1951, 12 801. 9 Patt, H. M., Swift, M. N., Tyree, E. B. and Johns, E. S. Amer. J. Physiol. 1947, 150 480. 1" Baker, B. E. and Ingle, D. J. Endocrinology, 1948, 43 422. 11 Selye, H. Brit. med. J. 1951, 10, 263. 12 Betz, E. H. and Jehotte, J. C.R. Soc. Biol., in press. 297 ACTION DE LA CYSTEAMINE SUR LES TUMEURS GREFFEES, IRRADIEES IN LOCO S. Neukomm*, Mme L. Peguiron* et A. HerveI * Service des Recherches Experimentales du Centre Anticancereux Romand, Lausanne. I Service de Radiotherapie et Centre Anticancereux, Universite de Liege. Depuis la decouverte par BAcq et ses collaborateurs^ de I'effet de radio- protection de la cysteamine (betamercaptoethylamine ou [3-M) chez la souris, un grand nombre de travaux ont ete consacres a I'etude des amines soufrees d'ou il ressort que la ,3-M et son derive oxyde sont a I'heure actuelle les protecteurs les plus efficaces puisqu'ils protegent a plus de 95 pour cent contre 700 r et entre 30 et 90 pour cent contre l.OOOr^. La grande efficacite du produit alliee a une faible toxicite (la dose mortelle 50 pour cent etant d'environ 0,35 mg par gramme de souris) a incite les chercheurs beiges a I'utiliser en clinique pour le traitement du mal des rayons dont I'origine se trouve en partie liee a Taction indirecte des hautes doses d'irradiation^. Administre apres la seance d'irradiation par I'injection intraveineuse lente, Herve'* a montre qu'une seule injection de 200 mg etait suffisante dans beaucoup de cas pour faire disparaitre les symptomes majeurs du mal des rayons. Nous avons recherche si la [3-M injectee par voie intraperitoneale etait capable de proteger une tumeur irradiee localement. II serait interessant d'autre part de determiner si la dose de ^-M, utilisee cliniquement contre le mal des rayons est aussi susceptible de proteger la lesion irradiee. C'est pour aborder ces problemes que I'un de nous (A. H.) entreprit des juillet 1952, au Centre anticancereux de Lausanne, la premiere serie d'ex- periences qui fait I'objet de la presente communication. MATERIEL ET METHODES Nous avons utilise pour ces recherches des souris de notre elevage (souche E et souche R^) males ou femelles ( Tableau I) greffees avec la tumeur de Caspari (adenocarcinome de la mamelle). La greffe est faite par injection sous-cutanee, au moyen d'lm trocart, de fragments de la grosseur d'une tete d'epingle. Le developpement apparent de la greffe commence 10-15 jours apres I'injection. Quelquefois, deux, voire trois, tumeurs se developpent simultanement, ce qui explique que le nombre de tumeurs observables pent etre plus grand que le nombre d'ani- maux greffes. L'accroissement de chaque tumeur est mesure en determinant au moyen d'un pied-a-coulisse le petit [a) et la grand [b) diametres. Le volume de ces tumeurs ovoides est directement proportionnel au produit a~b. Pour la tumeur de Caspari, le facteur de proportionnalite est 1,06, done negligeable. C'est-a-dire que la racine cubique du volume calcule [(a-^)^'''*] est tres sensiblement egale au diametre moyen de la tumeur. L'experience a 298 S. NEUKOMM, MME L. PEGUIRON ET A, HERVE l5 Si O 8 CO OQ. + Apres 1 1 1 1 M M M o 1 i o' o 0,100 CO. + 1 1 1 1 o M M M -4' o 1 1 M 1 1 1 O 00 o" in in o" 1 1 ,M o CM o" lO o" CM o" CO CO o" -r 1500 r + 1 MM M M M in o M M M 1 O o CO *^ o in o CO. + CM o MM o' CM_^ i 1 1 4, M M -H M M in ■n o' m o in CO 5 ' M -^ 1 o O M +1 M 1 CO o" o" o m o" in UO CO o CO. + o o" MM o o 1 ° M M CO r-- M M M 1 in in -+- o g 1 CM il 1 1 4^ M M 1 CM o" o % to CM m CO iction proteclrice de la {i-AI stir des tumeurs irradiees 1 bO ^ 1 CO CO X X 4x3mg 4x3mg 4x3mg bo bo bo bo esse XX XX 1 1 * a 1 s tC 'O -t CO r~- i^ ^ -f CO CM — Oi •+ CO CO O CO 'S fe3 cr> to tr> to Of VD ID t^ r^ fa] Of ^ o ^ — ' CM CO -* — <>J CO 'f lO «; — ' cvj CO ^ in 1^ r^ •-^i !:? 5 w 299 ACTION DE LA CYSTEAMINE SUR LES TUMEURS GREFFEES IRRADIEES, IN LOCO montre que raccroissement du diametre moyen suit une courbe lineaire en fonction du temps, tout au moins pendant la plus grande partie de revolution apparente des tumeurs {Figure 1). La vitesse de croissance est alors deter- minee par la pente de la droite interpolee qui relie les divers points des courbes d'evolution du diametre moyen en fonction du temps ; elle s'exprime en millimetres par jour. Les irradiations sont faites avec un appareil de contact-therapie en choisis- sant un localisateur approprie pour chaque tumeur*. Les tissus sains du voisinage sont proteges par des lames de plomb percees d'un trou de diametre correspondant a celui des tumeurs. Pendant I'irradiation, I'animal i Croissance tumeurs tB-mercaptoefhylamine I J I I L Figure 1 y 8 12 76 20 8¥ 28 32 jours Figure 2 Jmgr I /h- p-f1ercaptoethy/amine m 16 j'mqp 7^ 1 18 ISOOr 19Jui7/et1952 E 17 T 18 79 ' 20Decembre7952 3000 T 7 5 DOT <^mqp m _L 22 23,^„„ 2¥ 7500T 3000 V 25Jar)i^/er795¥ n'a pas besoin d'etre tenu, car il a re^u prealablement (30-45') une injection intraperitoneale de Numal 'Roche' (0,2cm^ sol. a 1 pour cent) qui provoque une narcose d'une heure environ. La ^-M a ete injectee 2h, 1 h et |h avant, ou \\\ apres I'irradiation par voie intraperitoneale a la dose de 3 ou de 4mg par animal. Les doses de roentgen par tumeur ont ete de 1.500 et 3.000. Dans les series / et //, les animaux ont re^u de la fi-M pendant 4 jours consecutifs, alors que dans la serie ///, une seule injection a ete faite avant ou apres I'irradiation {Figure 2). * Nous remercions M. le Prof. Babaiantz, chef du Service universitaire de radiologie, pour I'interet qu'il a bien voulu porter k ces travaux et les appareils qu'il a aimablement mis a notre disposition. 300 S. NEUKOMM, MME L. PEGUIRON ET A. HERVE RESULTATS Les resultats de nos trois series de recherches, exprimes en accroissement du diametre moyen (mm/jour), sont condenses dans le Tableau I. On constate que I'injection de [^-M a des animaux porteurs de tumeurs non irradiees (temoins) ne provoque pas de modification significative de la vitesse de croissance des tumeurs ; le chiflfre plus eleve de la moyenne ponderee com- parativement aux animaux non traites est du a I'absence d'un lot comparable dans la serie /// {Figure 3) . Pour les animaux dont les tumeurs ont re^u 1.500r, la [^-M injectee avant ou apres I'irradiation ne semble pas apporter de modifications au developpe- ment des tumeurs, tout au moins si Ton en juge sur la valeur des moyennes ponderees. II est probable toutefois que ce resultat soit la consequence d'un effet inhibiteur insuffisant de la dose de 1.500r qui n'est pas capable de 'reveler' la protection. Cette opinion est justifiee par le fait que I'applica- tion d'une dose de 3.000 r chez des animaux traites de la meme maniere que b So 0,8 0,7 0,6V 0.5 0,^\- 0,J 0,2- 0,1- 0 Te'moin 0 ' I^E A^ ^JJM at/ant RX P^+j3fi apres RX 0 = sons protecfeur 7500^ n^m precedemment n'entraine pas une diminution de la vitesse de croissance aussi importante que chez les animaux non traites par ^-M. La diflference entre les vitesses de croissance des tumeurs chez des animaux ayant regu de la ^-M avant ou apres I'irradiation et chez des animaux n'ayant pas regu de p-M est statistiquement significative. Tout se passe comme si, chez les animaux traitesa la [3-M, I'efficacite de la dose de 3.000 r avait ete diminuee de moitie, c'est-a-dire comme si Ton avait irradie avec une dose de 1.500r seulement. Nos resultats ne permettent pas d'etablir avec precision une relation entre le moment de I'administration de la fj-M et I'importance de Teflfet protecteur qu'elle exerce {Figure 4). COMMENTAIRES Chez la souris, I'injection intraperitoneale de [^-M protege les tumeurs irradiees localement par une forte dose de rayons X. Dans les experiences / et //, il faut noter que des injections ont aussi ete faites le jour precedant I'irradiation et les jours suivant I'irradiation {Figure 2). A protection obtenue dans ces cas ne semble pas influencee par les injections faites a des moments eloignes de I'irradiation mais bien par la seule injection 301 ACTION DE LA CYSTEAMINE SUR LES TUMEURS GREFFEES IRRADIEES, IN LOCO precedant d'une a deux heures le moment de I'irradiation. II apparait comme certain que Taction de la [3-M s'exerce dans un laps de temps tres court (de I'ordre d'une heure). Ce fait peut etre mis en correlation avec le metabolisme et I'elimination rapides du produit-"^. II faut noter que dans nos experiences, nous avons utilise une dose considerable de p-M ( 1 50 mg/kg) . Cette dose correspond a celle qui offre la meilleure protection contre le rayonnement X aux animaux irradies in toto par une dose de 750 r (100 pour cent mortelle pour tous les temoins'^). La dose active en clinique contre le mal des rayons est de 3 mg/kg, c'est- a-dire 50 fois moindre que celle utilisee dans nos experiences. L'effet d'une I 0,7 o,e 0,5 0,H 0,3 0.2 0,1 SonsjS-M Avecfi-M Avec ^-M AvanfX Apres X Voleurs moyennes ][ 1-Z \ avanf irradiation M 15 avant ou apres irradiation _L Figure 4. Effet protecteur de la ^-AI sur tumeurs irradiies 1500 3000 I _L 0 1500 3000 1500 3000 Dose telle dose chez I'animal n'a pas ete etudie. II n'est, par consequent, pas possible de transposer sur le plan clinique les conclusions de ce travail experimental. Les observations cliniques faites par I'un de nous et rapportees ailleurs^'^ n'ont jamais montre une resistance accrue de la lesion iiradiee. II semble certain que I'injection d'une petite dose de ^-M effectuee plus d'une heure apres I'irradiation soit incapable de modifier Taction des rayons X sur les cellules tumorales. Dans le cadre de nos experiences il est difficile d'apprecier quantitative- ment la protection chimique. Nos observations nous conduisent a admettre qu'un test quantitatif depend principalement : {a) de la dose de rayons utilisee pour ' reveler ' la protection, cette dose dependant en partie du volume des tumeurs a irradier ; {b) de la dose de protecteur administree ; {c) du moment de Tadministration du protecteur par rapport au moment 302 S. NEUKOMM, MME L. PEGUIRON ET A. HERVE de I'irradiation, ce facteur 'temps d'action du protecteur' etant etroitement lie au metabolisme du produit (destruction et elimination par I'organisme). Le role du volume des tumeurs dans le choix de la dose de roentgen a donner est particulierement evident dans I'experience /, lot 3 [Tableau I). Dans ce cas, la vitesse de croissance des tumeurs irradiees par 1500r n'a pas subi de modification par rapport a celle des temoins non irradies. Or les animaux de ce lot avaient tons des tumeurs tres grosses ; il est done tres vraisemblable cjue la dose choisie n'a pas ete suffisante povu' provoquer un arret de croissance rigoureusement comparable a celui obtenu dans le lot 4. CONCLUSIONS ET RESUME (7) La [3-M seule ne semble pas capable de modifier la croissance des tumeurs non irradiees. {2) La [3-M injectee a forte dose par voie intraperitoneale a des souris porteuses de tumeurs greffees protege les tumeurs centre I'irradiation locale. La protection est obtenue lorsque I'injection est relativement proche du moment de I'irradiation. La protection se manifeste d'autant mieux que la dose de rayons administree est plus grande, tout au moins dans les limites choisies pour nos experiences. {3) Les doses de [3-M utilisees dans nos experiences preliminaires sont 50 fois superieures a celles qui, en clinique, ameliorent les symptomes du mal des rayons. II reste a voir si les doses cliniques de ^-M et les conditions dans lesquelles elles sont administrees ont une incidence sur la regression des tumeurs irradiees. ADDENDUM This work was carried out in the Experimental Research Service of the Centre Anticancereux Romand at Lausanne because we knew the details of growing Caspar! tumour specially studied by Neukomm. The linear growth of tumour as a function of time seemed to be a good test of observation. Nevertheless, in this work, we have neglected the concentration of cyste- amine in the tumour itself Thanks to the use of labelled sulphur, Verly et al were able to follow the metabolism of the cysteamine and have showed that the concentration of this substance during the first twenty-four hours was larger in the liver and the intestine than in the other tissues. A similar research programme would be very interesting for ' spontaneous ' or 'experimental' tumours. It would be possible, in particular, to deter- mine the final protecting effect upon certain types of lesions by concentrating a more important quantity from the injected cysteamine. We intend to study this problem. REFERENCES ^ BAcq, Z. M., Herve, a., Lecomte, J., Fischer, P.. Blavier. J., Dechamp, G., Le Bihan, H. et Rayet, P. Arch. Intemat. Physiol. 1951, 59 442. ~ Bacq,, Z. M. et Herve, A. Bull. Acad. Royale Med. de Belgique, 1952, 17 13. 3 Herve, A. Revue medicale de Liege, 1952, 7 276. * Herve, A. et Bacq, Z. M. J. Radiol, et Electrol. 1952, 33 651. 5 Verly, W., Bacq, Z. M., Rayet, P. et Urb.'un, M. F. Biochimica et Physica Acta, 1954, 13 233. 303 ETUDE HISTOLOGIOUE DE LA REPARATION INTESTINALE CHEZ DES RATS IRRADIES SOUS DIVERSES CONDITIONS DE PROTECTION H. Maisin et C. Fievez Laboratoires de Recherches pour le protection de la population civile annexes a I'lnstitut du Cancer de Louvain Nous nous sommes surtout attardes a I'etude comparative des lesions et de la regeneration de I'intestin grele de rats irradies d'une fa^on standard et proteges de diverses manieres. Nous avons choisi cet organe comme premier but d'etude, car c'est lui que nous rendons responsable des morts constatees chez les animaux les premiers jours qui suivent I'irradiation. En effet, ceux-ci presentent une diarrhee extremement abondante et les seuls animaux qui survivent 4 a 5 jours apres I'irradiation sont ceux qui, pour une raison ou I'autre, protection mecaniquei- 2' ^ ou chimique par la betamercaptoethylamine (becaptan) ^^ ^ n'ont pas presente cette diarrhee. Notre materiel d'experimentation est represente par des rats blancs homozygotes. Tons nos animaux ont re^u 700 r sous 200 kV, |mmCu, 1 mm d'Al, a une distance F.P. de 55 cm. Notre appareil un Maximar de 250 kV de la General Electric Co. debite 34r/minute mesures dans I'air a I'integrateur Victoreen. Certains n'ont pas ete proteges (rats controles), d'autres ont ete proteges par un ecran de plomb de 5 mm d'epaisseur et de 2 cm de cote, place soit sur leur region sous-ombilicale^' ^ entrainant ainsi la protection d'une partie du gros intestin et des anses greles, soit sur leur region sus-ombilicale^- * entrainant alors la protection de la grosse partie des anses greles, d'autres enfin ont re(;u directement avant I'irradiation une injection intraperitoneale de 7,5 mg de becaptan par lOOg de poids sans aucune autre protection. Nous diluons lOmg de becaptan dans 1 cm^ d'eau distillee. Nous avons egalement injecte du becaptan a des animaux non irradies, mais ces injections nous semblerent sans effet. Nous avons sacrifie dans chaque serie au moins deux rats de 24 en 24 heures et nous nous limiterons aux resultats observes les 10 premiers jours qui suivent I'irradiation, c'est-a-dire pendant la periode dite intestinale et les jours qui lui font immediatement suite. Les pieces ont ete conservees 3 jours dans le Bouin et colorees a I'hemateine- eosine-safran. Nous avons essaye de chiffrer les lesions et de juger de la regeneration en comptant les mitoses des glandes de Lieberkuhn dans des conditions stan- dardisees (grossissement 800), leur nombre chez les rats normaux est en moyenne de 5 par champ. Chez les rats non irradies injectes de becaptan le nombre de mitoses ne varie pas, que cette numeration ait lieu 3 heures, 6 heures, 12 heures ou plusieurs jours apres I'irradiation. 304 H. MAISIN ET C. FIEVEZ I. RATS CONTROLES 1 jour apres V irradiation — repithelium de surface est relativement peu lese, la bordure ciliee est intacte dans la plus grande partie de sa longueur. Les glandes de Lieberkuhn sont nettement abimees. Elles paraissent tout d'abord rarefiees et la tunica propria plus lache. Leurs noyaux presentent des lesions varices de degenerescence. Les granulations des cellules de Paneth sont tres visibles, mais souvent liberees dans la lumiere glandulaire par effraction du pole secreteur. Le nombre de mitoses, au niveau des glandes de Lieberkuhn, est en moyenne de 1,25 par champ. 2 jours apres V irradiation — repithelium de surface presente des lesions plus serieuses ; les noyaux sont moins nombreux beaucoup plus irre- guliers, parfois tres volumineux. En outre, leur situation dans la cellule n'est plus aussi strictement polaire. Certaines cellules, rares a vrai dire, en sont depourvues. La bordure ciliee est respectee dans sa plus grande partie. Les glandes de Lieberkuhn montrent une anisonucleose de plus en plus nette. Les limites cellulaires sont peu precises et le protoplasme en est plus granuleux. Les grains des cellules de Paneth sont deverses presqu'en totalite dans la lumiere glandulaire et par consequent de moins en moins nombreux dans le protoplasme cellulaire. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 0,6 par champ. 3 jours apres Virradiation — la hauteur des villosites est reduite et I'epithelium de surface est de plus en plus aplati. L'anisonucleose et les alterations chromatiniennes sont de plus en plus manifestes. Les protoplasmes sont vacuolises. La bordure ciliee disparait a beaucoup d'endroits. Les glandes de Lieberkuhn montrent une lumiere nettement plus grande par aplatissement de I'epithelium glandulaire. Certaines glandes pre- sentent meme un epithelium endotheliforme. Les cellules de Paneth ne sont plus reconnaissables. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 0,38 par champ. 4 jours apres Virradiation — les villosites sont de plus en plus aplaties et I'epithelium de surface est tres altere, la bordure ciliee a disparu. Bon nombre des cellules presentent des vacuoles tres grosses, uniques ou multiples, refoulant le noyau ou ce qui en reste a un pole. La plupart des glandes de Lieberkuhn sont dans le meme etat que le jour precedent, mais par endroits apparaissent de rares glandes de Lieberkuhn mieux formees, avec un epithelium plus regulier cylindrique semblant recuperer une aptitude fonctionnelle, des cellules de Paneth sont a nouveau bien reconnaissables. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 1,15 par champ. Celles-ci n'existent en fait qu'au sein des glandes qui proliferent. 5 jours apres Virradiation — I'epithelium de surface redevient cylindrique, toutefois, les villosites restent encore assez effacees. A certains endroits la bordure ciliee reapparait. Les glandes de Lieberkuhn en proliferation sont de plus en plus nombreuses a la lumiere toujours plus elargie tandis que les glandes lesees se font de 305 X ETUDE IIISTOLOGIQUE DE LA REPARATION INTESTINALE CHEZ DES RATS plus en plus rares. Des cellules de Paneth existent dans la plupart des glandes neoformees. Le nombre de mitoses au niveau des glandes de Lieberkuhn tend a redevenir normal, il est actuellement en moyenne de 3,7 par champ. 6 jours apres Virradiation — I'epithelium intestinal et la hauteur des villosites redeviennent normaux. La densite des glandes de Lieberkuhn et leurs dimensions paraissent normales. Toutefois elles restent plus allongees, leur lumiere est presque fermee. Le nombre des cellules de Paneth et leurs granulations sont normalles. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 5,1 par champ. Le dernier rat de notre serie a ete sacrifie le dixieme jour ; la muqueuse intestinale y est redevenue entierement normale. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 6,5 par champ. II. RATS DONT LA REGION SOUS-OMBILICALE A ETE PROTEGEE Dans ce cas, la plus grande partie des anses greles n'a pas ete protegee et les lesions constatees a ce niveau sont superposables a celles observees chez les rats controles. III. RATS DONT LA REGION SUS-OMBILICALE A ETE PROTEGEE Dans ce cas la plus grande partie des anses greles a ete protegee mecanique- ment et ce sont ces parties que nous allons decrire. 1 jour apres r irradiation — contrairement a ce qu'on serait en droit d'attendre, I'intestin grele protege presente apres 24 heures, exactement les memes lesions que I'intestin grele des rats controles, memes alterations nucleaires, meme rarefaction des glandes de Lieberkuhn, meme sideration des mitoses. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 1,1 par champ. 2 jours apres rirradiatioti — les lesions constatees apres 24 heures n'ont pas evolue comme chez les controles. Si I'aplatissement des villosites est assez manifeste, par centre au niveau de I'epithelium de surface, les noyaux restent polaires et les alterations nucleaires sont relativement peu pro- noncees. Au niveau des glandes de Lieberkuhn, existent des alterations nucleaires certaines, mais I'epithelium glandulaire presente une activite mitotique considerable. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 6 par champ. Les cellules de Paneth sont intactes. 3 jours apres Virradiation — I'epithelium de surface est strictement normal a certains endroits, cependant sur de larges etendues, il presente des lesions comparables a celles decrites chez les animaux controles homologues, noyau plus central — anisonucleose — epithelium plus plat — perte de la bordure ciliee. Les cellules des glandes de Lieberkuhn sont cylindricjues, tassees les unes contre les autres, les lumieres glandulaires sont plus elargies et leurs lesions peu visibles. Les cellules de Paneth sont nombreuses et nettement visibles. 306 H. MAISIN ET C. FIEVEZ Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 7 par champ. 4 jours apres V irradiation— %i I'epithelium de surface est redevenu normal dans sa plus grande partie, il reste encore des endroits cependant, a epithe- lium lese. L'aspect de la sous-muqueuse de I'intestin grele reprend une apparence normale. Seule I'activite mitotique y est plus grande. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 8,4 par champ. Dans les jours qui suivent, I'intestin redevenu completement normal ne differe de I'intestin non irradie que par une activite mitotique plus intense. Celle-ci ne se regularise que vers le dixieme jour. IV. RATS INJECTES IMM EDI ATEMENT AVANT L ' I RR ADI ATION DE 7,5 MG DE BECAPTAN PAR 1 00 G DE POIDS 1 jour apres V irradiation — on peut dire que les lesions sont strictement sem- blables a celles observees chez les controles homologues. Les deux preparations peuvent etre superposees — meme integrite de I'epithelium de surface— memes lesions lieberkuhniennes. Le nombre de mitoses au niveau des glandes de Lieberkuhn est en moyenne de 1,4 par champ. 2 jours apres V irradiation— V €Tp'\^\\€\i\\m. de surface presente les memes alterations que les homologues controles. Les glandes de Lieberkuhn sont nettement lesees. Leur epithelium est aplati et la lumiere de ce fait tres visible. II montre de I'anisonucleose et des alterations nucleaires diverses. Les cellules de Paneth sont tres visibles. Le nombre de mitoses au niveau des glandes de Lieberkuhn est redevenu en moyenne de 1,7 par champ. 3 jours apres V irradiation— V CT^ithtWnm de surface peut toujours etre compare a celui des animaux homologues proteges mecaniquement. Quant aux glandes de Lieberkuhn, elles presentent une tendance nette a la regeneration. Geci peut etre objective par le nombre de mitoses qui est en moyenne de 5,2 par champ. 4 jours apres Virradiation — I'epithelium de surface reprend un aspect normal. II redevient plus cylindrique. II peut etre compare a celui des animaux homologues proteges mecaniquement et a celui des animaux controles du sixieme jour. Les glandes de Lieberkuhn comme celles des animaux proteges mecani- quement presentent une activite regeneratrice tres intense. Le nombre de mitoses y est de 7,4 par champ. 5 jours apres Virradiation — comme pour les animaux homologues proteges mecaniquement I'intestin grele reprend un aspect normal. L'activite mito- tique des glandes de Lieberkuhn est toujours tres intense. Nous avons denombre 9,3 mitoses par champ. Le sixieme jour apres I'irradiation l'activite mitotique au niveau des glandes de Lieberkuhn est toujours elev^e. Nous avons compte en moyenne 8,4 mitoses par champ. A partir du septieme jour l'activite mitotique au niveau des glandes de Lieberkuhn redevient normale. 307 ETUDE HISTOLOGIQ^UE DE LA REPARATION INTESTINALE CHEZ DES RATS Nous avons cru bon synthetiser nos numerations de mitoses dans un diagramme {Figure 1) qui nous permettra d'etablir des comparaisons et de tirer des conclusions. Rappelons que chez le rat normal le nombre de mitoses par champ microscopique au grossissement 800 est de cinq. En resume II a ete montre dans ce laboratoire que les rats dont I'intestin grele a ete protege mecaniquement^- -• * et que ceux qui ont re^u du becaptan^-^ ne presentent pas les diarrhees, habituelles chez les animaux controles ou dont I'intestin n'a pas ete protege. Actuellement, nous pouvons a la lumiere de nos constatations histologiques supposer que I'absence de diarrhee est liee a une regeneration plus rapide 70 9- 3 7 1 ^ 700 T — — Injection ME A a\/ant RX — Protection region sus-ombilica'le — Controle S 10 Jours apr'es f>X Figure 1. £tude histologique reparation intestinale chez rats irradies sous diverses conditions protectrices de I'intestin grele, regeneration obtenue tout aussi bien avec I'ecran de plomb qu'avec le becaptan. Rappelons que le premier jour apres I'irradia- tion, les lesions sont semblables chez tous nos animaux. Chez les animaux controles, la regeneration intestinale ne se produit qu'a partir du cinquieme jour. II en est de meme pour les parties d'intestin non protegees mecaniquement. La reparation presque complete ne s'ob- serve qu'au sixieme jour et les lesions sont maximales les troisieme et quatrieme jours. Chez les animaux dont Tintestin a ete protege mecaniquement ou qui ont regu une injection de becaptan immediatement avant I'irradiation, la regeneration debute, chez les premiers des le deuxieme jour, chez les seconds des le troisieme jour et la reparation presque complete est realisee des le quatrieme jour. Le maximum des lesions s'observe pour les rats proteges mecaniquement le premier jour, pour les rats injectes le deuxieme jour. Ceci est en contradiction avec les resultats obtenus par Van Lancker^ dans notre laboratoire. II croyait que I'intestin des animaux ayant re9u du becaptan avant I'irradiation ne se comportait pas differemment de celui des animaux controles. 308 H. MAISIN ET C. FIEVEZ CONCLUSION En faisant une revue de la litterature actuelle a ce sujet on pent voir que d'autres experiences avec d'autres substances donnent des resultats analogues. Ainsi Williams et de Long" trouvent au niveau de I'intestin de rats irradies par 700 r, une activite mitotique augmentee des la 73eme heure suivant I'irradiation apres leur avoir injecte du glutathion reduit ou du para-aminopropiophenone. Cronkite, Brecher et Chapman" trouvent cette stimulation mitotique apres injections de glutathion, au niveau de la rate, de la moelle et du thymus de souris irradiees. Des faits semblables sont decrits par Rosenthal, Goldschmidt et Picker- ing^ avec la cysteine, par Betz et Fruhling avec le cyanure de potassium** qui, tout en n'evitant pas les radiolesions des organes hematopoietiques, en rendent cependant leur reparation beaucoup plus rapide. Tons ces auteurs insistent done sur cette regeneration plus rapide. Doit-on assimiler Taction protectrice du becaptan (ou eventuellement d'autres substances) au niveau de I'intestin a celle realisee par un ecran de plomb ? L'histologie elementaire tend a nous faire repondre a cette question par I'affirmative. En effet, a part le leger retard de regeneration observee chez les animaux injectes, les lesions et les reparations sont identiques. Les deux types de protection realisent les memes aspects morphologiques, comme si tons les deux gardaient a un plus grand nombre de cellules leurs potentialites reproductrices avec comme consequence une reparation plus rapide et plus complete. Cependant, malgre cette identite morphologique il est peu probable et peu logique d'admettre que cette protection s'exerce au meme niveau. Mais ceci ne ressort plus des disciplines histologiques elementaires. REFERENCES 1 Maisin, J., DuNjic, A. et Maisin, H. C.R. Soc. Biol. Paris, 1954, 148 611. 2 Maisin, J., Maisin, H. et Dunjig, A. C.R. Soc. Biol. Paris, 1954, 148 743. 3 Maisin, J.. Maisin, H. et Dunjic, A. C.R. Soc. Biol. Paris, 1954, 148. * Maisin, J., Maisin, H. et Dunjic, A. Deuxieme Symposium de Radiobiologie, Liege, 1954. Butterworths Scientific Publications, London, 1955. 5 Van Lancker, J. C.R. Soc. Biol. Paris, 1953, 147 2057. « Williams, R. B., Jr. et de Long, R. P. Fed. Proc. 1953, 12 406. ' Cronkite, E. P., Brecher, G. et Chapman, W. H. Ptoc. Soc. exp. Biol. j\.T. 1951, 76 396. 8 Rosenthal, R. L., Goldschmidt, L. et Pickering, B. L Amer. J. Physiol. 1951, 166 15. 9 Betz, H. et Fruhling, L. C.R. Soc. Biol. Paris, 1950, 144 1013. 309 DISCUSSION TO PAPERS BY GEREBTZOFF AND BACQ, BETZ, NEUKOMM, PEGUIRON AND HERVE, AND MAISIN AND FIEVEZ Z. M. Bacq : To my mind, the facts gathered by Jacobson and Cole, by Lamerton, Loutit and Maisin, and by those who worked as chemists or cytologists with radiopro- tectors, are just parts of one general story. We must put all these facts together and try to formulate some kind of temporary logical idea which might be useful for further work in these fields. This is what I have attempted in a book with Alex- ander, which is now in press. ^ We think that the permeability of the nuclear membrane in haematopoietic tissues of mammals is much increased by ionizing radiations. The nuclear factor (Cole) escapes in large quantities in the cytoplasm, the activity of which is first very much increased (Altman, Richmond and Salomon ; Nizet, Herve and Bacq) ; this factor might escape in the blood ; it is rapidly destroyed and not resynthetized because the nuclei are heavily damaged, if the whole body has received a sufficient dose of radiation. If part of the haematopoietic system (bone marrow or spleen) has been shielded or if a chemical protector has been injected, the primary damage to the haemato- poietic system is decreased ; a substantial proportion of nuclei escape destruction ; in these nuclei, the growth factor is synthetized in larger quantities, and the regenera- tion of the haematopoietic system (about which everybody agrees) is activated. In my mind, chemical protectors are comparable to an imperfect physical screen which decreases the primary radiation damage. During the first years after the discovery of the phenomenon of chemical protection, many papers, even by our- selves, were published showing that the behaviour of rodents protected by cysteine, glutathione or cysteamine was exactly the same as that of the irradiated controls during the first 2 to 4 days after irradiation. A closer analysis shows that this idea is wrong ; Gerebtzoff and myself have just given evidence that the primary cellular damage (at 6 hours) is less in the spleen of mice irradiated under protection of cysteamine ; Devik, who is here (I hope that he will give us first-hand evidence), has observed that very soon ( 1 hour) after irradiation, the number of mitotic abnor- malities after X-irradiation is significantly reduced if cysteine or cysteamine has been injected before irradiation ; when looking with Gray through some of my published data^, I agreed that small differences which I had considered to be within the limit of error, might show a significant lesser damage in cysteamine injected animals. Thus our general idea seems to be consistent with the available observations. We would also like to suggest that the factor responsible for the polycythemia in chronic anoxia is identical with the factor which, according to Jacobson and Cole is active on haematopoietic tissues when injected after irradiation^ The behaviour of the adrenals is apparently complicated. We» have confirmed previous evidence published by Patt. There is a very rapid drop (already marked 1 hour after 800 r, maximal at the second hour) in cholesterol values of the supra- renals of the rat ; the values are normal at 24 hours and drop again to a very low level at 4 days {see Figure 1). The main interest of the figure is that during the first 24 hours, the behaviour of the adrenals of rats protected by cysteamine (10 mg/lOOg) is the same as that of the controls. Here again it looks at first sight that cysteamine has not protected against the primary action, but we think that further work may show that for a certain, lesser dose of X-rays, there will be a difference between chemically protected and control rats. But, as I said in the discussion following Court-Brown's paper, the first hours after irradiation are not a favourable period to observe the action of chemical protectors. 310 DISCUSSION The ascorbic acid of the suprarenals shows variations parallel to that of cholesterol both in controls and cysteamine-protected rats. More than 300 rats have been used in this study. We are not at all prepared to say that the cysteamine-injected rats survive because their adrenals are in normal condition from the first to the fourth day. It might Cholesterol in suprarenals of rats mg/ 1 OOg — Irradiated controls Irradiated under 7 23 S cysfeamme 2f f3 72 96 Hours after 800 r whole body — ~ Figure 1 very u ell be that the adrenals of these animals are in better shape because the liver and other important organs have been less damaged in the presence of cysteamine. The adrenals are not at all necessary for the demonstration of cysteamine action ; recent observations of Fischer show that cysteamine protects adrenalectomized rats against X-radiation. So far as I know, histological observation has not revealed changes in the supra- renals as early as 1 hour after irradiation. Irradiation increases rapidly the amount of .\CTH present in the hypophysis ; ACTH falls after 24 hours to subnormal levels*. In the pig, there is a marked increase of neutral steroid excretion in the urine during the first day only, after irradiation^. Thus the existence of a short hypophyso-adrenal reaction immedi- ately after a heavy dose of X-rays seems undeniable. After 24 hours, the story is indeed much more complicated. F. Devik®' " : In the discussion of morphological efTects of ionizing radiations it may be of interest to point out that the mitoses in the bone marrow of mice may show Table I Time in hours after 200 r whole body irr. 1 2 3 4 6 9 18 29 51 Cysteamine 3mg intra- perit. lOmin before irradiation 55 (2) 69 (5) 63 (2) 63 (5) 70 (5) 64 (5) 41 (20) 18 (4) 12 (4) Controls 85 (2) 78 (5) 83 (2) 82 (5) 87 (5) 80 (5) 62 (18) 21 (4) 8 (4) pronounced radiation effects 1 hour or less after 200 r whole body X-irradiation. These mitotic abnormalities, which mainly disappear in the course of 1-2 days, were found to be significantly reduced in frequency by hypoxia treatment during irradia- tion, and by pretreatment with cysteine, cystamine. and cysteamine as shown in Table I. The figures indicate the mean percentage of abnormal anaphases. Usually 311 HISTOLOGICAL CHANGES 50 anaphases were analysed in each specimen. The figures in parentheses indicate the number of mice which were investigated. Betz : Gerebtzoff a signale des differences dans I'extension des degenerescences cellulaires dans la rate de souris irradiees et de souris irradiees apres protection par la cysteinamine. Je voudrais lui demander si ces differences atteignent au meme titre les tissus myeloi'des et les tissus lymphoides de cet organe. M. A. Gerebtzoff : I did not study, from the quantitative point of view, the difference in reaction between lymphoid and myeloid tissues in spleen, but it appears that the protection of myeloid tissue by cysteamine is stronger than the protection of lymphoid tissue. Harvey M. Patt : Our studies*' * of the adrenal response to X-irradiation would seem to support, at least in part, the interpretation advanced by Betz. The data presented by Bacq are essentially a confirmation of earlier work. Ionizing radia- tions, in common with other noxious stimuli, induce changes that are presumed to reflect an increased demand for the adrenal hormones. The functional response of the adrenals is mediated by the pituitary and closely resembles that seen following a host of injuries. While the early decrease in adrenal lipids is probably indicative of an increased requirement for cortical secretions, the rise in adrenal cholesterol in the rat several days after median lethal irradiation may represent over-stimulation in excess of cortical hormone demand. That the elevated cholesterol is not a result of adrenal exhaustion is suggested by its absence with higher dosages. Although adrenal lipids are usually depleted before death, it is not known whether this is a cause or an effect of the more terminal events. It is particularly noteworthy that Edwards and Sommers'" were unable to detect any fundamental difference in radiation reactions as a consequence of adrenalectomy of shielded or irradiated parabionts and that Schneider et al^^ observed that the adrenals of irradiated rats were capable of sustaining non-irradiated adrenalectomized parabionts. Absence of the adrenals did not compromise protection of irradiated animals by parabiosis. These results strongly suggest that the adrenals of lethally irradiated rats suffer no basic impairment. Turning to the mechanism of protection by ^-mercaptoethylamine, there is, at present, no direct evidence and little reason to assume that the effect is a consequence of the protection of a specific restoration factor. Arguments for the latter fail to consider a very basic matter, namely the relative radio-sensitivities for different morphological and functional responses. We may note in particular (/) the difficulty of differentiating the extent of initial injury in the lethal range by ordinary histological criteria, (2) that many responses, e.g. lymphoid involution, are related more to the radiation dose than to the lethal or morbid effect, (3) that near maximal initial effects may occur at dosages considerably below those required for acute lethality. It is possible under appropriate experimental conditions to demonstrate prevention of injury in a variety of systems by agents such as p-mercaptoethylamine and cysteine. The sparing of comparatively few cells initially might exert a profound influence on the recovery pattern of the tissue as a whole. It should be remarked that it is neces- sary to consider another matter in interpretation of protective phenomena, namely, the temporal and spatial distribution of a given agent in the biological system. Localization of p-mercaptoethylamine to specific sites, as discussed by Eldjarn*, might preclude a more generalized protective action in the animal. Bacq has alluded to the possibility that the effects of fi-mercaptoethylamine on the one hand and of spleen shielding and homogenates on the other may be related. This is so to the extent that both modify the acute lethal action. In fact, cysteine has been shown by Simmons et al to synergize with spleen shielding. This, however, does not imply necessarily that the basic mechanisms underlying the protection are * See page 1 16. 312 DISCUSSION similar. The chemical agents under discussion are rather more general in their effects and are concerned apparently with the more immediate physico-chemical ramifications of energy transfer, probably because they serve as hydrogen donors. Tissue shielding and transplants may be thought of as procedures which do not modify the initial injury but rather encourage the recovery of specific physiological systems. Betz : Bacq has shown us his results on the depletion of the cortical cortex of irradiated rats and of those irradiated and protected by cysteamine. We made similar obser\ations 5 years ago, when studying the behaviour of the adrenal cortex of mice protected by injection of KCN. We saw indeed that the animals protected by KNC had a much less marked depletion of the cortical lipids than the controls. Our observations were made on sections on the adrenal stained with Soudan III. I would like to add to Bacq's comment that it is perfectly possible to detect by histological means the depletion of the adrenal cortex. Even the first phase of depletion occurring after 2 or 3 hours and lasting a short time is to be seen histologically. Bacq told us about a hypothesis on the mechanism of action of some chemical protectors. It is quite possible that some chemicals act by protecting a factor stimu- lating the haematopoietic regeneration, or by protecting some cells producing this factor. This is an interesting working hypothesis. So far as the mechanism of action of the spleen and bone marrow homogenates are concerned, I would like to suggest that they also act by the pathway of the nucleoprotein anabolism. Some results published by Kelly and Jones show that such preparations enhance the synthesis of DXA in irradiated animals. I know very well Patt's work on the adrenal depletion in irradiated rats and I agree entirely with him. I repeated his chemical studies and found the same results. The terminal drop of the ascorbic acid and cholesterol in the adrenals of animals submitted to a lethal dose of X-rays seems to be a sign of hyperactivity rather than a sign of exhaustion of the gland. I quite agree with Mole that the adrenals are necessary to the survival of irradiated animals. As I showed in my papers, our adrenalectomized rats were very sensitive to a whole body irradiation. Some amount of cortical hormones is necessary to maintain a normal resistance. But this does not rule out the possibility that a large excess of such hormone may be noxious to the haematopoietic tissues. We feel that the irradiated body produces an excess of cortical hormones ; as the results shown here indicate, this excess plays a role in the inhibition of the haematopoiesis we observe after a lethal dose of X-rays. J. S. Mitchell : Although the methods of classical histology show no obvious changes shortly after irradiation, studies w^hich I have carried out over a number of years with the use of ultraviolet photomicrography have shown changes in cellular nucleic acid metabolism in several types of cells within relatively short times after irradiation. In my early worki^^is on the accumulation of pentose nucleotides in cytoplasm after irradiation and inhibition of synthesis of deoxyribonucleic acid by irradiation, a substantial increase in cytoplasmic absorption at 2,537 A was found in a number of human malignant tumours at 80 minutes after irradiation. The magnitude of the change was consistent with the presence in the irradiated cytoplasm of ribonucleotides in local concentration often of the order of 3 per cent. In further work^*-^* experiments using ribonuclease in conjunction with quantitative ultraviolet photomicrography showed that the cytoplasmic nucleotides accumulating after irradiation were mainly ribo-polynucleotides closely resembling, and probably identical with, ribonucleic acid. Extraction methods showed characteristic nucleo- tide absorption with maximum at the wavelength 2,620 A. It is important to note that the increase in ribonucleotide absorption occurs almost entirely after irradiation with doses less than the region of 750-1,000 r ; at higher doses usually there is no 313 HISTOLOGICAL CHANGES accumulation of ribonucleotide absorption and often a definite decrease, presumably due to interference with less radio-sensitive processes in the disturbance of cellular nucleic acid metabolism (see also work to be published by L. D. Hamilton). Subse- quent experiments have detected increased cytoplasmic absorption in a biopsy of a human basal-celled carcinoma of the skin taken immediately after delivery of 40 r of gamma radiation in 43 minutes. Since 1940 I have been carrying out experiments on the irradiation of tissue cultures mainly of chick fibroblasts in situ on the stage of the ultraviolet microscope. Very little of this work has yet been published because of the difficulties of precise quanti- tative interpretation. It is of interest to include the result of an experiment {Figure 2) in which chick fibi^oblasts in culture (provided by Mrs. I. Simon-Reuss) received a dose of 435 r of X-radiation delivered in 6 minutes. There appears to be an increase in cytoplasmic absorption at 2,537 A and also an increase in diffusible absorbing material in the photograph taken at between 2-6 minutes after the end of the X-ray exposure. At about 87 minutes after the irradiation the characteristic absorption is lost, the cells are breaking down and there appears to be gross damage to the chromosomes visible. A great many control experiments were carried out which Figure 2. Serial ultraviolet photomicrographs of living cluck Jibroblasts before and after roentgen irradiation in situ on the microscope stage at the times given. 6mm quartz monochromat, N.A. 0-70, wavelength 2,537 A. Roentgen ray dose was 435 r delivered in 6 minutes {87kvp, no added filter ; irradiation through quartz coverslip). showed that the ultraviolet radiation used for these exposures did not produce comparable effects. Recently G. H. Hjort, working with me in Cambridge, has used the methods of ultraviolet photomicrography to study the effects of total body irradiation with doses between 100 and 600 r on the lymphocytes of lymphoid tissue in adult rats. The sections were prepared from frozen dehydrated tissue. It has been found that an increase in nucleotide type of absorption mainly in the cytoplasm can be detected as early as 15 minutes after irradiation and this biochemical change appears before any morphological changes can be detected. There is, however, wide variability in the behaviour of different cells in respect of these changes and much further quantitative examination of the experimental material is necessary. There is definite evidence that these early metabolic changes can be prevented by the adminis- tration of [3-mercaptoethylamine before irradiation. Details of this investigation will be published later. REFERENCES ^ Bacq, Z. M. and Alexander, P. Fundamentals of Radiobiology. London, Butterworths, 1955. Liege, Thone ; Paris, Masson. 2 BAC(i, Z. M., Herve, a. et Scherber, F. Arch. Mem. Pharmac. Therap. 1953, 94 93. 314 DISCUSSION Jr. Science, 1954, 120 112. 3BAcq, Z. M., Fischer, P. et Beaumariage, M. L. Bull. Acad. Med. Belg. 1954 * Mateyko, G. M. et al. Amer. Journ. Physiol. 1951, 167 808. 5 Brayer, F. T., Glasser, S. R. et Duffy, B. J « Devik, F. Brit. J. Radiol. 1954, 27 463. ' Devik, F. and Lothe, F. To be published. 8 Patt, H. M. et al. Amer. J. Physiol. 1947, 150 480. 9 P.A.TT, H. M. et al. Science, 1948, 108 475. 1" Edwards and Summers. J. Lab. Clin. Med. 1952, 40 342. 11 Schneider et al. Personal communication. Nature, Land. 1940, 145 105. Brit. J. exp. Path. 1942, 23 285, 296, 309. Brit. J. Radiol. 1943, 16 339. Brit. Ernp. Cancer Camp. 21st Ann. Rep. 1944, 62 in press. ^- Mitchell, J. S. ^^ Mitchell, J. S. 1* Mitchell, J. S. 1^ Mitchell, J. S. 315 CYTOLOGICAL EFFECT OF CHRONIC GAMMA IRRADIATION AND THE PROTECTIVE PROPERTY OF CERTAIN CHEMICALS AGAINST THE RADIATION INDUCED CHROMOSOME ABERRATIONS Knut Mikaelsen Institute of Genetics and Plant Breeding, Agricultural College of Norway, Vollebekk, Norway Most radiation effects are studied after acute radiation treatments over a short period of time. Effects of continuous or chronic radiation treatments over longer periods of time have been httle investigated until recent years. Some results on effects of chronic gamma irradiation have been reported from gamma field radiation or other use of ^"Co^--. At the Agricultural College of Norway we had a ^^-Ir isotope at work in 1953 and from 1954 a ^''Co source was operating in a gamma field. Some cytological results of chronic radiations and the protective property of certain chemicals against chromo- some aberrations induced by irradiation will be presented. Cuttings of Tradescantia paludosa were found most suitable for this study and were used as experimental material. Root development was initiated by placing the cuttings in tap water with continual aeration at ordinary green- house temperature. When the cuttings showed good development of primary roots, they were transferred to specially designed Incite vessels which contained Hoagland and Snyder's^ nutrient solution. After 24 hours a fresh nutrient solution was substituted for the old and the plants were exposed to chronic gamma radia- tion from ^^'Co. The roots were fixed immediately following the exposure period. Two different experiments were performed. In the first experiment the plants were irradiated at dose rate of 12-5, 25, 50 and lOOr/day. In another experiment the same dose rates were applied during a 48-hr expos- ure ; thus the roots received twice the total dose as in the previous experi- ment. Both acentric fragments and bridges were scored at anaphase, and the results are summarized in Table I. Both fragment and bridge frequencies seem to be almost linearly pro- portional to dose. It is noteworthy, that the bridge frequencies are so low after chronic irradiation. The ratios between the frequencies of fragments and bridges is approximately 6 in all series, while after acute exposures to X-rays and neutrons the formation of bridges is much more frequent. Thus, restitutions resulting in chromatid exchanges seem to be difficult during the chronic radiation exposures. By comparing the 24-hr and 48-hr exposure experiments, it seems obvious that the dose rate has been a more important factor in the yield of aberrations 316 KNUT MIKAELSEN than total dose given. The fragment frequencies were very similar at the same dose rate in the two experiments, while there is no correlation between fragments and total doses given. This chronic radiation method is used to test certain chemicals for their protective ability against radiation-induced chromosome aberrations. Table I. — Frequencies of chromosome aberrations in anaphase of meristematic root tissue o/Tradescantia paludosa after exposures to chronic gamma radiation from ^"Co Dose rate R/day Total dose in R No. of anaphase cells No. of fragments No. of fragments per loo cells No. of bridges No. of bridges per 100 cells {a) 48-hr e 12.5 'cposure 25 518 47 9-1 10 1-9 25 50 520 88 16-9 16 3-1 50 100 320 113 35-3 17 5-3 100 200 92 70 76-1 11 12-0 {b) 24-hr e 12-5 Kposure 12-5 396 26 6-8 5 1-3 25 25 388 61 15-7 9 2-3 50 50 417 122 29-3 22 5-3 100 100 293 175 59-7 15 5-1 Protective property of certain chemicals on chromosomes is reported by MiKAELSEN"*' ^' ^ Riley', Forssberg and Nybom^ Wollf^ and DevikI". The results from my experiments are summarized in Table II, where gluta- thione, cysteine, thiourea, sodium hyposulphite (NagSgOi) and sodium cyanide (NaCN) proved to have protective property. Table II. — Per cent reduction in fragment frequencies at different concentrations of the chemicals tested Concentrations Glutathione 0 Cysteine HCl low pH 0 Cysteine high pH 0 Thiourea Nai.S,0, 0 NaCN 0 0 0 lO-^M 13 15 45 5 X lO-^M 7 10 lO-'iM 36 44 44 41 11 3 X 10 *M 50 60 5 X lO'-'M 21 1-1-5 X 10-3M 53 53 48 Toxic 25 3 X 10-3 M Toxic Toxic 5 X lO-^M 30 10-2M 35 The chemicals were applied in the following manner. To the nutrient solution in which the Tradescantia cuttings were grown, the chemical com- pounds were added at certain concentrations. After one hour's preabsorp- tion, the plants, still in these solutions, were exposed to chronic gamma radiation for 48 hours at a dose rate of 25 r/day which yielded a convenient 317 CYTOLOGICAL EFFECT OF CHRONIC GAMMA IRRADIATION amount of fragments ( Table I) . By the use of the film badge method, it was indicated that the plants received a total dose of 50 r in all experiments. The number of both acentric fragments and bridges per 100 cells at anaphase was determined. Conclusions regarding the eflfects of chemical treatments, however, were based upon the fragment data only, since bridges occurred in such low frequencies that it was difficult to evaluate the data {Cf. Table I). Glutathione— In the presence of glutathione a marked and significant reduction in fragment frequency was obtained. The experiments included concentrations of glutathione ranging from lO^^M to 1 -5 X 10 =^M. None of these concentrations seemed to cause any serious disturbances to the roots not subjected to radiation. The protective property increased with increasins' concentrations and the maximum effect is obtained at 1 • 5 X 10-^M, where the fragment frequency was reduced 53 per cent. At a higher concentration (3 X 10~^M) the roots became soft and were obviously injured. They were unsuitable for study. Cvsteine— Cysteine was applied as cysteine-HCl in two different series of experiments. The presence of cysteine-HCl, lowered the pH-value of the nutrient solution, particularly with the two strongest concentrations. One series of experiments was performed with these acid solutions. Thus, the plants were grown and exposed to cysteine under quite different pH-values. Judging by mitotic activity, the different pH-values did not seriously disturb the roots since equal numbers of dividing cells were present in all series. The maximum effect of cysteine in acid solutions was 60 per cent reduc- tion in fragment frequency at 3xlO-*M. A concentration of 3xl0-3M could not be tolerated and proved to be toxic to the roots. In another series of experiments with cysteine-HCl, the solutions were neutralized with potassium hydroxide (KOH), so all cysteine and the control series had the same pH {^'6) during the radiation exposure. Compared with the previous series, less reduction in fragment frequency was obtained. The maximum effect was a reduction of 35 per cent at lO-^M. In both series of experiments the protective effect of cysteine increased with increasing concentrations. Thiourea— The chemical formula of thiourea is usually presented as NHg— S— NHg. In oxidation processes, however, it reacts ^^ as a sulphydryl compound NHg— SH=NH. Its effectiveness increased with concentrations and the maximum effect was 48 per cent reduction in fragment frequency at 10-3M. Sodium hyposulphite (Na2S204)— Sodium hyposulphite, a strong reducing agent, showed a remarkable and optimal effect already at a concentration of 10 "^M, where the reduction in fragments amounted to 45 per cent. At 10-^M no increase could be detected and lO^M proved to be toxic to the roots. Sodiu?n cyanide (NaCN)— With sodium cyanide a smaller but significant reduction of 25 per cent in fragment frequency was obtained at 10"^M. Conclusive evidence is presented of the modifying or protective property of glutathione, cysteine, thiourea, sodium hyposulphite, and sodium cyanide against chromosome fragmentation induced by gamma radiation. The mechanism by which these chemicals exert their protection is not clear. First, do these chemicals prevent chromosome breakage or do they heal broken 318 KNUT MIKAELSEX ends so that restitution of broken chromosomes is favoured ? A combina- tion of both is an alternative possibiUty. Prevention of breakage is con- sidered to be tlie most likely explanation. What arguments exist in favour of such an assumption ? Barron and Flood'"- presume that such oxidizing agents are responsible for the oxidation of aqueous solution of certain thiols by ionizing radiation, as they have demonstrated. Therefore, it seems probable that the mechan- ism of the sulphydryl compounds, glutathione, cysteine and thiourea, may be due to their reactions with oxidizing agents produced by irradiation in the cell nuclei. These oxidizing agents may be inorganic or organic free radicals or peroxides. Thus, the sulphydryl compounds compete with the chromosomes for free radicals or other oxidizing reactants and less breaks will be produced. It must also be considered that absorption of the sulphydryl compounds, as w^ell as the other chemicals tested, may change the metabolic state of the cell. Patt'^ has reported that his data suggest perhaps that cysteine action is related to the availability of intracellular oxygen. It may be justi- fiable, at least, to conclude that the sulphydryl compounds exert their effects through their — SH groups since the results are so similar with the three compounds. It is reasonable to assume that sodium hyposulphite, which is a strong reducing agent and combines rapidly with molecular oxygen in aqueous solution, decreases the amount of dissolved oxygen in the tissue. It is assumed that by removing oxygen from the tissue, the production of the secondary formation of O2H and H2O2 or other reactive products is prevented. King et al^^ claim, however, that in large measure oxygen exerts its action by altering the biological processes of the cell. The protective ability of sodium cyanide may be more complicated and obscure than was the case with the other compounds tested. It appears, especially as regards the two strongest concentrations, that cyanide reduces the mitotic rate. Considerably fewer anaphases were found in these roots and the reduction in fragment frequencies was small compared with the concurrent experiments with the other chemical agents. These facts indicate that metabolic inhibition or metabolic changes may be the most reason- able explanation of the protective effect of sodium cyanide. Such metabolic alterations, involving complicated enzyme systems, initiated by KCN and CO are discussed by King et al^^. The complicated effect of cyanide is demonstrated by D'Amato and Gustafsson'^ who showed that KCN treatment of seeds prior to X-irradiation increased visible mutations in barley with low concentrations. A stronger concentration of 10~-M de- creased the mutation rate, although the rate of chromosome breakage increased. BAcq'** found that cyanide decreased the mortality in X- irradiated mice. By comparing the effect of cysteine in acid and neutral medium, a marked difTerence in protection and tolerance is noticed. A similar response in the effect of cysteine, applied at different pH-values, on survival of mice after whole body X-irradiation was obtained by Patt et al'^'^ , ^^ and Goldie et al^^. They state that the probable explanation of this difference may be the fairly rapid oxidation of cysteine to cystine in a neutral medium before its administration. Marshak^" has shown that changes in intracellular 319 CYTOLOOICAL EFFECT OF CHRONIC GAMMA IRRADIATION acidity alter the radio-sensitivity of the cell. It seems justifiable, however, in this case to put the main emphasis on the oxidation of cysteine to cystine. Although the protective actions of these chemicals are not understood, indications of an indirect effect of radiation on chromosome fragmentation are demonstrated. This indirect effect seems to be of great importance in the production of chromosome breaks, since it is possible to reduce the radiation damage to about half in most of the cases. Unfortunately, how- ever, it will be difficult to determine the exact relation between direct and indirect radiation effect by chemical means, because of their toxic effects on roots at certain concentrations. REFERENCES 1 Sparrow, A. H. Science, 1951, 114 488. 2 Sparrow, A. H. and Singleton, W. R. Amer. Nat. 1953, 87 29. 3 HoAGLAND, D. R. and Snyder, W. C. Proc. Amer. Soc. Hort. Sci. 1933, 30 288. "MiKAELSEN, K. Science, 1952, 116 172. 5 MiKAELSEN, K. Proc. 9th Internat. Congress of Getvtics, 1953. «MiKAELSEN, K. Proc. Nat. Acad. Sci. 1954, 40 171. ' Riley, H. P. Rec. Genetics Soc. Amer. 1952, 21 62. 8 FoRSSBERG, A. and Nybom, A. Physiol. Plant. 1953, 6 78. 9 WoLLF, S. Genetics, 1954, 39 356. 10 Devik, F. Brit. J. Radiol. 1954, 27 463. 11 Whitmore, F. Organic chemistry, New York, 1951. 12 Barron, E. S. and Flood, V. J. Gen. Physiol. 1950, 33 229. 13 Patt, H. M. This Symposium. 1* King, E., Schneiderman, H. and Sax, K. Proc. Nat. Acad. Sci. 1952, 38 34. 15 D'Amato, F. and Gustafsson, A. Hereditas, 1948, 34 181. i« Bacq. Z. M. Science, 1950, 111 356. 1' Patt. H. M., Tyree, E. B., Straube, R. L. and Smith, D. E. Science, 1949, 110 213. 18 Patt, H. M., Smith, D. E., Tyree, E. B. and Straube, R. L. Proc. Soc. exptl. Biol. Med. 1950,73 18. 19 GoLDiE, H., Tarleton, G. T. and Hahn, P. F. Proc. Soc. exptl. Biol. Med. 1951, 77 790. 2« Marshak, a. Proc. Soc. exptl. Biol. Med. 1938, 39 194. 320 THE EFFECT OF RADIATION ON FROZEN TUMOUR CELLS Ilse Lasnitzki* Strangeways Research Laboratory, Cambridge It has been shown by several observers that the response to radiation can be influenced by the metaboHc activity of irradiated biological material. Glucksmann and Spear ^ demonstrated that in tadpoles the radiation effect was delayed if the animals were chilled during and after exposure. Gold- FEDER-, comparing the radio sensitivity of histologically similar mouse tum- ours found the radiation effect more marked in those showing a lower metabolic rate. A similar result was obtained by me^ with the S3 7 tumour exposed as ascites and subcutaneous form. For the ascites form, which according to Warburg and Hiepler* functions at very low energy levels, the MLD found was one-fifth of that for the solid sarcomas. In recent years Craigie^ and other workers have shown that certain tumours preserve their viability if frozen and kept at temperatures of dry ice and liquid air. This ability should make them a convenient material to study the effect of radiation in the frozen state at which metabolic activity is at a standstill, and in the present experiments the effects of radiation on tumours in the fresh and frozen state were compared. EXPERIMENTAL The material used was the Ehrlich ascites tumour. This tumour is frozen and stored on dry ice as a routine in our laboratory as an alternative to serial transplan- tations and samples stored, for instance, for several months proved to be viable on re-inoculation. The tumour cell suspensions were frozen at — 79° and kept at this temperature for 7 days prior to irradiation. Exposure took place in vitro. The dose was 1,500 r units at 75r/min. Two experiments were made. In the first one the frozen cell suspension was exposed on the laboratory bench and gamma rays were used ; in the second one it was irradiated on dry ice and X-rays were used. The frozen cells were thawed quickly immediately after exposure and injected subcu- taneously into C3H mice. For comparison of the radiation effect fresh cells obtained from the same tumour sample were exposed in an identical manner and inoculated subcutaneously into the same strain of mice. Each set had its own unirradiated fresh and frozen control grafts. Each inoculum contained approximately 15 X 10® tumour cells. The criterion of radiation effect was the number and size of .sub- cutaneous tumours obtained. RESULTS Figure I gives the percentage take obtained after inoculation of frozen and fresh unirradiated grafts. One week following inoculation it is 80 per cent for grafts derived from fresh and 90 per cent for grafts from frozen cells, an * Sir Halley Stewart Fellow. 321 THE EFFECT OF RADIATION ON FROZEN TUMOUR CELLS indication that freezing alone does not impair their viabihty. Figure 2 shows the percentage take of fresh irradiated and control grafts. The appearance of tumours derived from irradiated grafts is delayed for one 700 (Frozen) 100 (Fresh) Gamma rajs (Fresh) 2 3 Time in weelis Figure 1. Percentage take of frozen and fresh unirradiated grafts {gamma ray experiment) 12 3 1 Time in wee/7 \ / ~ ZO $7 /^ V - w / 1 1 \ 1 1 1 1 32 \ 16 \ 8 I 0 1 3 15 6 7 9 10 11 12 13 11 possible to study in detail the sensitivity changes occurring during this division and the subsequent stages of germination and mycelial growth. The results of a typical experiment are shown in Figure 1. A spore sus- pension was incubated in medium over a 14-hour period, samples being taken at hourly intervals. One part of each sample was irradiated with X-rays (approximately 8,000 r) and another with ultraviolet (200 ergs per mm-) these being plated at a range of dilutions, while a third was fixed and later stained for cytological study. For the sake of convenience the incuba- tion was interrupted after 7 hours and the suspension stored in the cold overnight. Samples taken before and after the storage, and treated in the usual manner, showed that the interruption had not caused any major change in response. 330 HOWARD B. NEWCOMBE The proportion of induced mutations was observed to increase to more than double when the spores had become binucleate at the time of irradia- tion (from 22 up to 79 per cent for X-rays, and from 19 up to 46 per cent for ultraviolet), and to decline to 10 per cent or less as multinucleate strands developed. As the two curves are based on approximately 6,000 mutants scored, out of a total of approximately 30,000 colonies observed, they are relatively accurate. Also, this general relationship between mutability and number of nuclei has been found consistently in other similar experiments. It should be noted also that when the cells are most sensitive to the mutagenic effects of the irradiation, they tend in addition to be most sensitive to the lethal effects. Table III. — Sensitivity to X-ray induced mutation Effect of pre-incubation in the absence of nutrient (Spores han'ested in saline ; diluted 1/10- in saline before incubation. from two experiments. Dose, 8,000 r) Combined data Induced mutation (hours) Mutant Total Per cent colonies colonies mutation 0 511 2,761 18-5 2 818 4,145 19-7 4 590 3,035 19-4 6 746 3,320 22-5 24 818 4,406 18-6 48 505 3.094 16-3 Growth during pre-incubation : at 3 days, out of 68 spores examined, 48 were still uni- nucleate, 2 binucleate, and 22 were very much shrunken and appeared dead. Unirradiated control : 0-5 percent mutation (18/3,439) ; no apparent difference between 0 hour and 9-1 1 day incubation. Sur\ival : in unirradiated controls, 40-50 per cent after 9-11 days' incubation; in irradiated samples, between 0-06 and 0-18 per cent throughout. The frequency of induced mutation eventually declines to 9-4 per cent (232/2,479) when irradiated after 9-1 1 days' incubation. Certain similarities in the actions of X-rays and ultraviolet should be noted. The peak sensitivity coincides in both cases with the binucleate state ; the extent of the increase is similar ; and so also is the extent of the subsequent decline in sensitivity. In addition there are minor irregu- larities so that certain of the points do not fall precisely on a smooth curve ; these are probably significant, and they deviate in the same directions for the two agents. The X-ray and ultraviolet survivals are also similar in their major features ; that is, sensitivity is high during the binucleate stage, and strikingly low when many nuclei are present. These variations in response could be due directly to the observed nuclear changes, or alternatively, the association could be fortuitous, and the result of some loosely associated physiological changes. However, our early attempts to separate the two failed. It was found that spores incubated in saline which lacked nutrient, showed neither the nuclear divisions nor 331 MECHANISMS OF MUTATION PRODUCTION IN MICRO-ORGANISMS the characteristic (X-ray) sensitivity changes, over a 48-hour period (see Table III). The test was carried out using a stock saline suspension of spores (which contained traces of nutrient leached from the agar on which the spores had been grown) and diluting by a factor of 1 /1 00 with pure saline to reduce the amount of nutrient. When a parallel experiment was carried out without dilution, a proportion of the spores (between a quarter and a half of them) were able to germinate and grow into short multinucleate strands, while the rest remained uninucleate. Under these conditions the characteristic rise and decline in sensitivity to X-rays could be observed, but was less striking than under the standard conditions {see Table IV). It seemed important at this stage to determine whether, as we supposed, the sensitivity changes were pecuHar to the growing spores, and absent in Table IV. — Sensitivity to X-ray induced mutation Effect of pre-incubation in the presence of traces of nutrient (Spores harvested from nutrient agar culture by washing with saline ; incubated undiluted. Combined data from two experiments. Dose, 8,000 r) Pre-incubation (hours) Induced mutation Mutant colonies Total colonies Per cent mutation 0 2 4 6 24 48 630 573 391 567 658 225 2,429 2,099 1,310 1,588 7,615 2.184 25-9 27-3 30-1 35-7 8-7 10-0 Growth during pre-incubation : at 1 day 20-24 per cent germination, average approxi- mately 10 nuclei per germinated spore ; at 2 days, no detectable change. Unirradiated control : 0-4 per cent mutation (3/857). Survival: 0-07-0 -27 in one experiment; 0-02-0 -04 in the other. (There was no appreciable difference between the two experiments with respect to mutation.) The frequency of induced mutation remains at a low level, 9-9 per cent (325/3285), when irradiated after 6-14 days' incubation. those which remained uninucleate. To confirm this, a suspension which had been incubated in this manner for 28 hours, and which had become relatively insensitive to the mutagenic effects of X-rays, was passed through very fine filter paper (Watman No, 3) to remove the mycelial strands. This filtrate, which contained mainly the ungerminated spores, showed a threefold increase in sensitivity to X-ray induced mutation {see Table V). A similar increase would also be expected as the result of filtering the suspension after it had been irradiated, and this was in fact observed {see Table V). Thus the eflfect could not be due to some subtle change in the condition of the suspension at the time of irradiation, and it is clear that the greater part of the sensitivity changes observed during the incubation must be associated with the growing cells, and that they are not a function of the suspension as a whole. 332 HOWARD B. NEWCOMBE Somewhat similar evidence was obtained from the changes which occurred under more nearly normal conditions. In most of the experiments where the spores were incubated in medium prior to irradiation, the sensitivity fell off rapidly as the average number of nuclei increased, to four or eight per strand. In one experiment, however, the decline was much more gradual in relation to the nuclear increase, and this was found to be associated with a more poorly synchronized growth, due apparently to an unusual degree of crowding ; the spores passed through the first nuclear division together, but a proportion of them lingered much longer than usual in the sensitive binucleate state. Thus all of the evidence so far, emphasizes a close association between nuclear number and response to radiation, which can be observed over a wide range of conditions of growth. The sensitivity changes do not seem Table V. — Sensitivity to X-ray induced mutation Effect of pre-incubation in the presence of traces of nutrient Effect of removal of germinated spores by filtration (Spores harvested from nutrient agar culture by washing with sahne ; incubated without dilution. Dose, 8,000 r) Treatment Induced mutation Pre-incubation Mutant Total Per cent colonies colonies mutation 0 hour Irradiated 586 2,425 24-2 28 hour* Irradiated! 45 956 4-7 28 hour* Irradiated t and Filtered 146 1,049 14-0 28 hour* Filtered and Irradiated 169 1.063 15-9 * Same incubated suspension. t Same irradiated suspension. Unirradiated controls : 0-hour, 0-7 per cent mutation (15/2,015) ; 28-hour, 0/9 per cent (13/1,509). Survival : in unirradiated control, 75 per cent after 28 hours' incubation ; in O-hour irradiated 0T2 per cent; 28-hour irradiated 0-5 per cent; irradiated-filtered and filtered-irradiated 0-05 per cent each. to be influenced by the particular stage in the nuclear division cycle, since the period of maximum sensitivity apparently extends over the whole of the binucleate state and not just a part of it, and the nuclear divisions subse- quent to the first are almost completely unsynchronized. Bvit it is still not certain that nuclear number directly determines the response, since changes in both nuclear number and response might have a common physiological cause. SENSITIVITY DIFFERENCES WHICH ARE NOT DUE TO NUCLEAR NUMBER It also appeared that other differences in the state of the cell at the time of irradiation could influence the proportions of induced mutant colonies, even where the nuclear number was constant. This was indicated by minor irregularities in certain of the experimental results, such as those 333 MECHANISMS OF MUTATION PRODUCTION IN MICRO-ORGANISMS noted in Figure 1. In addition the effects of various treatments which might influence the physiological state were tested using both multinucleate strands and uninucleate spores. Three such tests will be described. In the first, spore suspensions containing 6-5x10' spores per ml of medium were grown under the following sets of alternative conditions : {a) with 5 ml of air, and with 20 ml of air, in a 30 ml tube ; {b) incubating continuously for 32 hours, and incubating for four 8-hour periods with storage in the cold after each ; and (c) sealing the tubes lightly with plastic screw caps, and covering with loose aluminium caps ; these being applied in all combinations to make a total of eight different treatments. The amount of growth was very similar in all cases, the resulting strands being Table VI. — Effect on frequency of spontaneous mutant colonies of amount of air available during preliminary growth Other treatments Mutation Amount of Air (in 30 ml tube) Mutant colonies Total colonies Per cent Factor increase 5 ml 20ml A(CT5)* B (CL5) C (IT5) D (IL5) A (CT20) B (CL20) C (IT20) D (IL20) 10 7 16 8 52 45 29 34 644 633 591 345 790 378 591 430 1-6 1-1 2-7 2-3 6-6 12-0 4-9 7-6 4-1 10-9 1-8 3-3 * C=continuous incubation (32 hours). I=intermittent incubation (4 eight-hour periods, at 24-hour intervals). T=tube tightly capped (plastic screw cap). L=tube loosely capped (aluminium cap). Inoculum 6-5 X 10' spores per ml of medium. Growth : CT5 and CL5, strands 4-10 diameters in length ; in other suspensions 6-14 diameters in length. Nuclei stained clearly where the incubation was intermittent ; where the incubation was continuous, the nuclei were obscured by stainable material in the form of much larger cylindrical bodies which nearly filled the strands. for the most part more than four times their own diameter in length, and less than sixteen times. No difference was observed as the result of the two different kinds of caps used, and these treatments can be considered as replicates. Samples from all eight treatm.ents were plated without irradiation, and irradiated with standard doses of X-rays and of ultraviolet. The greater quantity of air resulted throughout in a considerably higher spontaneous mutant frequency (5 to 12 per cent, as compared with 1 to 3 per cent ; see Table VI) ; the numbers of ultraviolet induced mutants was not noticeably affected, but the X-ray induced mutants were reduced to about one half. Intermittent incubation on the other hand reduced the numbers of ultra- violet induced changes to half, but had little effect on the X-ray changes. There was also a consistent effect of intermittent incubation on the staining 334 HOWARD B. NEVVCOMBE of the cells, the nuclei being clearly visible in a lightly stained cytoplasm, whereas with continuous incubation the stainable material nearly filled the cell. Table VII. — Effect on X-ray mutability of amount of air available during preliminary growth f X-ray dose 16min at 50 cm) Mutation Amount of Air (in 30 ml tube) Other treatments* Mutant colonies Total colonies Per cent Minus control* Factor decrease 5 ml A (CT5) 40 475 8-4 6-9 B (CL5) 29 386 7-5 6-4 C (ITS) 39 362 10-8 8-1 D (IL5) 20 242 8-3 5-9 20 ml A (CT20) 59 547 10-8 4-2 0-61 B (CL20) 41 297 13-8 1-8 0-28 C (IT20) 49 394 12-4 7-5 0-93 D (IL20) 35 314 11-2 3-6 0-61 * For unirradiated controls, size of inoculum, other treatments, and amount of growth see Table f 7. Per cent survival = 73 -7, 58 •2, 61 -2, 70- 1, 69-4, 78-5, 66-7, 69-8, respectively. In a second experiment, spores were grown into somewhat longer strands, having lengths of about thirty times their own diameter and probably containing in the vicinity of thirty nuclei. The spores were incubated intermittently, that is for 7 hours each day up to a total of 35 hours, Table VIII. — Effect on ultraviolet mutability of continuous versus intermittent incubation during preliminary growth (Ultraviolet dose, 200 ergs per mm^) Continuous Other treatments* Alutation or intermittent Mutant Total Per cent Minus Factor colonies colonies mutation control* decrease Continuous A (CT5) 52 414 12-6 11-0 B (CL5) 83 442 18-8 17-7 C (CT20) 123 628 19-6 13-0 D (CL20) 68 377 18-0 6-0 Intermittent A fIT5) 61 629 9-7 7-0 0-64 B (IL5) 24 319 7-5 5-2 0-28 C (IT20) 54 475 11-4 6-5 0-50 D fIL20) 41 387 10-6 3-0 0-50 * For unirradiated controls, size of inoculum, other treatments, and amount of growth, see Tabic 17. Per cent survival = 64-3, 66-5, 79-5, 99-7, 100, 92-5, 80-4, 86-0, respectively. J\fote : With intermittent incubation, the nuclei were small and clearly stainable, whereas with continuous incubation, the cytoplasm seemed divided into large deeply staining bodies which nearly filled the mycelial thread. 335 MECHANISMS OF MUTATION PRODUCTION IN MICRO-ORGANISMS Storing overnight in the cold. Samples taken each day were tested for spontaneous mutation and for sensitivity to X-ray and ultraviolet induced change. Most of the growth was probably completed in the first 14 hours. Under these conditions the spontaneous mutations increased continuously with incubation {see Table IX). X-irradiation however induced only 1 or 2 per cent of additional mutants whereas ultraviolet was a remarkable effective mutagen despite the multinucleate state, inducing from 15 to Table IX. — Sensitivity to X-rays and to ultraviolet in the uninucleate and multinucleate states (Dose, 8,000 r. and 400 ergs per mm^ respectively) Mutation Survival Per cent Hours ^ — Pre- Mutant Total Per cent incubation colonies colonies mutation Incubated and X-irradiated (di luted 1/ 10 in medium L before X-irradiation) 0 152 700 21-7 2-1 7 126 3,563 3-5 10-7 14 94 3,160 3-0 9-5 21 142 2,821 5-0 8-5 28 128 3,890 3-3 8-7 35 187 2,490 7-5 7-5 Incubated and ultraviolet irradi iated (di iluted 1/10 in saline before ulti "aviolet) 0 75 298 25-2 6-7 7 649 2,196 29-6 8-9 14 388 2,485 15-6 7-6 21 243 435 55-9 1-0 28 294 749 39-3 1-6 35 389 911 42-7 1-8 Control (incubated and plated without irradiation) 0 62 7,813 0-8 7 53 6,189 0-9 14 128 7,072 1-8 21 152 3,957 3-8 28 147 3,376 4-4 35 213 4,055 5-3 Inoculum : 2 X 10' spores per ml. Incubation : in medium ; intermittent 7 hours out of each 24 ; stored in cold between incubations. Growth : all spores have germinated and grown into a multicellular mycelium (about 30 nuclei per infective unit) by 7 hours. The suspension probably reaches saturation shortly after. 55 per cent mutants {see Table IX). Thus, a state seems to have been achieved in which the sensitivity to the mutagenic action of the two agents differed widely. In addition there appear to have been significant fluctua- tions in sensitivity to ultraviolet induced mutation, and it would seem that such sensitivity must have been very much dependent upon uncontrolled physiological variables. Finally, spore suspensions in medium were bubbled with nitrogen and incubated, over a 24-hour period. During the treatment almost all nuclear 336 HOWARD B. NEWCOMBE division was suppressed so that few of the cells became binucleate, and a negligible proportion had more than two nuclei. These limited divisions might have been expected to increase the sensitivity to radiation induced mutation and killing, but instead the net effect of the treatment was to reduce both the ultraviolet and the X-ray mutability to about half, and to increase survival by 2- to 3-fold for X-rays and by 8- to 1 0-fold for ultraviolet {see Table X). The effects could not have been due to any differences in dis- soK^ed oxygen at the time of irradiation since all suspensions were diluted with the same chilled saline before being exposed to X-rays or ultraviolet. Table X. — Physiological control of sensitivity in the uninucleate state Effect of bubbling with nitrogen during 24-hour incubation in medium Experiment (Suspension) Hours' Incubatic with medium andW Mutation Survival Per cent Mutant colonies Total colonies Per cent mutation Factor reduction (24/0) Unirradiated A B 0 24 0 24 2 5 3 38 245 355 265 1,200 0-82 \ 1-41 1-13 3-12 No detectable - killing with incubation. Ultraviolet A 0 24 249 175 966 1,152 25-8 15-2 0-59 4-0 35-0 B 0 24 249 55 1,109 473 22-5 11-1 0-52 4-2 38-7 X-rays A 0 24 90 169 90 993 26-2 17-0 0-65 14-1 30-0 B 0 24 88 66 317 458 27-8 14-4 0-52 13-3 37-8 Note : Some of the spores were clearly uninuncleate (8 out of 49) ; some were uninucleate but the nuclei appeared 'dumb-bell' shaped as if incompletely divided (32/49) ; and a very few were binucleate (6/49) ; three out of 49 had 3-8 nuclei. Thus it is clear that various changes in the physiological state of a cell can influence the likelihood that it will produce a mutant colony as a result of irradiation. In the case of irradiated multinucleate cells it is altogether likely that mutant and non-mutant nuclei compete, and differences in the physiological state at the time of irradiation might persist long enough to influence the outcome of such internal competition. It is therefore difficult to show conclusively, using multinucleate cells, that tlie physiological state can influence the susceptibility of individual nuclei to induced mutation ; although certain of the present results could be most simply interpreted on this assumption. 337 z MECHANISMS OF MUTATION PRODUCTION IN MICRO-ORGANISMS However it is quite certain that the mutabiUty of individual nuclei must have been reduced to about one half in the experiments using nitrogen treated uninucleate (and binucleate) spores. And it must also be that the sensitivity of the individual nuclei to induced mutation is at least doubled in the binucleate cells arising under normal conditions of growth. Thus physiological differences are capable of resulting in very considerable variations in sensitivity to induced mutation. And furthermore, in the experiments demonstrating this, lethality and mutagenesis were similarly affected, indicating that the modifiable processes are in part common to both types of response. CONCLUSIONS Originally it was hoped that these experiments would yield information regarding the time of the induced mutations, and whether such changes were in any way dependent on the process of gene (and chromosome) replication. Both X-ray and ultraviolet induced mutations had appeared to be delayed until the time of gene replication in Escherichia coli, since irradiated cells gave rise to colonies sectored for induced changes, even where the so-called ' double selection ' technique had been used in an attempt to insure that each colony came from a single irradiated gene complement (Newcombe"' ^ ; WiTKiN^). Also, the mutagenic effects of ultraviolet in Streptomyces had been found to lose their capacity for photoreversal only under conditions favourable to nuclear division, and at a time shortly before the nucleus becomes visibly double (reported elsewhere). However, we have not been able to add to these observations any evidence of a critical period in the nuclear division cycle during which mutation might be taking place. Exceptional sensitivity to induced mutation appears to extend over the whole of the binucleate stage, and not just a part of it ; and the fractionation experiments appear to indicate that nuclear division brings no release from an earlier saturation (or impairment) of the capacity for induced mutation. What has been shown is that the physiological state of a cell at the time of irradiation can influence the likelihood that it will subsequently develop into a mutant colony. Where the cells are multinucleate at the time of irradiation it is not entirely certain whether such differences might not arise through effects influencing subsequent competition between mutant and non-mutant nuclei within the strands. However, where only one or two nuclei are present it is clear that the sensitivity of individual nuclei to induced mutation is subject to very considerable physiological control. This suggests the possibility that the non-linear dose-response relation- ships may also be physiological in origin. In support of this view, examples of non-linearity have been described which could not be the result of differential killing of induced mutants, and which would be very difficult to explain in terms of an initial population heterogeneity. In view of the evidence for a physiological control over the response of the genetic material of Strepto?nyces to mutagenic agents, it seems altogether possible that the physiological changes produced by high doses of the mutagens themselves may be effective in altering the capacity to respond to a further exposure. 338 DISCUSSION Two observations support the view that the non-linear X-ray dose- mutation curves observed in Streptomyces are the result of a genuine satura- tion or impairment of the capacity of individual spores to respond when the dose is high : First, at doses where a further increase is ineffective in producing muta- tions, it is also relatively ineffective in producing lethal changes. Second, cell suspensions have been obtained by pre-incubation which are very resistant to X-ray killing ; and in these the less-than-Iinear response to increasing doses could not be accounted for in terms of differential killing of cells which would otherwise produce mutant colonies. In addition, very considerable changes in sensitivity to X-ray induced mutation have been observed following the first nuclear division, and also as the result of various treatments affecting the physiological state at the time of irradiation. As the capacity for induced mutation is evidently dependent on the physiological state of the organism, it is not surprising that high doses of radiation should alter this capacity for mutagenic response. REFERENCES 1 Keener, A. J. Bad. 1948, 56 457. 2 Newcombe, H. B. J. Gen. Microbiol. 1953, 9 30. ^ Newcombe, H. B. and McGregor, J. F. Genetics, 1954, in press. * Sparrow, A. H. Atm. N.T. Acad. Sci. 1951, 51 1508. ^ McGregor, J. F. J. Gen. Microbiol. 1954, in press. « Newcombe, H. B. Genetics, 1951, 36 570. " Newcombe, H. B. ibid, 1953, 38 134. 8 WiTKiN, E. Cold Spring Harbor Symp. Quart. Biol. 1931. 16 357. DISCUSSION R. Latarjet : La chute du taux des mutations induites par les fortes doses de rayons ultraviolets a un caractere tres general, conformement aux indications du Newcombe. Aux cas qu'il a mentionnes, jepuisajouter celui que j'ai observe en obtenant les premieres mutations qui aient ete induites chez un virus* (Figure2). II s'agissait de muta- tions 'host range' obtenues chez le bacteriophage T2 en irradiant celui-ci pendant qu'il se multi- plie au sein de son bote Escheri- chia coli souche B. La courbe ci-jointe groupe les points experimentaux obtenus. EUe montre la chute tres brusque des mutations induites a partir d'une dose de 5.000 ergs mm"^. * Compt. rend. Acad. Sci. Paris, 1949,228 1354. 1 ! 1 1 1 1 • (235j 150 - 1 1 <^ 1 f 1 1 / 3 / .| 700 / - «o 1 • c; ••J 5 , / •/ 1 5 / 1 5; / • 50 ■ -.^ '- r^ ' .1 1 1 1 , . 3 It- Figure 2 5 6 jrlOOOerqs Tum^ 339 INFLUENCE DES DOSES ELEVEES DE METHYL- BIS-(2-CHLOROETHYL)AMINE SUR L'OVAIRE DE LAPINE ADULTE, AVEC OU SANS PREPARATION PAR LA BETAMERCAPTOETHYLAMINE Paul Desaive Laboratoire de Pathologic Chirurgicale Generale de rUniversite de Liege et du Centre National Beige pour I'Etude de la Croissance Normale et Pathologique L'appareil follicLilaire de I'ovaire adulte constitue, a notre avis, un excellent test biologique de Taction des radiations ionisantes et de celle des substances dites radiomimetiques ; si les effets des premieres sur ce systeme ont fait I'objet de nombreux et interessants travaux (parmi lesquels nous citerons tout specialement les remarquables publications de Lacassagne^ et de Gricouroff"), et nous ont personnellement servi pour etablir notre conception du mecanisme de revolution et de I'involution folliculaires^, I'influence des secondes n'a, par contre, suscite qu'un petit nombre de travaux originaux ; or ce probleme est important, en raison de I'extension croissante, en therapeutique humaine, de I'emploi des derives bi- ou tri- substitues du gaz moutarde ; c'est pourquoi nous I'avons aborde dans une note preliminaire recente*, dans laquelle nous avons signale que ' nitrogen mustard (Boots) ' {HN^i, a la dose unique de 0,3mmg/kg ou a cette meme dose suivie, a 8 jours d'intervalle, d'une nouvelle injection de 0,5mmg/kg, provoque une nette atresie des follicules murissants preovulatoires et surtout favorise I'apparition de formes atretiques anormales et d'un nombre in- habituel de follicules a ovocytes multiples et de complexes primordiaux polynuclees. Par ailleurs, les excellents resultats radioprotecteurs obtenus par Bacq et collaborateurs^ avec la betamercaptoethylamine (becaptan) nous ayant incite a etudier le comportement de cette substance a I'egard des effets de I'yperite a I'azote {HJ^^ sur les cellules generatrices de I'intestin grele du rat, nous avons pu constater^ que le becaptan, a la stricte condition d'etre introduit dans I'organisme quelques minutes avant //jVg, limite, dans une certaine mesure, les effets immediats de ce toxique. C'est pourquoi, nous avons tente d'etablir dans quel sens, la preparation de la lapine par le becaptan est capable de modifier la reponse de I'ovaire a Paction de HJS^- MATERIEL ET TECHNIQUES Nous avons utilise des lapines adultes d'un poids moyen de 2 kg, isolees en cage au moins un mois avant le debut des experiences. Six lapines ont re^u dans la veine marginale de I'oreille 2mmg de methyl- bis-(2-chloroethyl) amine (//.Vg), dilues extemporanement dans 3cm^ de serum physiologique. Cette dose de 2 mmg ( 1 mmg/kg) a ete bien sup- portee par les animaux ; elle represente cependant, a I'echelle humaine, une 340 PAUL DESAIVE quantite de 70mmg par kg, qui n'est toleree en une fois que par la seule voie intra-arterielle. Six autres lapines ont ete soumises au meme traitement, mais trois minutes apres avoir regu par voie intraperitoneal, 50 mmg de becaptan (soit 0,5 cm^ de la solution a 10 pour cent de becaptan de Labaz). Dans chacun de ces deux groupes experimentaux, les animaux ont ete sacrifies apres 12, 24, 36, 48, 72 et 96 heures. Les deux ovaires de chaque sujet ont ete preleves, fixes au liquide de Bouin, inclus dans la paraffine, debites en coupes seriees de 6 /x et colores soit a I'Azan pour I'etude topo- graphique de I'appareil folliculaire, soit a rhematoxyline ferrique-eosine pour Texamen cytologique. Puis, pour chacun des ovaires, une coupe sur vingt a ete dessinee au projecteur de profil SIP ; et sur le 'puzzle' constitue par 1 'ensemble de ces dessins, les differents types folliculaires evolutifs et involutifs, ont ete soigneusement comptes. Rappelons a ce sujet, que nous distinguons histologiquement^ 8 stades folliculaires successifs : 1 stade primordial (de reserve) P, 4 stades A, B, C et D correspondant a la periode de croissance de I'ovocyte, et 3 stades E, F et G contemporains de la preparation du follicule a I'eventuelle rupture (qui, chez la lapine, est provoquee par le coit). Les resultats de ces numera- tions ont ete reunis en tableaux permettant d'etablir une comparaison aisee entre les etats folliculaires des differents ovaires etudies. Enfin, par une technicjue particuliere de comparaison de deux ovaires d'une meme paire, nous avons recherche les effets eventuels du becaptan employee isolement. RESULTATS EXPERIMENTAUX Nos observations ont porte sur les points suivants : (7) L'aspect histologique et cytologique des ovaires et plus specialement la frequence des processus atretiques. (2) La formule folliculaire, c'est-a-dire la repartition des follicules en 7 stades successifs de A k G. {3) L'aspect et la frequence des organites atypiques, et plus particuliere- ment, des groupements anormaux de follicules primordiaux, des follicules a ovocytes multiples et des ruptures folliculaires intra-ovariennes. {A) Etude des ovaires de lapines traitees par une seule injection intraperitoneale de 50 mmg de becaptan Avant tout essai d'interpretation de Taction du becaptan sur les effets ovariens de HN^, il etait evidemment indispensable de determiner I'infiuence eventuelle du becaptan seule. Deux lapines ont subi I'ablation de I'ovaire gauche, utilise comme temoin, puis ont re9u 8 heures apres, en injection intraperitoneale, 50 mmg de becaptan. Les ovaires droits ont ete preleves 12 heures et 36 heures apres cette injection (soit 20 heures et 44 heures apres I'ovaire temoin). Voici Tableau /, la comparaison des formules folliculaires de ces ovaires et de leurs temoins. A la lecture de Tableau I, on constate que les differences entre les deux ovaires traites et leurs temoins sont legeres et repondent a la loi normale 341 INFLUENCE DES DOSES ELEVEES DE METHYL-B1S-(2-CHLOROETHYl) AMINE de la compensation (visible, a la SGeme heure, au niveau des stades, B, CetD). D'autre part, les ovaires preleves apres becaptan ne presentent aucune anomalie de structure ; les figures d'atresie s'y rencontrent en proportion normale et les follicules primordiaux y sont disposes comme d 'habitude ; on n'y rencontre pas de grandes formes atretiques anormales et enfin les folli- cules a ovocytes multiples qu'ils contiennent sont en nombre a peu pres egal a celui des temoins. En resume, le becaptan ne parait exercer par lui meme aucune action sur I'appareil folliculaire, constatation qui exclut pratiquement toute chance de provoquer par ce produit — a la dose employee — des lesions cytologiques serieuses de la glande genitale femelle. Cette observation est d'autant plus interessante que d'autres substances radioprotectrices sont, au contraire. Tableau I Follicules Follicules A B C D E F G To- toux A B C D E F G To- taux I 0 voire 2.485 612 '98 67 44 11 5 3.322 Ovaire dt 2.378 484 85 73 38 20 5 3.083 gauche preleve temoin I2h apres becaptan II 0 voire 2.188 368 48 48 31 12 5 2.700 Ovaire dt 2.367 647 88 72 16 8 7 3.205 gauche 1 1 prilevi temoin 36 h apres bicaptan nettement toxiques pour I'ovaire : par exemple, chez une lapine soumise a trois injections sous-cutanees journalieres de 2 mmg de cyanure de sodium, nous avons compte, dans un seul ovaire, 3 ruptures intra-ovariennes tradui- sant un bouleversement important de la physiologic folliculaire. {B) Etude des ovaires de lapines traites par HN^ seul on par HN2, precede de becaptan I. Aspects histologique et cytologique des ovaires traites — Outre une tres legere diminution du poids ovarien moyen, par rapport a la normale et une con- gestion assez considerable des hiles, nous avons, comme dans notre travail precedent, note une chute rapide du nombre de grands follicules preovu- latoires, et surtout une modification importante de I'etat des follicules primordiaux. Dans les toutes premieres heures (12° et 24°), ceux-ci montrent des lesions ovocytaires et folliculeuses importantes ; les minces cellules granuleuses se vacuolisent et se desunissent ; le cytoplasme de I'ovocyte gonfle, devient plus colorable et prend un aspect pommele ; la chromatine nucleaire — a 342 PAUL DESAIVE I'etat diplotene de H. de Winiwarter — se desagrege, se ratatine et se disperse finalement en grains pycnotiques. La frequence de ces images est beaucoup plus considerable que dans un ovaire normal, oil elles n'apparaissent que d'une maniere discrete et tres fugace ; dans I'ovaire yperite non protege par le becaptan, la proportion habituelle de 2/1.000 montea la douzieme heure a 369 1.000 et a la 24° heure a 549 1.000, puis s'abaisse progressivement a 180/1.000 a la 96° heure, en passant aux 36°, 48° et 72° heures par les valeurs 240/1.000, 261/1.000 et 251/1.000; dans I'ovaire protege, elle n'est a la douzieme heure que de 289/1.000 mais elle se releve a 542/1.000 a la 24° heure pour s'attenuer ensuite, en passant par les valeurs de 345/1.000 a la 36° heure, de 279/1.000 a la 48° heure, de 260/1.000 a la 72° heure et de 182/1.000 a la 96° heure. Nous signalons ici que I'atresie des follicules primordiaux se maintient a un taux relativement eleve jusqu'au huitieme jour, sans doute en raison du phenomene de ' mort retardee ' qui frappe les follicules primordiaux non immediatement tues par le toxique, mais suffisamment atteints pour etre incapables de poursuivre leur evolution. Notons pour memoire que dans un ovaire irradie a 2.500 r, Tatresie des follicules primordiaux, deja tres nette apres 2 heures, atteint 380/1.000 a la 16° heure et 824/1.000 a la 54° heure pour devenir pratiquement com- plete a partir de la 72° heure. Pour ce qui est des autres types folliculaires (de A a G) la comparaison des rapports : nombre de formes atretiques/nombre de formes evolutives, cal- cules dans les ovaires normaux d'une part et les ovaires yperites d'autre part, est extremement malaisee en raison du fait que, meme dans les con- ditions physiologiques, la valeur de chacun de ces rapports varie avec le moment du prelevement de I'ovaire par rapport a la duree de son cycle ; si ce moment est proche du debut de I'intervalle de 32,8 heures qui, selon nous, separe deux poussees evolutives successives, on trouve peu d'atresies ; si, au contraire, le prelevement a eu lieu a un instant proche du debut d'une nouvelle poussee, les seules follicules evolutifs persistants sont ceux destines, soit a passer au stade suivant, soit (s'il s'agit de follicules du stade G') a involuer definitivement, a moins que I'acte genital ne les ait prepares a I'ovulation. Nous avons reuni sur le Tableau II ci-apres les incidences atretiques moyen- nes relevees dans les deux series d'ovaires que nous etudions ici, en les rapprochant des resultats obtenus dans les deux series precedentes, ainsi que dans un groupe important d'ovaires normaux. De la lecture de ce Tableau II, il ressort immediatement que dans I'ovaire normal, le taux atretique est sensiblement le meme a chacun des 7 stades : I'appareil folliculaire est en equilibre, et les reductions numeriques parfaite- ment calquees sur le rythme des poussees de developpement, se font sans heurt pour produire, a partir d'environ 1.600 follicules A, 6 follicules G prets a I'ovulation. Au contraire, I'ovaire yperite est le siege de boule- versements importants : atresie massive initiale des follicules jeunes et des follicules murissants, suivie d'une hyperproduction reactionnelle d'elements jeunes et d'un retour progressif de I'appareil noblea une dynamique noi^male; des lors, les taux atretiques aux differents stades sont evidemment dissem- blables, et comme la regeneration — qui debute a la douzieme heure — est 343 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHLOROETHYl) AMINE necessairement dominante, la moyenne atretique generale, pour I'ensemble de nos observations, est inferieure a la moyenne classique (tout au moins pendant la phase folliculaire du cycle). Notons en passant qu'apres preparation par le becaptan, cette moyenne atretique est tres proche de la normale ; ce fait dont la valeur absolue ne doit pas etre exageree, en raison des reserves que nous avons emises plus haut, semble cependant indiquer que la radioprotection favorise un retour plus rapide de I'ovaire a son equilibre physiologique. Sur le plan de la simple observation microscopique, nous ajouterons ici, en confirmation de nos constations precedentes, que les elements immediate- ment touches par I'atresie sont, outre les follicules primordiaux, les types Tableau II Nombre de formes atretiques ^^ ' Nombre de formes evolutives A B C D E F G Moyennes generates Lapines nonnales 0,58 0,61 0,73 0,84 0,53 0,37 0,20 0,551 Lapines yperities (a) Dose unique : 0,6 mmg {de la 24° a la 192° heure) 0,325 0,325 0,381 0,451 0,391 0,281 pas de G ou pas de G atretique 0,359 (b) Dose repetee : 0,6 puis apres 8 jours 1 mmg {de la 24° a la 168° heure apres la seconde injection) 0,325 0,325 0,365 0,490 0,348 0,210 pas de G ou pas de G atretique 0,343 (c) Dose unique : 2 mmg {de la 12" a la 96° heure) 0,170 0,176 0,949 0,610 0,390 0,337 0,047 0,383 (d) Dose unique : 2 mmg {pre- ce'de'e de 50 mmg de be- captan {dela W a la 96° heure) 0,158 0,161 0,570 0,648 0,905 1,040 0,233 0,530 les plus avances de la chaine folliculaire, c'est-a-dire les follicules F et G ; les formes intermediaires — D et E particulierement — sont relativement epargnees. Enfin, notons encore que I'effort de reparation de I'ovaire est extremement precoce : a la douzieme heure, on voit apparaitre sous I'epithelium germin- atif, d'importants groupes de follicules jeunes en pleine activite. Nous reviendrons plus loin sur cette interessante constatation ; mais nous retenons des a present cjue les ovaires de lapines traitees par le becaptan avant de recevoir I'injection de HJ^2^ se caracterisent par une destruction moins brutale des follicules primordiaux et ensuite par une tendance de I'appareil folliculaire a reprendre plus rapidement son comportement involutif habituel. //. Formules foUiculaires — Nous avons groupe dans le Tableau lilies resul- tats des numerations foUiculaires aux differents stades A a. G, dans chacune des deux series ovariennes etudiees, et nous les avons fait suivre des resultats 344 PAUL DESAIVE moyens des numerations correspondantes obtenues a partir de 19 ovaires normaux preleves a des phases differentes du cycle. Dans les deux groupes experimentaux, nous relevons un premier fait important (que nous avions deja observe lors de nos experiences a doses plus faibles d'yperite) : a savoir, par rapport aux temoins, I'extreme elevation, des la douzieme heure, des nombres de follicules jeunes, A et B en ordre principal, en consequence de I'accroissement reactionnel de la quantite de Tableau III. Numerations Folliculaires A . Lapines traite'es par 2 mmg d'HNi Delais de Types folliculaires . Foi mes atreliques prilevement A B C D E F G speciales 12h 6.432 1.076 46 6 3 1 1 1 RO 24 h 3.541 593 89 23 5 5 4 IGTdet \ GMd 36 h 2.427 407 96 72 46 5 2 — 48 h 3.176 532 68 17 12 8 4 — 72 h 2.174 365 56 14 11 7 5 1 GH 96 h 4.578 762 334 124 76 16 7 Moyennes 3.721 622 115 43 25 7 4 0.83 B. Lapines traitees par 2 mmg d^HN^, apres 50 mmg de becaptan 12h 7.204 1.200 140 47 47 21 6 24h 2.465 411 54 13 8 5 4 2 GMd 36 h 2.789 462 55 43 34 7 3 — 48 h 4.500 750 84 26 9 4 3 — 72 h 1.878 312 124 87 60 24 6 1 RO 96 h 2.620 432 234 53 23 11 6 Moyennes 3,576 594 115 45 30 12 5 0,5 C. Lapines normales a differentes phases du cycle (19 ovaires) Moyennes 1.569 178 85 35 23 11 6 Pas de formes atre'ti- ques speciales follicules primordiaux sortis de la reserve quiescente et prets a evoluer ; nous n'avons pu, dans ce domaine, distinguer les ovaires proteges de levu's congeneres non proteges. Par contre, nous constatons que les follicules murissants des stades F et G, a I'autre extremite de la chaine evolutive, restent en quantite sensiblement normale dans les premiers, alors qu'ils diminuent considerablement dans les seconds ; il semble done que le becaptan soit capable de proteger, pendant un temps assez long pour leur permettre d'evoluer sans encombre, les 345 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHLORO£tHYL) AMINE follicules qui, au moment du traitement, se trouvaient aux stades inter- mediaires D et E, correspondant a la fin de la periode de croissance de I'ovocyte et au debut de la preparation a I'ovulation. D'ailleurs si nous comparons — pour eviter I'erreur due a d'eventuelles differences individuelles — les moyennes sur quatre jours de nos deux series et les moyennes normales, nous constatons effectivement que, malgre le retour a I'etat physiologique qui s'annonce des le troisieme jour dans les Figure 1. (a) Cortex ovarien {apres HJV^). (h) Foil. A. (c) Amas, d' all lire plasmodiale , de follicules primordiaux ovaires intoxiques par I'yperite, les nombres moyens de follicules F et G sont, dans ce cas, nettement inferieurs a la quantite ordinaire, alors qu'apres intervention de becaptan, ils lui sont egaux et meme superieurs. En conclusion, I'appareil folliculaire evolutif reagit a I'injection intra- veineuse de 2mmg d'HN^, dans le meme sens mais avec plus d'intensite qu'apres I'introduction de 0,6 mmg ; nous assistons en eff'et, a la meme atresie hative de^ grands follicules murissants, avec economic relative des stades intermediaires, mais avec declenchement d'une poussee plus impor- tante encore de follicules jeunes. 346 PAUL DESAIVE D'autre part, la preparation de la lapine par injection intraperitoneale de 50mmg de becaptan a pour consequence d'elever le nombre moyen observe de grands follicules murissants, ce qui semble conferer a cette substance une action protectrice immediate, mais limitee, vis a vis des stades les plus avances de I'echelle folliculaire. ///. Aspect et frequence des formations atypiques, a point de depart folliculaire — (7) A la suite de nos precedentes recherches, nous avons signale que la caracteristique essentielle de I'ovaire de lapines soumises a I'injection intra- veineuse de 0,3mmg/kg de HN^ est I'apparition dans le cortex ovarien, de groupements de follicules primordiaux, en cordons ou amas d"" allure plasmodiale (voir Figure 1) ; arrondies ou polygonales, limitees par un seul rang de cellules folliculeuses aplaties, ces formations contiennent dans un cytoplasme finement granuleux un nombre variable de noyaux au stade diplotene, caracteristique des ovocytes arrives a la fin de leur preparation prepuberale. Nullement specifiques de I'yperite, ces groupements — que nous avons rencontres — en quantite reduite — dans quelques ovaires pathologiques apres administration de doses elevees de gonadotrophines ou apres destruction de I'hypophyse par le radon, frappent ici par leur abondance et leur volume. Nous les avons observes a partir du troisieme ou du quatrieme jour apres 1 mmg d'yperite et des la 24° heure lorsque I'injection a ete repetee apres huit jours ; dans les deux modes experimentaux, ces groupements particu- liers se maintiennent pendant quatre jours puis disparaissent a une epoque qui correspond approximativement au retour de I'ovaire a la normale. Dans les experiences relatees ici, nous avons retrouve ces groupements a la 96° heure apres 2 mmg d'yperite ; mais, fait remarquable, nous en avons notes des la 12° heure dans les ovaires proteges, en petit nombre il est vrai et assez pauvres en ovocytes (trois ou quatre en moyenne) ; il semblerait done que le radioprotecteur utilise favorise la production de ces organites, qui est vraisemblablement liee a un processus regeneratif, interpretation sur laquelle nous reviendrons bientot. (2) Une autre observation a traite a la frequence elevee dans les ovaires yperites, des follicules a ovocytes multiples. L'ovaire de lapine est d'ordin- aire pauvre en ces elements particuliers ; nous en avons trouve dans 102 ovaires sur 159 (soit dans 64,16 pour cent de ceux-ci) en quantite variant de 1 unite a 121 par organe et dans la proportion de 1 follicule multiovulaire pour 200 follicules mono-ovulaires. Or I'examen de 13 ovaires traites par //.Yg a faible dose nous a permis de decouvrir chez chacun d'entre eux, des organites pluri-ovocytaires a I'un ou I'autre des sept stades folliculaires evolutifs, avec une frequence de 1 pour 180 et des nombres totaux de 1 a 58 unites. Nous avons fait la meme constatation dans nos deux series actuelles et nous reproduisons ici nos numerations a ce sujet (Tableau IV). Non seulement, tons les ovaires enleves chez des lapines traitees par 2 mmg d'HX^ renferment des follicules multiovulaires, mais encore, est-ce parmi eux que nous avons trouve celui qui, de tons les ovaires de notre collection, en contient le plus grand nombre : 169. Signalons en outre que, dans ces deux groupes, la valeur moyenne des rapports numeriques entre les follicules multiovulaires et les follicules ordinaires depassent de loin celles observees dans nos autres series d'ovaires ; 347 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHLORO£tHYl) AMINE dans le groupe des non proteges, le nombre moyen de follicules pluriovocy- taires est de 30,5 pour un nombre folliculaire total moyen de 4.528 (soit un follicule multiovulaire pour 145 follicules mono-ovulaires) et dans le groupe des proteges, ce nombre s'eleve a 45,3 pour 4.376 (soit un follicule plurio- vulaire pour 96 follicules ordinaires). Ces faits confirment d'une part, la frequence elevee des follicules multi- ovulaires dans les ovaires yperites et, d'autre part, I'influence favorable que le becaptan exerce sur la genese de ces elements qui, au meme titre que les formations plasmodiales, nous semblent constituer un indice de regeneration ovocytes ovarienne hyperactive. Enfin, signalons en passant que les deux ruptures intra-ovariennes {RO) que nous avons observees, I'une a la douzieme heure, chez un non protege, Tableau IV A. Lapines traitees par 2 mmg HN^, Follicules multiovulaires 12" h 98 [56 J, 31 5 {do?n 1 triple), 9 C, 1 D, 1 RO] 24° h 3 (2.4, 1 B) 36° h 2 (I .4, 1 B) 4&"h 6 (4.4, 2B) 72° h 13 (8.4, 5 5) 96° h 61 [37 A {dont 1 triple), 19 B, 2 C {dont 1 triple), \ D, \ E, \ F] Moyenne : 30,5 B. Lapines traitees par 50 mmg de becaptan puis par 2 mmg d'HN2 12° h 24° h 36° h 48° h 72° h 96° h Follicules multiovulaires 169 [19 A {dont 1 triples), 50 B {dont 5 triples), 23 C {dont 2 triples), 2D, IE, \F] 3 (2 A, 1 B) 3 (2 A, 1 B) 22 (10.4, 6B, 5C, 1 D) 33 [10 .4, 7 5, 5 C, 4 Z) {dont 1 triple), 3E,3F {dont 1 triple) 1 RO {quad- ruple)] 42 (23.4, \0B, 5 C, 2 D, \ E, I F) Moyenne : 45,3 I'autre a la soixantedouzieme heure chez un protege, concernaient des follicules G a ovocytes multiples : 1 biovocytaire et un quadriovocytaire. (5) II nous reste un dernier point a envisager ; celui de la presence dans les ovaires yperites de types atretiques anormaux, et en particulier de ces ruptures intra-ovariennes auxquelles nous venous de faire allusion et que nous avons ete le premier a signaler chez la lapine. II est certain que I'intoxication brutale de I'ovaire par I'yperite precipite de maniere excessive I'atresie des follicules preovulatoires : nous avons vu, a la 24° heure, aussi bien apres que sans protection, apparaitre de grands follicules tres dilates (GT, qui ne se voient habituellement que dans les ovaires de lapines gonadotropisees ou dans les ovaires greffes dans la rate apres castration), ainsi que des meroxanthosomes (GM) exceptionnellement rencontres dans les ovaires normaux ; nous avons meme observe, a la 72° heure, chez un non protege, un follicule hemorragique (GH). 348 PAUL DESAIVE Nous avons egalement note, surtout chez les lapines non protegees, la naissance prematuree de rantrum dans les follicules des stades jeunes C et meme B. Mais le fait avant tout remarqiiable est la frequence reellement anormale, apres yperite, des ruptures folliculaires intra-ovariennes. On sait que chez la lapine, comme chez la chatte, I'ovulation n'est provoquee que par le coit, sterile ou non, par I'excitation electrique du tractus genital externe, ou par I'administration d'une quantite importante de gonadostimuline. II pent cependant arriver qu'un follicule se rompe a I'interieur de I'ovaire ; normal chez les monotremes et certains oiseaux, ce processus atretique est considere comme rarissime chez les mammiferes. Nous I'avons observe 24 fois sur 358 ovaires (soit dans 6,7 pour cent des cas) et seulement dans 2 ovaires sur 56 appartenant a des lapines n'ayant subi aucune influence experimentale^ (soit dans 3,5 pour cent des cas). Or sur les 13 ovaires yperites a dose faible, nous avons olDserve deux fois ce phenomene ; et sur les 12 des presentes series nous I'avons egalement constate deux fois (1 a la douzieme heure chez un ovaire non protege et 1 a la 72° heure chez un ovaire protege). Nous obtenons ainsi un total de 4 RO sur 25 ovaires yperites, soit un pourcentage de 16, de loin superieur a la proportion normale et qui n'est egale que par ceux — 15,70 et 21,5 pour cent — que nous avons etablis chez des lapines, respectivement hypophyseoprives et thyreoprives. Les RO que nous avons vues dans la serie actuelle sont, en outre, tres particulieres ; la premiere, a la douzieme heure, concerne un follicule gauche biovulaire qui s'est rompu en deux endroits ; I'un des ovocytes est deja accole a I'une des breches par laquelle s'est engagee sa corona radiata ; I'autre semble se dinger vers la seconde ouverture, plus largement creee en direction du stroma voisin. Quant a la seconde RO, a la 72° heure, elle est plus extraordinaire encore ; le follicule mur devenu dehiscent contenait quatre ovocytes : le premier est situe au fond de la neocavite cree dans le pourtour folliculaire, le second et le troisieme (celui-ci atretique) sont dans un chenal qui se dirige vers le cortex, et le quatrieme est sorti de I'ovaire, et est attache a la sereuse ovarienne par un reste de corona. La nature atretique de ce phenomene est indeniable ; sa cause principale reside dans une alteration de I'enveloppe folliculeuse dont certains elements peuvent avoir subi specifiquement Taction necrosante du HN^, a moins cjue cette lesion ne soit indirectement le resultat d'une modification vasculaire localisee, en relation avec I'alteration, sous I'influence du toxique, du metabolisme ovarien de I'acide ascorbique. Les ovaires des animaux traites par le becaptan ne sont pas tres differents de leurs congeneres soumis a Taction de HN^ seul ; nous y retrouv^ons en effet, quelques formes atretiques atypicjues — meroxanthosomes et ruptures intra- ovariennes — mais en nombre moindre (3 au total, au lieu de 5) ; cette derniere constatation serait de pen d'interet (des differences individuelles quant au nombre de follicules susceptibles de subir ces formes d'atresie pouvant exister, au depart de Texperience, entre les ovaires utilises), si elle ne coincidait avec un comportement inverse des grands follicules evolutifs, qui sont nettement plus nombreux chez les lapines protegees ; des lors, le becaptan pent etre considere comme tres probablement capable de limiter Tatresie des follicules murissants. 349 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHL0R0ETHYL) AMINE Ell conclusion — L'injection intraveineuse, chez la lapine, de 2mmg de HN^ apporte une confirmation des alterations que nous avons anterieurement observees, avec les doses plus faibles, au niveau de I'ovaire, et qui sont en ordre principal : (7) La perception d'un trouble profond dans le mecanisme — rythme par la succession, a intervalles de 32,8 heures, des poussees folliculaires — de revolution et de I'involution de I'appareil noble de I'ovaire. {2) L'accroissement considerable, au cours des premieres heures, de la proportion (ordinairement tres faible) des formes atretiques par rapport aux formes normales de follicules primordiaux P. (3) La diminution du nombre moyen des grands follicules murissants et leur passage premature en atresie par des mecanismes brusques — tels que transformation meroxanthosomiale ou rupture intra-ovarienne — exception- nels dans les conditions normales. A cote de ces phenomenes degeneratifs, s'inscrivent des manifestations reparatrices, parmi lesquelles nous citerons essentiellement : (7) Une brutale mise en chantier, des la douzieme heure, de follicules jeunes de types A et B surtout, issus d'un important groupe d'elements primordiaux qui normalement aurait suffi a alimenter trois ou quatre poussees successives. [2) La presence d'une quantite excessive de follicules pluriovocytaires. (5) L'apparition, dans le cortex ovarien, de groupes polynuclees, formes d'cvocytes de type primordial, au noyau diplotene, loges dans une enveloppe folliculeuse commune, et qui, bien que non specifiques de Taction de I'yperite, frappent ici par leur abondance et leur volume. Si l'injection intraveineuse d'yperite a I'azote est precedee de I'introduc- tion intraperitoneale de 50mmg de becaptan, les lesions ci-dessus decrites perdent de leur acuite, tandis que les manifestations regeneratrices s'ac- centuent ; cette 'protection' relative s'exteriorise par les faits suivants : (7) Une allure d'ensemble plus normale des processus folliculaires et notamment une moyenne atretique generale proche de la moyenne habituelle. (2) Une legere diminution, au cours des toutes premieres heures, du taux d'atresie des follicules primordiaux. (3) Le maintien a un niveau plus eleve du nombre moyen des grands follicules du type de Graaf (4) La meme considerable hyperproduction de follicules jeunes que dans I'ovaire simplement yperite, mais, avec en plus, (5) la decouverte d'un nombre plus important encore de follicules multiovulaires et enfin, (6) la precocite extreme de l'apparition des groupements primordiaux polynuclees, dont des exemples sont deja observables a la douzieme heure. REVUE La connaissance des effets biologiques du gaz moutarde ([3 ^'-dichloro- diethyl sulfide) remonte aux travaux d'AuERBACH et Robson^ qui ont pour la premiere fois signale, en 1942, les alterations chromosomiales que cette substance provoque, in vivo, chez la Drosophile. Plus tard, Koller^", puis 350 PAUL DESAIVE Darlington et Koller'^ employant le meristeme de la racine d'Allium et le pollen de Tradescantia, ont demontre que la centromere des chromo- somes en meiose ou en mitose etait particulierement sensible au gaz moutarde, et que le pourcentage des fragmentations chromosomiales s'accroissait de la 6 heure a la 72° heure apres le traitement. En utilisant comme test des embryons d'amphibien, Gillette et Bodenstein^^^ puis Bodenstein^^ ont observe au niveau de la crete epidermique un arret des mitoses a I'inter- phase, mais avec maintien de la croissance et de la differenciation de certaines cellules qui deviennent anormalement grandes puis se detruisent. Oilman et Philips^* ont considere que I'yperite et ses derives stoppent les cellules en interphase tout en permettant aux cellules en mitose de poursuivre le cycle de celle-ci. Ross^-', partant de la similitude des effets mutagenes et chromosomiaux produits sur Viciafaba, d'une part par les diepoxydes et les yperites (Loveless et Revell^^) qui reagissent par I'intermediaire d'un ion carbonium G + , et d'autre part, par les rayons X et les peroxydes, qui interviennent par le moyen de radicaux libres, a etabli differents points d'analogie entre les deux modes d'action. La similitude entre ces inecanismes n'est cependant pas absolue. Ford^^ a pu preciser, sur les cellules du meristeme Vicia faba, que la methyl-bis- (2-chloroethyl) amine {HA^'2) agit electivement sur I'heterochromatine alors que celle des radiations ionisantes (des photons gamma en I'occurence) se distribue de maniere egale aux chromosomes courts ; Ford a de plus constate que les effets mitoclasiques de //.Ag sont legerement plus tardifs que ceux des rayons gamma ; cette difference a ete egalement observee par Koller et Casarini^^ au niveau de la moelle osseuse et du carcinome de Walker du rat ; de plus, ces auteurs au meme titre que Loveless et Revell^^ puis Revell^^, considerent qu'au contraire des photons X, dont I'influence est maximale a la fin de la periode de repos du noyau ou au debut de la prophase, HA 2 agit essentiellement au debut de I'intercinese c'est-a-dire au moment ou la cellule se trouve en pleine phase metabolique preparatrice a la division. Pour ces derniers auteurs, HA'2 et ses analogues ne sont nullement ' radiomimetiques', leur mode d'action etant vraisemblablement different de celui des radiations ionisantes. Une excellente mise au point de ce probleme difficile a ete publiee recem- ment par Roller^" qui a notamment insiste sur le fait, signale par Auer- BACH et Moser-'^ que chez la Drosophile, I'abaissement de la tension d'oxygene diminue les effets genetiques des rayons X mais n'influence pas ceux de I'yperite. Enfin, tout recemment Hicks--, comparant au niveau des cellules nerveuses de I'embryon de sauterelle, les effets des R.X., des poisons radiomimetiques et de certaines hormones, a constate que HA2, agissait sur ce substrat de maniere nettement moins selective que les radia- tions ionisantes. L'actiori de I'yperite et de ses derives sur les glandes genitales a ete peu etudiee. Dans une revue generale des lesions provoquees par cette substance, dans I'espece humaine, Sophie Spitz -^ n'apporte aucun element concernant I'ovaire, mais signale un certain nombre d'alterations testiculaires, qui ont ete decrites en detail par Landing et collaborateurs^* ; ces lesions consistent essentiellement, dans les 24 premieres heures, en deformations chromatiques 351 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHL0R0ETHYL) AMINE dans les noyaux au repos des spermatogonies et des spermatocytes I et dans line inhibition des mitoses reductionnelles ; plus tard, les tubules apparaissent desorganisees et ne montrent plus I'orientation classique des differentes couches, depuis les spermatogonies jusqu'auxspermatozoides acheves. Pour Kindred-^, les elements les plus sensibles de la lignee sont les spermatides. Comme nous I'avons fait observer anterieurement, il existe, entre ces alterations testiculaires et les modifications que nous avons constatees dans I'ovaire traite par HN^, une certaine analogic, en ce sens que la desinte- gration des parois tubulaires du testicule correspond a la desorganisation du mecanisme de revolution et de I'involution folliculaires. Mais d'importantes differences sont a signaler entre les deux organes en ce qui concerne les types de cellules germinales presentant le maximum de sensibilite a //A'a- Dans le testicule, en effet, ce sont, d'apres Kindred '^^ les spermatides qui presentent ce caractere ; or ces elements n'ont pas de corres- pondants dans I'ovaire de lapine normale, puisque I'ovotide (formee par elimination des deux globules polaires a la suite des deux mitoses reduction- nelles) n'apparait que dans la trompe apres ovulation et fecondation. En fait, I'ovaire adulte ne renferme que des ovocytes de premier ordre qui vont s'accroitre regulierement, du stade folliculaire P a la fin du stade D, puis se preparer lentement du stade E au stade G a la prophase de la premiere mitose de maturation. Par consequent, la sensibilite particuliere, observee par nous, des follicules primordiaux et des follicules murissants doit tenir a un autre facteur que I'etat nucleaire ou cytoplasmique de I'ovocyte ; et ce facteur nous croyons pouvoir ie situer dans les cellules folliculeuses. Lorsque un follicule P, faisant partie de la reserve corticale de ces elements est incite (pour une raison que nous cherchons depuis longtemps a situer de maniere precise, mais que nous savons avec certitude ctre liee a I'ovaire lui-meme — car elle continue a intervenir apres hypophysectomie, thyroid- ectomie, destruction chimique ou chirurgicale des attaches nerveuses de I'ovaire etc.) a prendre la voie qui le conduira au premier stade evolutif ^4 et, s'il ne s'atresie pas en cours de route, aux stades suivants jusqu'a la forme preovulatoire G, les minces cellules aplaties, en couche unique, de son enveloppe, deviennent cubiques, se multiplient et construisent en feuillets successifs la parol interne granuleuse du follicule, secretent le liquide follicu- laire, produisent Tantrum et assurent par la corona radiata, la nutrition ainsi que le soutien de la cellule oeuf par la corona radiata. Or il est bien connu (particulierement depuis les experiences d'irradiation de I'ovaire) que la sensibilite maxima du follicule correspond aux moments ou ses cellules folliculeuses traversent les deux periodes critiques qui se situent d'une part au passage du stade P au stade A et d'autre part, a la fin du stade G, lorsque la parol, amenuisee par la distension antrale, est prete a la rupture. C'est pourquoi, nous estimons, qu'en sus d'une possibilite de lesion directe de certains ovocytes fragilises, I'atteinte preferentielle, par HN2, des follicules P et des follicules G, est due, pour les premiers, a une lesion par ce toxique des minces cellules folliculeuses, pretes a entrer en division pour constituer la future parol multistratifiee du follicule A, et pour les seconds, a une destruction soit de certains elements de la corona avec comme consequence la mise a la parol de I'ovocyte et I'entree en atresie meroxanthosomiale du 352 PAUL DESAIVE follicule, soit, plus rarement, de groupes granuleux sous thecaux, qui, en s'atrophiant, creent une zone de moindre resistance avec rupture intra- ovarienne. Cette maniere de voir nous conduit sans difficulte a I'une des deux interpre- tations qui peuvent etre donnees de la genese des formations polynuclees, pseudoplasmodiales, que nous avons observees dans le cortex ovarien, du quatrieme au septieme jour apres HN^ seul. Rappelons au prealable que des elements de structure comparable observes par Landing et collabo- rateurs-"* dans les testicules de souris traitees par diverses moutardes nitro- genees ont ete tout specialement etudies par Kindred ^^ dans les canaux seminiferes du rat traite par le compose tris(p-chloroethyl) amine ; ces groupes plurinuclees derivent quelquefois de spermatocytes de premier ordre, mais le plus souvent ils ont pour origine soit les spermatocytes de deuxieme ordre (d'apres Landing et collaborateurs), soit les spermatides, qui de tons les elements de la lignee, sont d'apres Kindred les plus sensibles aux effets de HJV^. Kindred a note I'existence en abondance de ces cellules geantes chez quatre animaux sur 18 ; il en a rencontre mais en beaucoup plus petit nombre, chez certains temoins. Pour expliquer la genese de ces elements, cet auteur suppose que HN^ (comme //Ag d'ailleurs) qui est un alkyl soluble dans les graisses, provoque une modification de la tension superficielle et finalement la degenerescence des barrieres qui separent les cytoplasmes des cellules seminales et entraine ainsi leur confusion. Le fait qu'un certain nombre d'obstacles proteiques ou autres, peuvent s'opposer avec un succes, variable d'un animal a un autre, au passage du toxique du plasma sanguin vers les cellules testiculaires, serait, d'apres Kindred, la raison des differences individuelles constatees quant au nombre present de cellules geantes. D'indiscutables analogies morphologiques existent entre ces groupements speciaux des testicules de souris et de rats et les formations plasmodiales que nous avons vues dans les ovaires de lapine. Notons cependant que dans ces derniers, I'element en cause est et ne pent etre que I'ovocyte I, alors que dans le testicule il s'agit de spermatocytes II ou de spermatides, n'ayant pas de correspondants dans la grande femelle. En outre, les spermatocytes et les spermatides ne sont separes les uns des autres que par leurs membranes cytoplasmiques, tandis que les ovocytes primordiaux sont deja entoures d'un fin treillis protecteur de quelques cellules folliculeuses aplaties ; de sorte que, pour realiser par un mecanisme analogue a celui admis par Kindred, le groupement des vesicules germinatives au sein d'une masse cytoplasmique commune, //.Vg devrait assurer la des- truction, non pas d'une simple lame cytoplasmique condensee, mais d'une veritable barriere cellulaire ; or, nous avons vu plus haut que cette destruc- tion de la granuleuse est responsable de la mort des follicules P isoles ; rien ne s'opposerait done a ce que cette meme lyse granuleuse — pour autant qu'elle soit limitee aux regions d'accolement d'un groupe de follicules P et respecte la region qui separe ce groupe du stroma ovarien — soit a I'origine des groupes plasmodiaux. Toutefois, la chronologic assez tardive de ce processus (qu'on ne voit clairement apres HJV^ seul qu'a partir de la 96° heure) et I'integrite remar- quable de la plupart des ovocytes contenus dans les amas, nous ont amene 353 2a INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHL0R0ETHYL) AMINE a reconsiderer son origine, dont I'essence degenerative — probable dans le testicule — nous parait ici moins evidente. Nous avons en effet signale que I'une des caracteristiques essentielles de I'ovaire yperite est une hyperproduction tres precoce (des la douzieme heure) de follicules jeunes A et B principalement, ce qui presuppose la mise en chantier rapide de nombreux follicules primordiaux ; cet effort a pour consequence un hatif 'depart en ligne' de groupes primordiaux dont les elements n'ont pas le loisir d'organiser une parol granuleuse individuelle ; seules, se divisent les cellules folliculeuses des elements du groupe situes a la peripheric, tandis que les autres — qui devraient assurer la formation des 'septa' entre les differents follicules P, s'atrophient et realisent ainsi la forma- tion de pseudoplasmodes. Quant a la raison pour laquelle ces groupes ne sont visibles qu'au dela de la 72° heure, alors que I'accroissement des elements A et B est deja tres net a la douzieme heure, nous la situons dans le systeme interpretatif suivant. Lorsqu'a lieu une poussee folliculaire, le nombre des elements P qui partent en evolution est conditionne par le nombre de follicules A qui se sont atresies lors du passage au stade suivant B ; si cette atresie est excessive (et c'est le cas sous I'influence de HN^, les follicules P se degagent de la reserve en beaucoup plus grand nombre, lyiais a partir d'elements dejd prepares a cet avenir. Et ce n'est que plus tard que I'effort de reparation demande au cortex ovarien met en jeu des formes primordiales non encore liberees les unes des autres et groupees en paquets dans les lacis conjonctifs qui occupent I'espace situe directement sous I'epithelium germinatif; le depart de ces follicules primordiaux se fait hativement et en groupes compacts, dont les elements peuvent aisement se confondre sous une enveloppe folliculeuse commune. Vue de cette maniere, la production de groupes primordiaux polynuclees serait done d'essence purement regeneratrice et traduirait simplement la hate excessive que met I'ovaire a preparer ceux de ses elements de reserve qui sont destines a combler les vides crees par I'atresie au sein des lignees de follicules evolutifs proprement dits. Une troisieme hypothese — d'ailleurs seduisante — nous a ete fort courtoise- ment soumise a Genes par Aron-*^ qui, frappe de I'analogie morphologique entre les amas plurinuclees decrits par nous et les cordons sous corticaux que montre I'ovaire prepuberal a la fin de I'ovogenese, nous a demande si nous ne devious pas considerer ces pseudoplasmodes comme la manifestation d'une neoformation d'ovocytes a partir de I'epithelium germinatif adulte. Nous accepterions volontiers cette suggestion si, malheureusement, elle ne se heurtait a la tres serieuse objection que nous avons de tout temps faite a I'idee — deja rejetee par de Winiwarter^' — de la persistance de potentia- lites ovogenetiques chez la lapine adulte, a savoir I'impossibilite de mettre en evidence, dans cette espece animale, les phases nucleaires successives qui conduisent du stade protobroque de I'ovogenie jeune au stade diplotene de I'ovocyte de premier ordre. Bien que puissent etre opposees a cette objection la notion probable de la fugacite de ces images transitionnelles et la demonstration par Gaillard^s d'une ovogenese indiscutable a partir de I'epithelium germinatif de frag- ments en culture d'ovaires de lapines adultes, nous croyons devoir nous 354 PAUL DESAIVE limiter — pour I'instant — a conferer aux formations polynuclees de Tovaire yperite une signification regeneratrice, en attribuant leur production a I'acceleration excessive de leur mise en chantier ; mais il est evident que la decouverte de figures de passage entre I'epithelium germinal et ces groupes pseudoplasmodiaux modifierait immediatement notre opinion actuelle an sujet de la genese de ces interessantes formations. II n'est pas sans interet de rappeler ici que dans le duodenum du rat, irradie (Engelstad-^. Warren et Friedman^") ou traite par HN^ (Desaive et Varetto-Denoel^) on pent voir, en doublure du fond des villosites, des formations pseudo-syncytiales analogues a celles observees dans le testicule et Tovaire, et dont I'origine — a partir des cellules generatrices en multipli- cation acceleree — est egalement de signification reparatrice. Nous avons deja signale que les ruptures folliculaires intra-ovariennes, exceptionnellement frequentes apres yperite, ont pour cause une alteration localisee de la parol granuleuse des grands follicules murissants. Quant aux elements a ovocytes multiples, le tableau de leur distribution dans les ovaires yperites qu'ils sont particulierement nombreux tout au debut de I'experience, a la douzieme heure, et a la fin de celle-ci, a la 96° heure. La premiere de ces deux poussees a vraisemblablement pour origine le brusque depart vers les stades A et suivants de follicules P prepares a cette evolution ; comme ce changement d'etat affecte un nombre de ces elements de loin superieur a la normale, on con^oit ainsement que deux ou trois follicules, ou d'avantage puissent rester accoles pendant les premieres phases de leur croissance, favorisant ainsi la production d'elements multiovulaires, moyens ou grands. La seconde poussee trouve une explication dans les groupes primordiaux polynuclees, qui, comme nous I'avons decrit plus haut, apparaissent aux environs du troisieme ou du quatrieme jour. II nous reste a discuter le comportement des ovaires chez des lapines preparees par le becaptan, avant d'etre injectees de HX^- Depuis la publication des recherches de Colter et Quastel-*^, on connait I'effet protecteur qu'exerce in vitro la trimethylamine a I'egard de Taction inhibitrice d'HX^ sur certains enzymes. Pour notre part, nous avons precedemment observe que le becaptan protege dans une certaine mesure, centre les effets de //Ag, I'intestin grele du rat, et nous venons de montrer qu'injecte avant HJV2, le becaptan abaisse, dans les premieres heures, le pourcentage atretique des follicules primordiaux et attenue par la suite le moindre passage, observe apres //.Ng, des stades moyens aux stades preovu- latoires F et G. En d'autres termes, le becaptan se comporte au niveau de I'ovaire comme un limitateur de I'atresie ; et il est remarquable c^ue ce meme antagonisme s'observe a I'egard des radiations ionisantes puisque nous av^ons signale anterieurement^'-, que le becaptan releve notablement le seuil de sterilisa- ion roentgenienne de I'ovaire. Ces observations nous paraissent etre en faveur d'une protection directe exercee par le becaptan, a la fois vis a vis des yperites azotees et des radiations ionisantes. En ce qui concerne I'ovaire, ce phenomene se situe soit dans I'ovocyte (bien cjue I'etat de repos nucleaire de cette cellule ne plaide pas en faveur de cette hypothese), soit plus vraisemblablement dans les cellules granuleuses qui, en constante multiplication a tous les etages de revolution 355 2a 2 INFLUENCE DES DOSES ELEVEES DE METHYL-BIS-(2-CHLOROETHYl) AMINE folliculaire, forment le feuillet trophique du follicule, et consiste probable- ment dans un processus de competition entre le becaptan et I'agent toxique employe, a I'egard des reactants cytoplasmiques ou nucleaires des cellules sensibles. Par ailleurs, nous devons reconnaitre que cette action directe du radio- protecteur n'aboutit qu'a des resultats protecteurs tres limites ; de nom- breuses experiences (que nous avons resumees et discutees dans notre travail precedent et sur lesquelles nous ne reviendrons pas ici) ont indiscutablement etabli que la radioprotection se traduit d'avantage par une accentuation du processus de reparation ; au niveau de I'ovaire, nous avons vu plus haut que ces manifestations regeneratrices se manifestent avant tout par I'appari- tion tres precoce de groupements polynuclees sous corticaux ; et nous esperons que de prochaines recherches permettront d'etablir de fa^on certaine les parts respectives que prennent, dans la couverture de I'organ- isme a I'egard des radiations ionisantes et des poisons radiomimetiques, I'intervention directe des substances protectrices a I'echelle de la cellule sensible, leur influence sur la genese d'eventuels facteurs humoraux regener- atifs ou encore leur participation a la liberation d'agents hormonaux, stimulants de la reparation. RESUME Les ovaires de lapines soumises a I'injection intraveineuse de 2mmg de methyl-bis-(2-chloroethyl) amine presentent d'importantes alterations qui comprennent essentiellement, une part de manifestations atretiques (trouble general du mecanisme de revolution et de I'involution folliculaires, accroisse- ment considerable de I'atresie des follicules primordiaux, diminution du nombre moyen des follicules murissants et atresie atypique-meroxanthoso- miale ou par rupture intra-ovarienne — de ces memes follicules) et une part de phenomenes regenerateurs (elevation des la douzieme heure du nombre de follicules jeunes, exces important de follicules pluriovulaires et apparition dans le cortex de groupements primordiaux plurinuclees). La preparation des animaux par I'introduction intraperitoneale de 50mmg de p-mercaptoethylamine (Becaptan Labaz), trois minutes avant I'injection intraveineuse de 2mmg de HJ\\, se traduit par : un trouble plus discret du mecanisme complexe de revolution et de I'involution folliculaires, une moindre atresie des follicules primordiaux dans les premieres heures, le maintien a un taux moyen plus eleve des grands follicules preovulatoires, la surabondance de follicules multiovocytaires et I'extreme precocite d'appa- rition dans le cortex de groupements primordiaux polynuclees. Les experiences ci-dessus relatees confirment done les resultats que nous avons obtenus au niveau de I'ovaire au moyen de doses plus faibles de HN^ ; bien que les ovaires leses par les doses — non lethales — de HM^ qi^ie nous avons employees soient capables de retrouver rapidement une apparente integrite, il ne parait pas douteux que les derives bi- ou tri-methyles du gaz moutarde soient en mesure de provoquer chez la femme des alterations plus ou moins profondes de la fonction ovarienne dont la possibilite d'apparition doit etre connue des cancerologues cliniciens. La p-mercaptoethylamine, introduite a la dose de 50mmg dans la cavite intraperitoneale quelques moments avant I'injection intraveineuse de 2 mmg 356 PAUL DESAIVE de HJ\\, s'oppose dans une certaine mesure aux effets atresianls de ce poison et accentue, en les hatant, les processus folliculaires reactionnels a caractere reparateur. .Xous adressons tons nos remerciements a Messieurs les Docteurs A. Herve et J. Closon ainsi qua Madame Varetto-Denoel, Mademoiselle T. Francis et Monsieur G. Thiry pour leur assistance technique. REFERENCES 1 Lacassagne, a. £tude physiologique et histologique des effets produits sur I'ovaire par les rayons X — These de Lyon — 1913. 2 Gricouroff, G. Radiophysiologie et radiotherapie, 1931, 2 fasc. 1. 3 Desaive, p. Contribution radiobiologique a I'etude de I'ovaire — These 1940. * Desaive, P. Communication — 41 e Reunion de I'Association des Anatomistes — Genes, avril 1954, sous presse. 5 Bacq, Z. M. et coll. Science, 1953, 117 633. ® Desaive, P. et Varetto-Denoel, Jos. Communication — 6e Congres Inter- national de Lutte contre le Cancer, Sao Paulo, juillet 1954. ^ Desaive, P. Arch. Biol., Tome LVHI, fasc. 4, 1947, et Tome LIX, fasc. 1, 1948. 8 Desaive, P. Arch. Biol., Tome LX, fasc. 4, 1949. ^ Auerbach, C. et Robson, J. Rev. Min. Suppl. no. W.3979, 1942. (Cites par Butler et Randall, Progress in Biophysics, Londres, 1954, vol. 4.). 1" Roller, P. C. Proc. Roy. Soc. (Edimbourg), 1943, 61 398. 11 Darlington, C. D. et Roller, P. C. Heredity, 1947, 1 187. 12 Gillette, R. et Bodenstein, D. J. Exp. ^ool. 1946, 103 no. 1. 13 Bodenstein, D. J. Exp. Zool. 1947, 104 31 1, et 1948, 108 93. 14 GiLMAN, A. et Philips, F. S. Science, 1946, 103 409. 15 Ross, W. C. J. Nature, 1950, 165 108. i« Loveless, A. et Revell, S. Nature, 1949, 164 938. 1" Ford, C. E. Brit. J. Radiol. 1952, 25 189. 18 Roller, P. C. et Casarini, A. Brit. J. Surg. 1952, 6 173. 1^ Revell, S. Heredity, 1953, 6 suppl. 107. -° Roller, P. C. In Progress in Biophysics, 1954, 4 195. London, Pergamon Press Ltd. -1 Auerbach, C. et Moser, H. Experientia, 1951, 7 341. 22 Hicks, S. Journ. of Cellular and Comparative Physiology, 1954, 43 suppl. 1, 151. 2 3 Spitz, S. Cancer, 1948, 1 383. 2* Landing, B. H., Goldin, A., Noe, H. E., Goldberg, B. et Shapiro, D. N. Cancer, 1949, 2 1055. 25 Rindred, J. Arch. Pathol. 1947, 43 253. 2^ Aron, M. (Communication verbale.) 2- De Winiwarter, H. Arch. Biol. 1910, 25 169, 683. 28 Gaillard, J. P. (Communication verbale.) 29 Engelstad, R. B. Amer. J. Rontgenol. 1938, 40 243. 30 Warren, S. et Friedman, N. B. Cites par Friedman, Arch. Pathol. 1942, 34 749. 31 Colter, J. S. et Quastel, J. H. Nature, 1950, 166 773. 32 Desaive, P. Acta Radiologica, 1954, 41 545. 357 HISTOLOGICAL CHANGES IN MAN AND RABBITS AFTER PARENTERAL THORIUM ADMINISTRATION Charles Johansen Finsen Institute, Copenhagen Thorium has a half-life of 1 -4 X 10^" years, the decay products have an appreciably shorter life, and the activity of 4 g of thorium is fairly equivalent to that of l[jig of radium. 90 per cent of the activity of thorium is due to the alpha activity, 9 per cent to the beta and 1 per cent to the gamma activity. The alpha radiation is thus completely dominant. Thorium is generally used as a 20 per cent colloidal suspension of thorium dioxide, that is to say 20 ml of this suspension are equivalent to about ly,g of radium. It is well known from the literature that thorium is practically not excreted from the organism and, in the case of intravenous and intra-arterial adminis- tration, is deposited in the cells of the reticulo-endothelial system, and therefore especially in the spleen, the liver and the active bone marrow ; furthermore, it is known that in and around the thorium deposits, degenerative changes and fibrosis are found. When thorium is injected outside the blood-stream, the greater part remains at the site of injection and may here produce more distinctive changes, as will be shown here in the vascular wall at the site of injection, in paravascular deposits and in the articular capsule when it has been used for arthrography. It has been discussed in the literature whether the effects of thorium are so detrimental that its use is dangerous. A few cases of aplastic anaemias have thus been mentioned (Spier and co-workers^, Schmidt and co-workers-), and the occurrence of a few tumours has been reported (MacMahon and co-workers^, Ludin*, Horta^, Zollinger*', Abrahamson and O'Con- nor'), and animal experiments on rats have shown an indubitable carcino- genic effect of thorium. Other authors (for instance Lima^) consider that they have seen no harmful sequelae of the use of thorium, while Thomas^ and co-workers adopt a view between these two extremes ; on the basis of a series of 4,300 patients they consider that they have seen no severe sequelae but, owing to a tendency to fibrosis of the liver and the spleen and to cica- tricial changes, they nevertheless think that thorium should only be used when the indications weigh much in favour of such treatment. Our series comprises about 250 patients who received doses equivalent to from 0-5 to 5[ig of radium. Four of the patients died of diseases of the blood. These were : (1) A man, aged 40, who 1 1 years earlier had been given doses of thorium equiv- alent to about 1 (xg of radium. He now had a severe, aregenerative anaemia with a 358 CHARLES JOHANSEN Hgb. percentage of about 30. In the course of 2 years he received about 65 blood transfusions, but then died of agranulocytosis with sepsis and severe haemosiderosis. (2) A woman, aged 37. who 7 years earlier had received an injection of a quantity of thorium equivalent to 1 to 2iJig of radium. She died of typical myelosclerosis. (3) A man. aged 60, who 10 years earlier had had a dose of thorium deposited which was equivalent to 1 jjig of radium. He died of chronic myelogenic leukaemia. (4) A man, aged about 40, who died of stem-cell leukaemia 14 years after he had been contaminated with a thorium dose equivalent to 2[i.g of radium. On the other hand, our series showed no increased incidence of cancer and contained no cases of maHgnant tumours whose occurrence could be reason- ably attributed to the thorium contamination. In experiments on rabbits I have given intravenous injections of doses of thorium equivalent to from 0 • 005 to 0 • 1 25 [xg of radium per kg. Hitherto, none of these animals have presented leukaemic changes, but so far the cause of death in 8 animals has been a disseminated, malignant tumour, a reticulo-endothelio-sarcoma. These animals had all been given doses of thorium equivalent to 0-07(j.g of radium per kg, or more, and they died between 2 and 3 years after the injection. Presumably a much greater number of animals in the groups will present this form of tumour, since, so far, all animals which received these doses and died more than 2 years after the injection had this tumour as cause of death. None of the controls has presented tumours of any kind. With regard to the malignancy of the tumour, there can be no doubt, as it can be transplanted. After intra- muscular injection of tumour tissue, cortisone-treated animals showed tumour- cell emboli in the pulmonary capillaries a few days later, and a few animals have had tumours in the lungs. Exactly the same form of tumour has been found in human beings in three cases, all following injection of thorium into the blood-stream. Horta's^ and Liidin's* patients thus died 3 and 14 years respectively after injections of thorium doses equivalent to about ly.g of radium, and MacMahon's^ patient died 14 years after injection of a dose corresponding to about 4[xg of radium. This last-mentioned patient died of intraperitoneal haemorrhage after spontaneous rupture of the tumour, and I may mention that 2 of my rabbits showed the same cause of death. This form of tumour may thus occur in both man and rabbits, and is presumably one that is specific of thorium deposition in the parenchymatous organs. It may therefore seem strange that we have not observed one case of this tumour in our series of about 250 persons. In my opinion this is just a question of time. Our series exclusively comprises persons who were contaminated with thorium from 4 to 16 years ago, and we know nothing definitely about the length of the period that will pass before radiation-induced tumours in man manifest themselves clinically. Several scientists, however, suggest that radiation-induced cancers will appear from 10-30 years after the contamination with an optimum time of 16 years. If we acknowledge this assumption the following calculations may give an idea of the cancer problem for our patients. In Figure 1 the block diagram to the left gives absolute numbers of patients as ordinates and years after contamination as abscissa ; it is first noted that only 3 of the patients have passed the 16-years limit. To the right a Gaussian-like curve 359 HISTOLOGICAL CHANGES AFTER PARENTERAL THORIUM ADMINISTRATION for assumed cancer incidence has been drawn ; the optimum is at 16 years and the ordinate is arbitrary as nothing is known about the percentage incidence ; for the uranium mine workers the cancer incidence was about OVr- Possible cancer incidence 10 ;^ n m m eo ze Duration of observation Figure 1 Sf 26 28 30 ^jt Unknown 50 per cent, and for the New Jersey patients the cancer incidence has been about 10 per cent so far in a non-selected material*. It is evident from Figure 1 that we must wait about 10 years before we can answer the cancer question and about 20 years before it can be fully cartographed. REFERENCES 1 Spier,.!., Cluff, L. E. and Urry, W. D. J. Lah. Clin. Med. 1947, 32 147. 2 Schmidt, W., Schultz, A. and Lapp, H. Strahlentherapie, 1950, 81 93. 3 MacMahon, H. E., Murphy, A. S. and Bates, M. I. Amer. J. Path. 1947, 23 585. * LiJDiN, M. Schweiz. Z- Allg. Path. 1953, 16 987. 5 HoRTA DA SiLVA, J. Ckirurg. 1953, 218. 6 Zollinger, H. V. Schweiz. Med. Wsch. 1949, 52 1266. * Abrahamson, L., O'Connor, M. H. Irish J. med. Sci. 1950, 6 229. * Lima, P. A. Cerebral Angiography, London, 1950. » Thomas. Cited Loeb, V., Jr., Seamann. W. B. and Moore, C. V. Blood, 1952, 7 915. DISCUSSION M. A. Gerebtzoff : Errera has asked me to present a document showing the long persistence of thorium in the human organism. It is an autoradiography of a liver preparation taken from a man who had received an injection of thorotrast some 20 years previously. One still sees thorium granules from which escape a-particles. * AuB, Evans, Hempelmann and Martland [Medicine 31, Sept. 1952) describe a cancer-incidence of 33 per cent in their selected material of patients with deposits of 0 • 7-7 [ig C Ra± MsTh. 360 INDEX TO CONTRIBUTORS Bold figures refer to papers read ; ordinary figures indicate contribution to discussion Abbatt, J. D., 229 Alexander, P., 49, 119 Alper, T., 39 Bacq, Z. M., 25, 116, 117, 118, 150, 224, 232, 290, 310 Barnes, D. W. H., 134 Bekkum van, D. W., 201 Belcher, E. H., 136 Betz, E. H., 117, 169, 228,292,312, 313 Bonet-Maury, P., 75 Burg, C, 1, 67, 168 Butler,J. A. v., 46, 118,280 Buu-Hoi, N. P., 64 Charlesby, A., 49 Chevallier, A., 1, 25 Cole, L. J., 141, 133, 147 Court-Brown, W. M., 229, 234 Craig, Doris L., 262 Desaive, P., 340 Deschner, Eleanor E., 275 Devik, F., 313 Doudney, C. O., 112 Dunjic, A., 154 Duplan, J.-F., 64, 67 Ebert, M., 30 Ehrenberg, L., 74, 104, 285 Eldjarn, E., 1 16 Ellis, F., 216 Ellis, Marie E., 141 Elson, L. A., 235 Ephrussi-Tavlor, H., 79 Errera, M., 74, 93 Fievez, C, 304 Forssberg, A., 148 Fox, M., 61 Gaston, Evelyno, 122 Gerebtzoff, M. A., 290, 312, 360 Gray, L. H., 277 Gregoire, S., 110 Gros, C. H., 210 Gustafsson, A., 282, 284 Haigh, M. V., 92 Herve, A., 194, 232, 298 Hevesy, G., 148 Hollaender, A., 112 Holmes, Barbara E., 220 Horgan, V, J., 26 Howard, A., 224 Jacobson, L. O., 122 Johansen, C, 358 Jolles, B., 140, 151, 169, 190 Kirby-Smith, J. S., 262 Koch, G., 110 Lacassagne, A., 64 Lajtha, L. G., 216 Lamerton, L. F., 136 Lane, G. Roy, 265 Laser, H., 68 Lasnitzki, Use, 321 Latarjet, R., 79, 120, 339 Laws, J. O., 190 Lourau-Pitres, Mme., 225, 228 Loutit, J. F., 134 Magat, M., 38, 59, 118 Maisin, J., 154, 304 Mandel, P., 117, 210, 228 Marks, Edna K., 122 Mee, Lorna K., 45, 220 Mikaelsen, K., 316 Mitchell, J. S., 170, 313 Mole, R. H., 104, 150, 209 Mouton, R. F., 147 Munson, R. J., 104 Neary, G. J., 104 Neukomm, S., 189, 298 Newcombe, H. B., 120, 326 Nizet, A., 194 Oliver, R., 216, 219 Ostergren, G., 280 Patt, H. M., 105, 117, 312 Peguiron, L., Mme., 298 361 INDEX TO CONTRIBUTORS Philpot, J. St. L., 26 Sheppard, C. W., 262 Pollard, E., 70 Simson, Miss P., 46 Sobels, F. H., 69, 284 Suit, H., 199 Rajewsky, B., 81 Swanson, C. P., 254 Revell, S. H., 243, 279 Rodesch, J., 210 Verly, W. G., 110 362