Karolinska Inatitutet) Stockholm, Sweden

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NO. IAEA-H-764-F • '

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TITLE I. '- Experimental Investigations on; the Interplay between natural and artificial radiation protection

FINAL REPORT FOR THE PERIOD

1 .July; 1969; - 3OvJune 0.972

' AIJTHOH(S)

• !. L.Révésis

INSTITUTE

Dept. cf Tumor Biology Karolinslra Institutet (inst. Fiir TumiSrbiologi Karolinska Inatitutet) Stockholm, Sweden

-#" IHTERMTIOML ATOMIC ACEWCY lÉMÉÍffiafeÉ^ i -

INST. FOR TUMORBIOLOGI KAROUNSKAINSTITUTET m 10401 STOCKHOLM 60 DEPT. OF TUMOR BIOLOGY KAROLINSKA INSTÍ.TUTET S-104 01 STOCKHOLM 60 SWEDEN

. CERTIFIED

FINAL REPORT

Research Contract Ho.: 764 Title of Research Project: Experimental investigations o'n the interplay between natural and artificial radiation protection. Institute where research is being carried out: Department of Tumor Biology» Royal Karolinska Institute Medical School, Stockholm, Sweden. Principal scientific investigator: László Révész, H.D., Professor. Time period covered: 1 July 1969 - 30 June 1972.

The experimental investigations, carried put during the timé period covered by the original contract and the subsequent renewal contracts, closely followed the program outlined in our original grant application of 1968 and the complementary plans indicated in the"subsequent renewal applications. The work carried out, the results obtained and:.the conclusions drawn are described in details in the publications which are listed in;this report (vide infra) and of which copies have been submitted to the Agency. The work can be summarized in the following.: . :::' í '::.,~>: "-r^-••'••'•' '.•••"'".'•' - .'•"•-'.••" "•'' • •"". • "".-.'"-•-' -.''ii~:: In general, the investigatibnsTeprésént á contri bütiónto studies of the - factors which determine the radiation sensitivity and -resistance in mammalian cells. In particular, the interaction between natural and artificial protection was studied in line with the recommendations of a panel convened by IAEA in Vienna in October 1968 and which emphasized the importance of thiols in the function of^bfological inaciomolecules and in-the contfcol^ot resulatqry processes in the cell. Accordingly, effort was made to investigate, on the one hand, the effect of known protectors on the intrinsic protection afforded by thiols inherent to cells and, on the other hand, to study the effect of

imm

• ••^«^¡úlKij treatment of such protectors on the recovery process from sublethal radiation damage and on the DNA repair processes. Fro this purpose, several of the methods and the techniques we had developed in the past and many observations vje had made in our "previous experiments could be used with advantage. Thus, the correlation between radiosensitivity and thiol content in closely related cell-lines, observations on the changes in the amount of the cellular sulphydryl-groups which indicated the release of internal protectors by treatment with internal protectors, the oxygen dependency of the recovery process, and related observations served as the main basis of our investigations. One main line of the study concerned a closer characterization of the content and interrelated chc.iges in the amount of different intrinsic cellular sulphydryl-grpup-containing substances after treatment with various radioprotective agents in relation to the sensitivity variations to X-rays. Accordingly, answer was sought to such questions as to the composition of the sulphydryl- containing substances which are liberated by different radioprotectors; the localization of the liberated substances; the correlation between the liberated ¡1" substances and the extent of radioprotection; the time scale of the liberation and subsequent renewed binding of the cellular thiols released by the protectors; and the extent to which variation of the oxygen tension influences this proce'ss. Another main line of research concerned the variation in the extent of cellular recovery and DNA repair in connection with split-dose exposure of protector-treated cells to X-rays in the presence or absence of oxygen. In these investigations answer was sought to questions, in the first hand, on the relationship between the recoverabi1>ty of the radiation damage, on the relationship betv/een the prolongation of the recoverable period of radiation injury and the dosage of the protector, the interference of the protectors with oxidative metabolism required by the recovery process and the influence of radioprotectors upon the fixation of the radiation damage. In the preliminary experiments, a series of methodological problems were solved. Among others, a series of cell-line: with varying intrinsic sulphydryl content were developed taking advantage of a mutation-selection process in connection with sublethal irradiations. A similar procedure was used in deve- loping closely related cell-lines with varying radiosensitivity. For studies on the effect of external protectors on the physiological protection, an in vitro method was worked out implying the incubation of cell suspensions in a medium supplemented with different protectors in varying concentrations. A chromatographic technique by gel-filtration used for the identification of different sulphydryl-group-containing^compounds. A special system was built and particular techniques developed fqr.the control of the oxygen concentra- tion during irradiation of cells in vitro which permitted treatments in complete anoxia or in varying grades of hypoxia. Severa.1 methods were modified to adapt them to our particular, experimental conditions. Such modifications concerned tests on the clonogenic survival of. irradiated cells¿isotope labeling of'the cellular sulphur atoms or the method of identifying the redox: state of the respiratory chain components, by taking advantage of the difference in their absórbante or their induced fluorescence. "These methodological-studies have been described in détails'irj our publications listed below. .1" 1 ;The question to "what extent cysteaminé: influences the JcenularprocessC of recovery from radiation damage was studied by an analysis of the" survival, > parameters of oxically and anoxically irradiated cells pretreated1with gystea- - mine. The data indicated that cysteamine decreases the induction of lethalr í~ ; ri^Jltí^^^gej^J^^tí^^^lJ a it enhancesennces? the rrecovere y Jftrpm sublethal damage. ; : Particular experiments wéfe^deTrgnédTiñ'ísíuddeTrgnédTiñ'ísíudy 'imé^Tás'eTy^thVn^nani^insL^^'^y^i^ ^ underlying these effects. A chemical{exchange reaction was found between cySteamine and the cellular mixed disulphidesresuTting; in an intracellular ^ release of low-molecular sulphydryl-group-containing compounds. This exchange reaction was shown to occur also in deoxyribonucjeoproteins isolated from cell

/ -l-i 1 ~¥V>£3&tRB& nuclei to which cysteamine was bound. The substances released by the exchange reaction were identified to consist almost exclusively of glutathione. When the free thiol groups in the deoxyribonucleopro^eins were blocked specifically by NEM, the exchange reaction, though to a lesser extent, still occurred indicating that cysteamine interacts also with disulphide bonds in addition to the free sulphydryl groups. In regard to the recovery process, cysteamine v/as found to prolong the period during which radiation damage remains recoverable. Without cysteamine treatment, fixation of the radiation damage was found to occur within a few minutes. The damage of the anoxically irradiated and cysteamine treated cells v/as shov/n to remain amenable for repair for at least one hour. The data suggested that the fixation of the damage is in itself an enzymatic process, and the prolongation of the recoverable period may be due to an inhibition of the enzyme activity by cysteamine. Different experimental systems involving radiation exposures to split- doses of X-rays were used in the study of the oxygen dependence of the recovery process. Oxygen was shown to act as an modifying agent to lethal damage as well as to the -recovery from sublethal' damage. The summated result of the oxygen dependent recovery and oxygen dependent sensitization was shown to give an oxygen protecting or sensitizing ratio the magnitude of which is related to the radiation dose. This relationship was studied in great details in regard to its practical importance in the radiation therapy of tumours. It was con- firmed in mcdel experiments that, after irradiation with small doses, recovery from radiation damage due to oxygen can compensate for the increase of the radiation sensitivity which is attributable to oxygen as-well. On the other hand, after large doses, the extent to which recovery of the oxically irradiated cells can contribute to the protection, is decreased. These observations served as the basis for the development of a new dose fractionation regime which is tested clinically at the Oncology Institute in Bratislava. The preliminary data which have been published indicate that the regression pattern of tumours does not differ appreciably from the pattern noted after conventional treatment but healthy tissue reactions are considerably decreased suggesting an improved therapeutic ratio by the new treatment regime. The alkalyne sucrose gradient technique was used in studies concerned with rejoining of single strand breaks in the DNA after irradiation of cells in the presence of absence of oxygen with or without added cysteamine. The data indicated that cysteamine protected the DMA when irradiation v/as made under oxic but notwhen irradiation was delivered under anoxic conditions. It could be concluded that cysteamine probably protects against the induction of single strand breaks by oxygen removal. The post-irradiation repair process was not influenced appreciably by cysteamine either oxic or anoxic condition prevailed during irradiation.. . Besides the main research lines of investigations sunmiarized above, answer was sought alsoto several related, :,minor problems the clarification of v/hich was considered essential in the course of these studies. Such Was; the question of the. penetration of cysteamine over the cell membranes. It was found that it is largely dependent upon the py of the medium. At morei alkaline p\\ the irK creased dissociation of cysteannne-facilitated the passage through cell.membranes. :.'••"'';: In conclusion, these studies indicate an intimate interplay between arti- ;o pf ieíal=and:^naturá^ shjpwn^tqr^cóncern V :: protection both from the 1 éthai: and súblethál effects ofHradijafifnTTRRancemlrit"?: of."tWe intrinsic protecting mechanism may represent á main action of treatment' with chemical radioprotectors. The work summarized in this final report is described in detail in fifteen papers which have been published or are in the press. Reprints, >•.- galley-proofs or manuscripts of these papers have previously been submitted to the Agency with the exception of, three articles whichhave been^completed since pur previous progress report was written and which are attached to this final report. Upon the present completion of the research project agreed in the Contract-* I wish to express my sincere thanks and hiqh appreciation for the generous support received from the Agency and which was a most valuable contribution in permitting us to perform this study.

Stockholm, July 12, 1972

L.: Réyészy H.D. Professor

3 • , !

I?^ 5.

PAPERS PUBLISHED ON THE WORK DONE UNDER THE CONTRACT

SIRACKA, E., LITTBRAfJD, B., REVESZ, L., Effect of oxygen on the recovery of lethal and non-lethal radiation damage. Exptl. Cell Res. 58 (1969) 191-194.

REVESZ, L., LITTBRAND, B., Grov/th of tumor cell populations after single or fractionated exposures to X-rays in the presence or absence of oxygen..Gann Monograph, Tokyo, £ (1970) 255-261.

REVESZ, L., LITTBRAND, B., Chemical protection of the recovery process. "Biological Aspects of Radiation Protection" (SUGAHARA, T., HUG, 0., Editors), Igaku Shoin Ltd., Tokyo, (1971) 195-200. ** '

REVESZ, L., LITTBRAND, B., Radiation damage and repair in cysteamine treated cells. Acta RadioL 10 (1971) 257-266.

REVESZ, L.f LITTBRAND, B., Izmenenie velichin kislorognogo effekta pri razlichnyh dozah Rentgenovskogo oblicheniya i ego vozmozhnoe klinicheskoe znachenie (The change in the extent of the oxygen effect at different doses of Roentgen irradia-

tion and its clinical importance). Radiobio1ogiya,;Moscow¿ 21 (1971) 383-386.

F.0DIG, H.G., EDGREN, M., RLVESZ, L., Effect of pH on the uptake of cysteamine in vitro and in,vivo. Int. J. Radiat. Biol. 20 (1971) 297-299.

REVESZ, L.s A sugarserulesek cellularis reparacioja (Cellular recovery from radiation injury). Magyar Radiología,'Budapest, 23_ (1971) 129-137.

REVESZ, L., LITTBRAND, B., Energy dependence of cellular recovery from sublethal radiation injury: its possible importance in fractionated radiotherapy. Proc. First European Biophysics Congress 2_ (1971) 155-159.

SIRACKA, E., DURKOVSKY, J., JANCINA, J., REVESZ, L., Radiotherapy of tumors by a dynamic dose fractionation on regime combined with oxygen inhalation. Neoplasma US (1971) 607-616. 6.

LITTBRAND, B., Oxygen dependent radiosensitivity variation of mammalian cells. Balder Press, Stockholm (1971).

MODIG, H.G., EDGREN, M., Repair of single strand breaks in the DNA of cysteamine treated Chinese hamster cells after irradiation under oxic and anoxic conditions. Studia Biophys. 29 (1971); 117-124.

MODIG, H., EDGREN, H., REVESZ, L., Release of thiols from cellular mixed disul- phides and its possible role in radiation protection. Int. J. Radiat. Biol. (1972), in the press.

REVESZ, L., LITTBRAND, B., Variation of the oxygen enhancement ratio at different X-ray dose levels and its possible clinical significance. Proc. Fourth Internat. Radiation Research Congress (1972), in the press.

- * • HAOT, J., HIESCHE, K., REVESZ, L., Sensitivity of irradiated and normal bone marrow to H-2 antiserum. Arch. Pathol. (1972), in-the press.

REVESZ, L., Dependence of dose-fractionation on the oxygenation of tumors. Fraction Size in Radiobiology and Radiotherapy (REVESZ, L., SCOTT, O.C.A., SUGAHARA, T., Editors). Igaku Shoin, Ltd., Tokyo (1973), in the press.

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RESEARCH CONTRACT No, 764 Summary of final report

TITLE Experimental, investigations on the interplay between natural and artificial radiation protection. RESEARCH INSTITUTION Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden CHIEF SCIENTIFIC INVESTIGATOR

L. Révész.

PERIOD OF CONTRACT

1 July 1969 - 30 June 1972

SCIENTIFIC BACKGROUND AND SCOPE OF PROJECT „ A panel convened by IAEA in Vienna 21 - 25 October, 1968 and which reviewed the progress in our knowledge of radiation damage to the biological molecular informations system with special regard to the role of SH-groups, emphasized the importance of thiols in the function of biological ma.cromoleculesand in the control of regulatory processes. In-line with the conclusion and recommenda- tion of this panel to intensify the study of the influence of SHrgroup-containing radioprotectors upon the repair and recovery mechanisms, the general purpose of our investigations was to examine the interaction between natural and artificial protection. In particular we planned to investigate, on the one hand, the effect of known protectors on the.intrinsic protection afforded by the SK-group-containing compounds inherent to cells and, on the other hand, the effect of treatment by known protectors upon the recovery process from sublethal radiation :.¡l\ injury as well as on the repair processes¿especially, those concerned with damaged DNA. The' cbrrélatiórKbetween radiosensitivity and thiol content in closely.related cell-lines,^observations on the changes in the amount of the sulphydryl-grbups in the cells indicating an extensive mobilization of a reser- voir of internal protectors by treatment with artificial protectors, the «xygen dependency,of the recovery process served, among other observations, as the main basis of the investigations¿ The character, content, absolute and interrelated changes in. the amount cf different, intrinsic cellular sulphydryl-group-con-' taining:substances after/treatment with various radioprotective agents, as . related to the sensitivity variations to X-rays, represented the immediate research objectives of one main line of the investigations. The objectives of an other line of research were the Variaticasin the extent of cellular recovery : and.DNA repairL jn connection-:withTSpl4t-d6se;exposure.of protector-treated cells\'. to X-rays under oxic and,ahoxic conditions with: the purpose to obtain quantita- *: tive; information, among other questibns, on the relationship, between the pro- ; longátioñ of the recoverable period of radiation injury and the dose of protector, the influence of the radioprotectors upon the fixation of the radiation damage; and the interference af the protec£ors_with the oxidative metabolism required by the recovery process. " ~ - EXPERIMENTAL METHOD Methods, worked out previously and implying the incubation of cell suspensions in a medium supplemented with the particular protector in varying concentrations were used, in general, to study the effect of external protectors on the physiological protection. Cell-lines with varying intrinsicSH content • and varying radiosensitivity have been developed by serial, sublethal irradiations for this type ot'experiments. Sulphydryl-group-cpntainirig compounds were identified chromotographically by gel-filtration, while the radiosensitivity changes were determined by establishing the clonogenic survival of the irradiated cells. A special technique was worked out to control the oxygen concentra- tion during irradiation of cells, which permitted treatments in complete anoxia on in varying grades of hypoxia. These techniques were combined with the alkalyne sucrose gradient method in studies concerned with DNA damage and repair. The method developed by Chance and associates taking advantage of the differences •;. in absorbance between oxidized and reduced forms of the respiratory chain components viere used in the studies of the intracellular redox states in the cells. Advantage was taken of cells prelabeled with S35 cystein for the identification of the innerent thiols in experiments with cold extrinsic thiols. Isotope labeled protectors in combination with cold cells viere used in the complementary experiments for the study of the chemical exchange reaction between the internal, natural and external, artificial protectors. , RESULTS OBTAINED Analysis of the survival parameters of oxically and. arioxically irradiated, cysteamine-treated ceils indicated that this sulphydryl-group-containir.g radiation protector decreases the induction of lethal radiation damage as well as it contributes, at the same time, to enhance recovery from sublethal damage. The intracellular release by an exchange reaction of low:molecular, sulphydryl- group-containing compounds was suggested, in part, as the mechanism of action for the reduction of lethal damage. The great majority of .these compounds was identified as glutathione. Experimental proof was obtained that deoxyribonucleo- proteinsare also involved in the exchange reaction. Further data indicated;that cystéamine interacts also with disulphide bonds, in addition to the sulphydryl groups. As for the mechanism of effect on the recovery process,,the.data were ; in agreement with the assumption that cysteamine prolongs the period during which radiation damage remains recoverable arid, thus, amenable for repair; With- ... out cysteamine treatment, fixation of the radiation damage was found;to be very rapid,. Following: anoxic irradiation, it is completed within a few minutes after which access to oxygen does not any longer permit recovery. ! Oxygen was shown to.act as a modifying agent to lethal damage arid to; rer covery from sublethal damage. The oxygen dependence ,of the recovery process, :. ,: was established;in;;several¿.different experimental Systems inyolving=exposures ;. to split-doses; ofX-rays. The sunnated result of efficient repair (protection) . J?, and increased cell damage (sensifization) by oxygen was shown to give an oxygen protecting or sensitizing ratio the magnitude of which is related to the radiation dose. Cysteamine protected DNA against breaks when irradiation was made under oxic but not under anoxic conditions. This protector did not influence the extent of repair when either oxic or anoxic conditions prevailed under irradiation. The data suggested that protection against single strand breaks is due probably to the removal of oxygen. Methodological experiments demonstrated that the penetration of cysteamine over the cell membranes depends largely upon the pH of the medium. Increased dissociation of cysteamine at more alkaline pH may facilitate the passage through the membranes.

<- 1- CONCLUSION Our study revealed an intimate interplay between artificial and natural radiation protection. The interplay concerns protection both from the lethal and < sub-lethal' effects of radiation. Enhancement of the intrinsic protecting mechnisms may. represent a;niain: action of treatment with chemical radiopro- tectqrs. •-,•'• ."-.. •',•/••>":'• '•'/•". '.' ''•'-'-':- •''••' PAPERS PUBLISHED ON THP; WORK DONE UNDER THE CONTRACT . SIRACKA, E., LITTBRAND, B., REVESZ, L., Effect of oxygen on the recovery of lethal and non-lethal radiation damage. Exptl. Cell Res. 58 (1969) 191-194. REVESZ, L., LITTBRAND, B., Growth of tumor cell populations after single or fractionated exposures to X-rays in the presence or absence of oxygen. Gann Monograph, Tokyo, £ (1970) 255-261. REVESZ, L., LITTBRAND, B., Chemical protection of the recovery process. "Biological Aspects of Radiation Protection" (SUGAHARA, T., HUG, 0., Editors), Igaku Shoin Ltd., Tokyo, (1971) 195-200. REVESZ, L., LITTBRAND, B., Radiation damage and repair in cysteamine treated cells. Acta Radio!. j[0 (1971) 257-266. REVESZ, L., LITTBRAND, B., Ismenenie velichin kislorognojo effekta pri razlichnyh dozah Rentgenovskcgo oblicheniya i ego vozmozhnoe klinicheskoe znachenie (The change in the extent of the oxygen effect at different doses of Roentgen irradiation and its clinical importanre). Radiobiologiya, Moscow, VT (1971) 383-386.

MODIG, H.G,, EDGREN, M., REVESZ, L., Effect of pH on the uptake of cysteamine in vitro and in vivo. Int. J. Radiat. Biol. 20 (T971) 297-299. REVESZ, L., A sugarserulesek cellularis reparacioja (Cellular recovery from radiation injury). Magyar Radiologia, Budapest, 23_ (1961) 129-137. REVESZ, L., LITTBRAND, B., Energy dependence of cellular recovery from sublethal radiation injury: its possible importance in fractionated radiotherapy. Proc. First European Biophysics Congress £ (1971) 155-159. ; • SIRACKA, C, DURKOVSKY, J., JANCINA, J., REVESZ, L., Radiotherafiy.,of tumors by a dynamic dose fractionation on regime combined with oxygen, inhalation. ; ; Neoplasma ljj (1971) 607-616. , •,£ . LITTBRAND, B., Oxygen dependent radiosensitivity variation of mammalian cells. Balder Press, Stockholm (1971). " ;v MODIG, H.G., EDGREN, M., Repair of single strand breaks in.the DNA of cysteamine treated Chinese hamster cells after irradiation under oxic and anoxic conditions. Studia Biophys. 29 (T971) 117-124. MODIG, H.G., EDGREN, M., REVESZ, L., Release of thiols from cellular mixed disul- phides and its possible role in radiation protection. Int. J. Radiat. Biol. (1972), in the press. REVESZ, L., LITTBRAND, B., Variation of the oxygen enfaancement ratio at different X-ray3dose levels ancTits possible'clinical significance. Prec. Fourth Internat. Radiation Research Congress (1972), in the press. - ~ ' ,í^aiiKsÉS§3^í^^

HAOT, J., HIESCHE, K., REVESZ, L., Sensitivity of irradiated and normal bone marrow to H-2 anti serum. Arch. Pathol. (1972), in the press. REVÉSZ, Lv, Dependence of dose-fFactionation on the oxygenation of tumors. Fraction SizeinRadiobiology and Radiotherapy (REVESZ, L., SCOTT, O.C.A., SUGAHAR, T., Editors). Igaku Shoin, Ltd., Tokyo (1973), in the press.