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Karolinska Inatitutet) Stockholm, Sweden NO. IAEA-H-764-F • ' •/•••', 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 comple- mentary 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 protec- tion 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 sul- phydryl-groups which indicated the release of internal protectors by treat- ment 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 sul- phydryl-grpup-containing substances after treatment with various radiopro- tective 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 pro- tectors; 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 in- dicating 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 in- dicated 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
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