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ORGAN, TISSUE, AND CELL DAMAGE
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[76] R a d i o p r o t e c t i o n b y A s c o r b i c A c i d , D e s f e r a l , and Mercaptoethylamine
By A J I T
SINGH,* HARWANT SINGH,*
and JAMES S.
HENDERSON*
Introduction It is well known that exposure to high-energy radiation can cause damage to biological systems. ~ This effect has been exploited in treating cancer, since the cancer cells can be killed by exposure to high-energy radiation. 2,3 H o w e v e r , in the process, the adjacent healthy tissue is also damaged. The protection of healthy tissue during radiotherapy for cancer has been one of the strong motivations for continuing research on exogenous radioprotectors.~,*-7 The main aim of this chapter is to focus on the methodology o f determining radioprotection by chemical agents using the mouse as a model animal system. F r o m this point of view, (1) some of our recent results on radioprotection of mice 8 are shown, (2) the mechanisms of radiation damage and their multiplicity are briefly discussed, and (3) the rationale for choosing three chemical agents [ascorbic acid (AA), Desferal (DF), and mercaptoethylamine (MEA)] for radioprotection studies is presented. Understanding of radiobiological effects in humans requires studies involving model chemical systems, microorganisms, tissue culture, and animals. Experimentation with animals ultimately provides assurance that conclusions from experimental results obtained in simpler systems have a biomedical reality. L a b o r a t o r y mice, available in many well-de* Atomic Energy of Canada Limited. i A. P. Casarett, "Radiation Biology." Prentice-Hall, Engelwood Cliffs, New Jersey, 1968. 2 K. N. Prasad, "Human Radiation Biology." Harper and Row, Hagerstown, Maryland, 1974. 3 F. A. Mettler, Jr., and R. D. Moseley, Jr., "Medical Effects of Ionizing Radiation." Grune & Stratton, Orlando, Florida, 1985. 4 A. Singh and H. Singh, Prog. Biophys. Mol. Biol. 39, 69 (1982). 5 G. E. Adams, in "Advances in Radiation Chemistry" (M. Burton and J. L. Magee, eds.), Vol. 3, p. 125. Wiley, New York, 1972. 6 D. L. Klayman and E. S. Copeland, in "Drug Design" (E. J. Ariens, ed.), Vol. 6, p. 82. Academic Press, New York, 1975. 7 A. M. Michelson and K. Puget, in "Oxygen Radicals in Chemistry and Biology" (W. Bors, M. Saran, and D. Tait, eds.), p. 831. de Gruyter, Berlin, 1984. A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. von Sonntag, eds.), p. 587. Plenum, New York, 1989. METHODS IN ENZYMOLOGY,VOL, 186
Copyright© 1990Governmentof Canada
[76]
USE OF EXOGENOUS RADIOPROTECTORS
687
Ionization and excitationa i
i
Reactions with .H, .OH, eaq and Bio .a i Reactions with 0~, RO~., sec-8io a I
i
Reactions with H202 t ROOH a Biochemical e f f e c t s o i
Acute
i
pathological effects at high doses b
Late pathological e f f e c t s at I 0
I I0 "f2
I I0 -9
I I0 -6
I I0 "3
I I0 °
I 10 3
low doses !
I 10 6
b i
I
I0 9
TIME (s) FIG. 1. Time scale of damaging events from the time of an energy deposition event, leading to biological effects of irradiation; a, for details, see A. Singh and H. Singh, Prog. Biophys. Mol. Biol. 39, 69 (1982); b, for details, see F. A. Mettler, Jr., and R. D. Moseley, Jr., "Medical Effects of Ionizing Radiation." Grune & Stratton, Orlando, Florida, 1985.
fined inbred strains, are large enough for easy handling but not too large for economic husbandry and whole-body irradiations in large numbers. However, the experiments should be carefully designed to keep the wastage of animals to a minimum and relevant animal care legislation9 should be followed. Mechanisms of Damage and Protection Radiation damage in biological systems is initiated by the primary ionic, excited, and free radical species formed during the energy deposition events. 4,5 Several mechanisms contribute to the subsequent radiation damage over a wide time scale, as shown in Fig. 1. For example, the primary free radicals [hydrogen atom (. H); hydroxyl radical (. OH); hydrated electron (eaq), and bioradicals (Bio ")] induce damaging reactions 9 Foundation of Biomedical Research (U.S.A), Clin. Res. Pract. Drug Regul. Aff. 5, 265 (1987).
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ORGAN, TISSUE, AND CELL DAMAGE
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within a few microseconds of their formation. These reactions are, in turn, followed by reactions induced by the secondary free radicals [superoxide anion (O2~), peroxy radicals (ROz .), and secondary bioradicals (sec-Bio .)] and the metastable species (hydrogen peroxide and organic hydroperoxides), which may continue over minutes and days, respectively. Biochemically altered macromolecules (DNA, RNA, enzymes) may ultimately engender gross pathological lesions (e.g., neoplasia) over a much longer period. 2-5,1° Most of the chemical and biochemical components of biological systems are sensitive to and reactive with the primary species and secondary free radicals formed on exposure to high-energy radiation.l~-~4 Because of the biochemical complexity of biological systems, a very large number of secondary free radical species form. Many of the reactions between the free radical species and the biochemicals present may protect biological integrity. For example, the reactions of free radicals with sulfhydryl compounds like cysteine4,5 and the reaction of superoxide anions with superoxide dismutasey are protective. However, damage also results from the reactions of the primary and secondary species, through a variety of mechanisms. Results of many radioprotection studies, two of which are mentioned here, also point to the conclusion that radiobiological damage occurs through more than one mechanism. Storer ~5investigated radioprotection of A/J and C57BL/6J mice, by S-2-aminoethylisothiouronium bromide hydrobromide, mercaptoethylamine (MEA), p-aminopropiophenone (PAPP), and 5-hydroxytryptamine creatinine sulfate. The results showed that (1) although the protection of the bone marrow by each of these chemical agents was similar, the protection of the life span was dependent on the sex and strain of the mice; (2) the protection against lethality offered by the different agents was different for mice of the same strain and sex; and (3) the protection by each agent varied in a different manner with increasing radiation dose. We have also reported similar dose-dependent effects: In the case of BALB/c mice, DF acts as a radiosensitizer at 10 L. Packer, (ed.), this series, Vol. 105. l~ B. H. J. Bielski, D. E. Cabelli, R. L. Arudi, and A. B. Ross, J. Phys. Chem. Ref. Data 14, 1041 (1985). ~2G. V. Buxton, C. L. Greenstock, W. P. Helman, and A. B. Ross, J. Phys. Chem. R e f Data 17, 513 (1988). t~ p. Neta and A. B. Ross, in "Chemical Kinetics of Small Organic Radicals, Vol. 4: Reactions in Special Systems" (Z. B. Alfassi, ed.), p. 187. CRC Press, Boca Raton, Florida, 1988. 14p. Neta, R. E. Huie, and A. B. Ross, J. Phys. Chem. Ref. Data 17, 1007 (1988). t5 j. B. Storer, Radiat. Res. 47, 537 (1971).
[76]
USE OF EXOGENOUSRADIOPROTECTORS
689
700 rads (7 Gy), but becomes a radioprotector at 1000 and 1300 rads (10 and 13 Gy). s Biological systems have intrinsic protective components, for example, the sulfhydryl compounds like cysteine, and the DNA repair enzymes. 4 Exogenous administration of the same, or similar, compounds that provide inherent protection would be expected to provide additional radioprotection. Many studies have confirmed this expectation. Information is now available on the radioprotection of microorganisms and animal systems by many chemicals and biochemicals. 4-7 Choice of Radioprotectors Since a variety of mechanisms are responsible for radiobiological damage, mixtures of protectors, each aimed against a different damaging mechanism, should be more effective than the same protectors used individually. Results with mixtures of radioprotectors have been reported z6 which justify this expectation. The concept of the additive effect of a mixture of protectors guided our further work on radioprotection of BALB/c mice s using the following three different types of radioprotectors.
Ascorbic Acid The oxygen effect in radiation biology is well known. 5 Since oxygen enhances radiation-induced biological damage, antioxidants should be radioprotectors. Indeed, many of the radioprotectors are antioxidants. 6 Ascorbic acid is an antioxidant, ~7 an essential vitamin for humans, ~s and a prime factor in biological defense. ~9Though the mechanisms of action of AA are not fully understood, it is known to reduce ferric ions to ferrous ions and to react with free radicals.~7,2°,2~ It is important to understand its role, if any, in modifying radiobiological damage. Aseorbic acid has been used in radiation protection studies in cells, mice, and rats, with varying results. Radioprotection of E. coli, Chinese
16j. R. Maisin, G. Mattelin, and M. Lambiet-Collier, Radiat. Res. 71, 119 (1977). t7 T. L. Dormandy, Lancet 1, 647 (1978). is j. Drummond, Biochem. J. 13, 77 (1919). 19A. Szent-Gyorgyi, "The Living State." Academic Press, New York, 1972. 2o B. H. J. Bielski, in "Ascorbic Acid: Chemistry, Metabolism and Uses" (P. A. Seib and B. M. Tolbert, eds.), p. 81. Advances in Chemistry Series 200, American Chemical Society, Washington, D.C., 1982. 21 R. L. Willson, in "Radioprotectors and Anticarcinogens" (O. F. Nygaard and M. G. Simic, eds.), p. 1. Academic Press, New York, 1983.
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ORGAN, TISSUE, AND CELL DAMAGE
[76]
hamster ovary cells, and rats by AA has been reported. 22-24In the case of mice (NMRI males, Swiss, and CF0, however, it was found to be a radiosensitizer rather than a radioprotector. 25,26
Desferal Part of the radiobiological damage by radiation-induced hydrogen peroxide and organic hydroperoxides could be due to the Fenton reaction, Fe2 + + H202 --> • OH + OH- + Fe3 +
(1)
and its equivalent in the case of the hydroperoxides, Fe2 ÷ + ROOH --~ RO" + OH- + Fe3 +
(2)
brought about by free transition metal ions, particularly iron, in the biological systems. 4,272s Desferal forms a very stable water-soluble complex with free ferric ions. The chelated ferric ions are then removed from the body by renal excretion. 29,3° Based on work on inhibition of lipid peroxidation by DF, Gutteridge et al. 28 suggested that DF may act as a radioprotector by chelating iron.
Mercaptoethylamine Thiols are very reactive toward free radicals4,SA2'13: RSH + R ' - ~ RS" + R ' H
(3)
The thiyl radicals formed are relatively nonreactive and they mainly produce nontoxic disulfides: RS. + RS. --~ RSSR
(4)
22 M. Naslund, L. Ehrenberg, and G. Djalali-Behzad, Int. J. Radiat. BioL Relat. Stud. Phys. Chem. Med. 30, 95 (1976). 23 M. K. O'Conner, J. F. Malone, M. Moriarty, and S. Mulgrew, Br. J. Radiol. 50, 587 (1977). 24 L. Ala-Ketola, R. Varis, and K. Kiviniitty, Strahlentherapie 148, 643 (1974). 25 j. Forsberg, M. Harms-Ringdahl, and L. Ehrenberg, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 34, 245 (1978). 26 H. H. Tewfik, F. A. Tewfik, and E. F. Riley, in "Vitamin C: New Clinical Applications in Immunology, Lipid Metabolism and Cancer" (A. Hanck, ed.) p. 265. Huber, Bern, 1982. 27 A. Singh, in "CRC Handbook of Free Radicals and Antioxidants in Biomedicine" (J. Miquel, A. T. Quintanilha, and H. Weber, eds.), Vol. 1, p. 17. CRC Press, Boca Raton, Florida, 1989. 28 j. M. C. Gutteridge, R. Richmond, and B. HalliweU, Biochem. J. 184, 469 (1979). 29 Desferal, Desferrioxamine, Product Information, Ciba-Giegy Limited, Basel, Switzerland, 1988. 30 M. Aksoy and G. F. B. Birdwood (eds.), "Hypertransfusion and Iron Chelation in Thalassaemia." Huber, Bern, 1985.
[76]
USE OF EXOGENOUSRADIOPROTECTORS
691
These reactions probably constitute the main mode of the radioprotective mechanism of thiols, though other mechanisms have also been suggested. 4 Mercaptoethylamine (cysteamine, 2-aminoethylthiol), which is a thiol, is a widely used and very effective radioprotector, 6,16,z5 in mice, against radiation-induced lethality. Experimental
Radioprotection Studies wRh Mice Mice used in various radiation protection studies s,15,zS,z6,3~ include NMRI, Swiss, CFI, A/J, BALB/c, BALB/c +, C57B1, HA(ICR)f, and C57BL/6J strains. Results in experiments testing such complexities as the effects of irradiation on mice vary with sex, age, strain, and husbandry. For example, the LDs0 (radiation dose which causes death of 50% of the irradiated mice I in 30 days) values for CF~, Swiss, NMRI, and HA(ICR)f mice have been reported as 429, 525, 610, and 679 rads, respectively. Thus, the CFI mice appear to be the most radiosensitive of the four strains, and the HA(ICR)f the most radioresistant. The differences in radioprotection of different sexes are exemplified in the study of A/J and C57BL/6J mice by Storer, 15 in which it was found that while MEA protected females better than males in both strains, PAPP protected A/J females more than the males, but C57BL/6J males better than the females. Colonies of the same strain may also differ in their radioresponse significantly, on account of environmental factors, such as the bacterial flora resident in the gut. It is also important to use experimental mice within as narrow a range of age as possible, since the radiation effects can be age-dependent.l The details about the BALB/c mice used by us 8 have been published) 2 Radioprotection studies entail all the complexities of radiation effects plus those from the putative protectors, which are not necessarily simple. Careful planning and rigorously disciplined execution of the experiments are therefore needed to obtain information when dealing with complex biological effects and their modification by exogenous radioprotectors. In the case of inbred mice, it is usual to allot, from animals of the same age and sex, a subset of 10 for each treatment. To obtain reliable results on the effect of each protective protocol, all factors except the controlled variation in the concentrations, and possibly modes of administration, of the radioprotector(s) should be kept constant. 31 W. F. Ward, A. Shih-Hoellwarth, and P. M. Johnson, Radiat. Res. 81, 131 (1980). 32 j. S. Henderson and J. L. Weeks, Ind. Med. 42, 10 (1973).
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ORGAN, TISSUE, AND CELL DAMAGE
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It is possible to obtain mice for such experiments from established suppliers. It is advisable to quarantine them upon receipt to allow adaptation to their new environment. Indeed for the month required for an LDs0/30 assay it would be best to keep them quarantined to minimize deaths from unfamiliar exposures. In such cases, the recommendations of the suppliers or the procedures given in standard handbooks 33 should be followed. The irradiated mice should be returned to their own cages and held there in equable conditions with their accustomed diet and water ad libiturn. Dead mice should be removed and counted during twice daily inspections of the cages. Where possible, necropsies should be done to monitor any deaths that may not be radiation-induced. Administration o f Radioprotectors
Ascorbic acid taken orally is satisfactorily absorbed from the gut but can be injected as necessary. In mice about 0.27 mg/day of AA is endogenously produced. 26 The pharmacokinetics of AA has been discussed by Basu and Schorah. 34 We injected 6 mg of AA per mouse (in 1 ml saline solution alone or in combination with the other radioprotectors) approximately 15 min before irradiation, s In comparison, Tewfik et al3 6 administered AA in drinking water before and after irradiation, and Forsberg et al. 25 injected AA 1 hr before irradiation. We preferred injections since administration of radioprotectors in drinking water or food can change the drinking and eating patterns of treated mice, compared to the controls. Our choice of the injection time of approximately 15 min before irradiation allowed us to administer all three radioprotectors, alone or in mixture, in single injections. Desferal is poorly absorbed when taken orally. When injected, however, it is quickly distributed through all body fluids and then gradually lost (over several hours 29) to renal glomerular filtration and tubular secretion. Though DF has not been used as a radioprotector before, Ward et a l ) 1 administered another chelator, penicillamine, intraperitoneally, 15 min before irradiation of mice. The dose used in our work s was 1.5 mg/ mouse. Mercaptoethylamine is distributed quickly, when injected, and is found throughout all organs (except the testes) within 15 min. It is rapidly 33 C. S. F. Williams, "Practical Guide to Laboratory Animals." Mosby, St. Louis, Missouri, 1976. T. K. Basu and C. J. Schorah, "Vitamin C in Health and Disease." AVI, Westport, Connecticut, 1982.
[76]
USE OF EXOGENOUSRADIOPROTECTORS
693
degradedin the tissues and rapidly excreted in the urine; hence, most of it is lost within 2 hr. It is toxic to humans in the doses required for radioprotection, but its beneficial effects in animals and in cultures of animal cells are well documented. 6,~6,22,25 Our injection of 6 rag/mouse is consistent with the previous usage of this radioprotector, tS,t6 In our work, s each mouse receives the desired dose in 1 ml of fluid, injected into the peritoneal sac through the belly wall, which had been shaved and cleansed with alcohol. The control group of mice is injected in the same way a n d at the same time as the treated groups but with the saline carrier only. The time of administration of the radioprotectors can be optimized by changing the time interval between the administration of the protector and irradiation, keeping all other factors unchanged. Timing must allow a balance between the attainment of uniform distribution and elimination through normal metabolic processes. Where information is not available from previous studies in the literature, simple tests on the time profile of the level of the radioprotector in the body fluids and organs of mice, subsequent to its administration, may allow the optimum time for the administration of the protector to be determined with economy of time and mice. The concentration of a radioprotector can be optimized by changing its concentration and monitoring the effect, keeping all other factors unchanged. Again, it may be useful to establish first how the in vivo concentration increases with increased amounts of the radioprotector being administered. Many radioprotectors may be toxic to mice at very high concentrations, so their levels should be kept low enough to avoid toxicity but high enough to get the maximum possible benefit. Once the concentrations of various radioprotectors have been individually optimized, their optimization in mixtures can be accomplished by variations in the levels of each one. Irradiation
Mice can be irradiated with X-rays, y-rays (6°C0), electron beams from an electron accelerator, or heavier energetic particles such as protons, neutrons, helium ions, and other heavier ions. 35,36 The use of the first three is most prevalent. It is important that there be a zone for uniform irradiation of 10 mice at a time, housed in a suitable, ventilated holder.
35 I. G. Draganic and Z. D. Draganic, "The Radiation Chemistry of Water." Academic Press, New York, 1971. W. M. Saunders, Radiat. Phys. Chem. 24, 365 0984).
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ORGAN, TISSUE,
AND CELL DAMAGE
[76]
They should not be unduly warm; however, the holder should not be large enough to permit them too much movement. The mouse holder should preferably be of organic material, e.g., polycarbamate, polycarbonate, or polystyrene, so that the absorption of radiation by it is similar to that of the mice. A metal holder may introduce complex dose contours and shadows owing to its higher density. The uniformity of irradiation and the dose rate in the mouse holder, at the precise site for irradiation, can be determined with many dosimeter systems, 37 the Fricke dosimeter being the most common. We used Theraton F (Manitoba Cancer Foundation) for the y-irradiation of mice. Its output is frequently checked and calibrated for therapeutic purposes. The 6°Co source of the Theratron emits ~/-rays as a collimated beam for precise periods by the carefully timed opening of its port. The rate at which the power of the ~/-emission decays is well known ( - 1 % per month). From the dosimetry data, the known decay rate for 6°Co, and the exposure time, the dose delivered to the mice is calculated) For irradiation the mice are loaded in groups of 10 into a 10-celled polycarbamate holder, at room temperature. Each cell of the holder is 56 x 28 × 43 mm and is ventilated with three 7-mm holes in the lid and two in the floor. The cells are arranged in two parallel banks of five so that all are encompassed within a cross section of 17.5 × 16.0 cm. The mice receive the required dose, uniformly, within 5 min. Since all the irradiations are done in one evening, the dose rate is constant, for all practical purposes. The entire holder is collared by a 31-mm thickness of solid polycarbamate. This keeps scatter from the incident radiation uniform throughout the holder. Four identical holders are used, one at a time, to enable a large number of mice to be irradiated in a continuous session.
Generation of Survival Curves The curves obtained by plotting the animal survival data at a given dose against time are called survival curves.~ Our work is done in the range of 700 to 1300 rads (7 to 13 Gy). 8 For the purpose of discussion here, our detailed data at 1000 rads is given in Fig. 2. These data show that (1) both DF and MEA protect but AA sensitizes mice under the conditions used; (2) the protection offered by a mixture of MEA and AA is less than that offered by MEA alone; and (3) the protection offered by MEA and DF is much greater than that offered by MEA alone. In the case of results such as shown for saline and AA in Fig. 2, it is advisable to obtain a 37 N. W. Holm and R. G. Berry, "Manual on Radiation Dosimetry." Dekker, New York, 1970.
[76]
USE OF EXOGENOUSRADIOPROTECTORS Lz
I00
/1
i
AA
80
Q\
I
SALINE ~
I
MEA 4-
0........MEA o ~,... -
DF
\,E,.A,
4o
0
695
0
// zr
I
8
~/
12
16
J
28
32
DAYS AFTER IRRADIATION
FIG. 2. Survival of BALB/c mice treated with exogenous radioprotectors, on irradiation to 1000rads (10 Gy): AA, ascorbic acid; DF, Desferai; MEA, mercaptoethylamine, x, ©, e , l , Data from A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. yon Sonntag, eds.), p. 587. Plenum, New York, 1989. [3, A, Unpublished data (A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, 1985).
greater number of points for statistical analysis) 8to increase the degree of certainty of the conclusions being drawn. In Fig. 2, the radioprotection seen with MEA is similar to that reported by others.15,2YThe radiosensitization seen with AA is consistent with that reported by Tewfik et al. 26 and Forsberg e t ai. 25 However, the data in Fig. 2 suggest radiosensitization at a dose of I000 rads, whereas Tewfik et al. 26 reported radiosensitization at doses below 700 rads. This difference could be due to the different strains of mice used in the two studies and due to the different modes of administration of AA (injection8 versus orally in drinking water26). The drastic reduction of the protection offered by MEA by the simultaneous administration of AA is also consistent with the previous work by Forsberg e t al.25; however, the effect of AA seems to be greater according to the data in Fig. 2, again perhaps owing to the different strains of mice in the two studies. The radioprotection offered by DF (Fig. 2) supports the view4,28that reactions (I) and (2) play important roles in radiobiological damage. The much larger protection offered by DF and MEA administered together as compared to MEA alone is very promising and warrants extension of this work. The survival curves shown in Fig. 2 can be reproduced, or similar curves obtained with other mice and other radioprotectors, by following 3s p. Armitage, "Statistical Methods in Medical Research." Blackwell, Oxford, 1971.
696
ORGAN, TISSUE, AND CELL DAMAGE
[77]
the details given above. Our investigation of three radioprotectors and their two mixtures were conducted simultaneously on a nearly homogeneous population of mice irradiated during one session. Therefore one control group satisfactorily served all the protective protocols tested. From the data on mortality of mice in 30 days as a function of dose, LDs0 values for the mice chosen can be obtained. 1 Published LDs0 values should not be used as a benchmark reference for such work, since many factors can influence them. They should only be used as a guide in the choice of the doses to be used.
Concluding Remarks The mouse provides a good model animal system to investigate radioprotection by chemical and biochemical agents. A promising approach is to investigate radioprotection by mixtures of protectors, each agent aimed against a different mechanism of radiobiological damage. A good example of this approach is the result obtained with a mixture of MEA and DF, where chelation of ferric ions by DF seems to enhance the protection offered by MEA at a dose of 1000 rads.
[77] I n f l u e n c e of Thiols on T h e r m o s e n s i t i v i t y of M a m m a l i a n Cells in Vitro
By ROLF D. ISSELS and ARr~ONAGELE Introduction The cytotoxic effects of a number of chemotherapeutic agents have been reported to be enhanced by heat. 1,2 The exact mechanisms underlying thermosensitization are not understood in detail and might differ with the various compounds. We recently demonstrated that the enhanced toxicity at 37 and 44° of aminothiols like cysteamine is due to the generation of activated oxygen species during the autoxidation of these compounds and is significantly reduced in the presence of c a t a l a s e 3 (see Fig. 1). When studying the thermosensitizing capability of thiols, the following observations have to be taken into account. As demonstrated for Chinese G. M. H a h n , Cancer Res, 39, 2264 (1979). 2 G. M. H a h n and G. C. Li, Natl. Cancer Inst. Monogr. 61, 467 (1982). 3 R. D. Issels, J. E. Biaglow, L. Epstein, a n d L. Gerweek, Cancer Res. 44, 3911 (1984).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
ORGAN, TISSUE, AND CELL DAMAGE
[76]
[76] R a d i o p r o t e c t i o n b y A s c o r b i c A c i d , D e s f e r a l , and Mercaptoethylamine
By A J I T
SINGH,* HARWANT SINGH,*
and JAMES S.
HENDERSON*
Introduction It is well known that exposure to high-energy radiation can cause damage to biological systems. ~ This effect has been exploited in treating cancer, since the cancer cells can be killed by exposure to high-energy radiation. 2,3 H o w e v e r , in the process, the adjacent healthy tissue is also damaged. The protection of healthy tissue during radiotherapy for cancer has been one of the strong motivations for continuing research on exogenous radioprotectors.~,*-7 The main aim of this chapter is to focus on the methodology o f determining radioprotection by chemical agents using the mouse as a model animal system. F r o m this point of view, (1) some of our recent results on radioprotection of mice 8 are shown, (2) the mechanisms of radiation damage and their multiplicity are briefly discussed, and (3) the rationale for choosing three chemical agents [ascorbic acid (AA), Desferal (DF), and mercaptoethylamine (MEA)] for radioprotection studies is presented. Understanding of radiobiological effects in humans requires studies involving model chemical systems, microorganisms, tissue culture, and animals. Experimentation with animals ultimately provides assurance that conclusions from experimental results obtained in simpler systems have a biomedical reality. L a b o r a t o r y mice, available in many well-de* Atomic Energy of Canada Limited. i A. P. Casarett, "Radiation Biology." Prentice-Hall, Engelwood Cliffs, New Jersey, 1968. 2 K. N. Prasad, "Human Radiation Biology." Harper and Row, Hagerstown, Maryland, 1974. 3 F. A. Mettler, Jr., and R. D. Moseley, Jr., "Medical Effects of Ionizing Radiation." Grune & Stratton, Orlando, Florida, 1985. 4 A. Singh and H. Singh, Prog. Biophys. Mol. Biol. 39, 69 (1982). 5 G. E. Adams, in "Advances in Radiation Chemistry" (M. Burton and J. L. Magee, eds.), Vol. 3, p. 125. Wiley, New York, 1972. 6 D. L. Klayman and E. S. Copeland, in "Drug Design" (E. J. Ariens, ed.), Vol. 6, p. 82. Academic Press, New York, 1975. 7 A. M. Michelson and K. Puget, in "Oxygen Radicals in Chemistry and Biology" (W. Bors, M. Saran, and D. Tait, eds.), p. 831. de Gruyter, Berlin, 1984. A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. von Sonntag, eds.), p. 587. Plenum, New York, 1989. METHODS IN ENZYMOLOGY,VOL, 186
Copyright© 1990Governmentof Canada
[76]
USE OF EXOGENOUS RADIOPROTECTORS
687
Ionization and excitationa i
i
Reactions with .H, .OH, eaq and Bio .a i Reactions with 0~, RO~., sec-8io a I
i
Reactions with H202 t ROOH a Biochemical e f f e c t s o i
Acute
i
pathological effects at high doses b
Late pathological e f f e c t s at I 0
I I0 "f2
I I0 -9
I I0 -6
I I0 "3
I I0 °
I 10 3
low doses !
I 10 6
b i
I
I0 9
TIME (s) FIG. 1. Time scale of damaging events from the time of an energy deposition event, leading to biological effects of irradiation; a, for details, see A. Singh and H. Singh, Prog. Biophys. Mol. Biol. 39, 69 (1982); b, for details, see F. A. Mettler, Jr., and R. D. Moseley, Jr., "Medical Effects of Ionizing Radiation." Grune & Stratton, Orlando, Florida, 1985.
fined inbred strains, are large enough for easy handling but not too large for economic husbandry and whole-body irradiations in large numbers. However, the experiments should be carefully designed to keep the wastage of animals to a minimum and relevant animal care legislation9 should be followed. Mechanisms of Damage and Protection Radiation damage in biological systems is initiated by the primary ionic, excited, and free radical species formed during the energy deposition events. 4,5 Several mechanisms contribute to the subsequent radiation damage over a wide time scale, as shown in Fig. 1. For example, the primary free radicals [hydrogen atom (. H); hydroxyl radical (. OH); hydrated electron (eaq), and bioradicals (Bio ")] induce damaging reactions 9 Foundation of Biomedical Research (U.S.A), Clin. Res. Pract. Drug Regul. Aff. 5, 265 (1987).
688
ORGAN, TISSUE, AND CELL DAMAGE
[76]
within a few microseconds of their formation. These reactions are, in turn, followed by reactions induced by the secondary free radicals [superoxide anion (O2~), peroxy radicals (ROz .), and secondary bioradicals (sec-Bio .)] and the metastable species (hydrogen peroxide and organic hydroperoxides), which may continue over minutes and days, respectively. Biochemically altered macromolecules (DNA, RNA, enzymes) may ultimately engender gross pathological lesions (e.g., neoplasia) over a much longer period. 2-5,1° Most of the chemical and biochemical components of biological systems are sensitive to and reactive with the primary species and secondary free radicals formed on exposure to high-energy radiation.l~-~4 Because of the biochemical complexity of biological systems, a very large number of secondary free radical species form. Many of the reactions between the free radical species and the biochemicals present may protect biological integrity. For example, the reactions of free radicals with sulfhydryl compounds like cysteine4,5 and the reaction of superoxide anions with superoxide dismutasey are protective. However, damage also results from the reactions of the primary and secondary species, through a variety of mechanisms. Results of many radioprotection studies, two of which are mentioned here, also point to the conclusion that radiobiological damage occurs through more than one mechanism. Storer ~5investigated radioprotection of A/J and C57BL/6J mice, by S-2-aminoethylisothiouronium bromide hydrobromide, mercaptoethylamine (MEA), p-aminopropiophenone (PAPP), and 5-hydroxytryptamine creatinine sulfate. The results showed that (1) although the protection of the bone marrow by each of these chemical agents was similar, the protection of the life span was dependent on the sex and strain of the mice; (2) the protection against lethality offered by the different agents was different for mice of the same strain and sex; and (3) the protection by each agent varied in a different manner with increasing radiation dose. We have also reported similar dose-dependent effects: In the case of BALB/c mice, DF acts as a radiosensitizer at 10 L. Packer, (ed.), this series, Vol. 105. l~ B. H. J. Bielski, D. E. Cabelli, R. L. Arudi, and A. B. Ross, J. Phys. Chem. Ref. Data 14, 1041 (1985). ~2G. V. Buxton, C. L. Greenstock, W. P. Helman, and A. B. Ross, J. Phys. Chem. R e f Data 17, 513 (1988). t~ p. Neta and A. B. Ross, in "Chemical Kinetics of Small Organic Radicals, Vol. 4: Reactions in Special Systems" (Z. B. Alfassi, ed.), p. 187. CRC Press, Boca Raton, Florida, 1988. 14p. Neta, R. E. Huie, and A. B. Ross, J. Phys. Chem. Ref. Data 17, 1007 (1988). t5 j. B. Storer, Radiat. Res. 47, 537 (1971).
[76]
USE OF EXOGENOUSRADIOPROTECTORS
689
700 rads (7 Gy), but becomes a radioprotector at 1000 and 1300 rads (10 and 13 Gy). s Biological systems have intrinsic protective components, for example, the sulfhydryl compounds like cysteine, and the DNA repair enzymes. 4 Exogenous administration of the same, or similar, compounds that provide inherent protection would be expected to provide additional radioprotection. Many studies have confirmed this expectation. Information is now available on the radioprotection of microorganisms and animal systems by many chemicals and biochemicals. 4-7 Choice of Radioprotectors Since a variety of mechanisms are responsible for radiobiological damage, mixtures of protectors, each aimed against a different damaging mechanism, should be more effective than the same protectors used individually. Results with mixtures of radioprotectors have been reported z6 which justify this expectation. The concept of the additive effect of a mixture of protectors guided our further work on radioprotection of BALB/c mice s using the following three different types of radioprotectors.
Ascorbic Acid The oxygen effect in radiation biology is well known. 5 Since oxygen enhances radiation-induced biological damage, antioxidants should be radioprotectors. Indeed, many of the radioprotectors are antioxidants. 6 Ascorbic acid is an antioxidant, ~7 an essential vitamin for humans, ~s and a prime factor in biological defense. ~9Though the mechanisms of action of AA are not fully understood, it is known to reduce ferric ions to ferrous ions and to react with free radicals.~7,2°,2~ It is important to understand its role, if any, in modifying radiobiological damage. Aseorbic acid has been used in radiation protection studies in cells, mice, and rats, with varying results. Radioprotection of E. coli, Chinese
16j. R. Maisin, G. Mattelin, and M. Lambiet-Collier, Radiat. Res. 71, 119 (1977). t7 T. L. Dormandy, Lancet 1, 647 (1978). is j. Drummond, Biochem. J. 13, 77 (1919). 19A. Szent-Gyorgyi, "The Living State." Academic Press, New York, 1972. 2o B. H. J. Bielski, in "Ascorbic Acid: Chemistry, Metabolism and Uses" (P. A. Seib and B. M. Tolbert, eds.), p. 81. Advances in Chemistry Series 200, American Chemical Society, Washington, D.C., 1982. 21 R. L. Willson, in "Radioprotectors and Anticarcinogens" (O. F. Nygaard and M. G. Simic, eds.), p. 1. Academic Press, New York, 1983.
690
ORGAN, TISSUE, AND CELL DAMAGE
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hamster ovary cells, and rats by AA has been reported. 22-24In the case of mice (NMRI males, Swiss, and CF0, however, it was found to be a radiosensitizer rather than a radioprotector. 25,26
Desferal Part of the radiobiological damage by radiation-induced hydrogen peroxide and organic hydroperoxides could be due to the Fenton reaction, Fe2 + + H202 --> • OH + OH- + Fe3 +
(1)
and its equivalent in the case of the hydroperoxides, Fe2 ÷ + ROOH --~ RO" + OH- + Fe3 +
(2)
brought about by free transition metal ions, particularly iron, in the biological systems. 4,272s Desferal forms a very stable water-soluble complex with free ferric ions. The chelated ferric ions are then removed from the body by renal excretion. 29,3° Based on work on inhibition of lipid peroxidation by DF, Gutteridge et al. 28 suggested that DF may act as a radioprotector by chelating iron.
Mercaptoethylamine Thiols are very reactive toward free radicals4,SA2'13: RSH + R ' - ~ RS" + R ' H
(3)
The thiyl radicals formed are relatively nonreactive and they mainly produce nontoxic disulfides: RS. + RS. --~ RSSR
(4)
22 M. Naslund, L. Ehrenberg, and G. Djalali-Behzad, Int. J. Radiat. BioL Relat. Stud. Phys. Chem. Med. 30, 95 (1976). 23 M. K. O'Conner, J. F. Malone, M. Moriarty, and S. Mulgrew, Br. J. Radiol. 50, 587 (1977). 24 L. Ala-Ketola, R. Varis, and K. Kiviniitty, Strahlentherapie 148, 643 (1974). 25 j. Forsberg, M. Harms-Ringdahl, and L. Ehrenberg, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 34, 245 (1978). 26 H. H. Tewfik, F. A. Tewfik, and E. F. Riley, in "Vitamin C: New Clinical Applications in Immunology, Lipid Metabolism and Cancer" (A. Hanck, ed.) p. 265. Huber, Bern, 1982. 27 A. Singh, in "CRC Handbook of Free Radicals and Antioxidants in Biomedicine" (J. Miquel, A. T. Quintanilha, and H. Weber, eds.), Vol. 1, p. 17. CRC Press, Boca Raton, Florida, 1989. 28 j. M. C. Gutteridge, R. Richmond, and B. HalliweU, Biochem. J. 184, 469 (1979). 29 Desferal, Desferrioxamine, Product Information, Ciba-Giegy Limited, Basel, Switzerland, 1988. 30 M. Aksoy and G. F. B. Birdwood (eds.), "Hypertransfusion and Iron Chelation in Thalassaemia." Huber, Bern, 1985.
[76]
USE OF EXOGENOUSRADIOPROTECTORS
691
These reactions probably constitute the main mode of the radioprotective mechanism of thiols, though other mechanisms have also been suggested. 4 Mercaptoethylamine (cysteamine, 2-aminoethylthiol), which is a thiol, is a widely used and very effective radioprotector, 6,16,z5 in mice, against radiation-induced lethality. Experimental
Radioprotection Studies wRh Mice Mice used in various radiation protection studies s,15,zS,z6,3~ include NMRI, Swiss, CFI, A/J, BALB/c, BALB/c +, C57B1, HA(ICR)f, and C57BL/6J strains. Results in experiments testing such complexities as the effects of irradiation on mice vary with sex, age, strain, and husbandry. For example, the LDs0 (radiation dose which causes death of 50% of the irradiated mice I in 30 days) values for CF~, Swiss, NMRI, and HA(ICR)f mice have been reported as 429, 525, 610, and 679 rads, respectively. Thus, the CFI mice appear to be the most radiosensitive of the four strains, and the HA(ICR)f the most radioresistant. The differences in radioprotection of different sexes are exemplified in the study of A/J and C57BL/6J mice by Storer, 15 in which it was found that while MEA protected females better than males in both strains, PAPP protected A/J females more than the males, but C57BL/6J males better than the females. Colonies of the same strain may also differ in their radioresponse significantly, on account of environmental factors, such as the bacterial flora resident in the gut. It is also important to use experimental mice within as narrow a range of age as possible, since the radiation effects can be age-dependent.l The details about the BALB/c mice used by us 8 have been published) 2 Radioprotection studies entail all the complexities of radiation effects plus those from the putative protectors, which are not necessarily simple. Careful planning and rigorously disciplined execution of the experiments are therefore needed to obtain information when dealing with complex biological effects and their modification by exogenous radioprotectors. In the case of inbred mice, it is usual to allot, from animals of the same age and sex, a subset of 10 for each treatment. To obtain reliable results on the effect of each protective protocol, all factors except the controlled variation in the concentrations, and possibly modes of administration, of the radioprotector(s) should be kept constant. 31 W. F. Ward, A. Shih-Hoellwarth, and P. M. Johnson, Radiat. Res. 81, 131 (1980). 32 j. S. Henderson and J. L. Weeks, Ind. Med. 42, 10 (1973).
692
ORGAN, TISSUE, AND CELL DAMAGE
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It is possible to obtain mice for such experiments from established suppliers. It is advisable to quarantine them upon receipt to allow adaptation to their new environment. Indeed for the month required for an LDs0/30 assay it would be best to keep them quarantined to minimize deaths from unfamiliar exposures. In such cases, the recommendations of the suppliers or the procedures given in standard handbooks 33 should be followed. The irradiated mice should be returned to their own cages and held there in equable conditions with their accustomed diet and water ad libiturn. Dead mice should be removed and counted during twice daily inspections of the cages. Where possible, necropsies should be done to monitor any deaths that may not be radiation-induced. Administration o f Radioprotectors
Ascorbic acid taken orally is satisfactorily absorbed from the gut but can be injected as necessary. In mice about 0.27 mg/day of AA is endogenously produced. 26 The pharmacokinetics of AA has been discussed by Basu and Schorah. 34 We injected 6 mg of AA per mouse (in 1 ml saline solution alone or in combination with the other radioprotectors) approximately 15 min before irradiation, s In comparison, Tewfik et al3 6 administered AA in drinking water before and after irradiation, and Forsberg et al. 25 injected AA 1 hr before irradiation. We preferred injections since administration of radioprotectors in drinking water or food can change the drinking and eating patterns of treated mice, compared to the controls. Our choice of the injection time of approximately 15 min before irradiation allowed us to administer all three radioprotectors, alone or in mixture, in single injections. Desferal is poorly absorbed when taken orally. When injected, however, it is quickly distributed through all body fluids and then gradually lost (over several hours 29) to renal glomerular filtration and tubular secretion. Though DF has not been used as a radioprotector before, Ward et a l ) 1 administered another chelator, penicillamine, intraperitoneally, 15 min before irradiation of mice. The dose used in our work s was 1.5 mg/ mouse. Mercaptoethylamine is distributed quickly, when injected, and is found throughout all organs (except the testes) within 15 min. It is rapidly 33 C. S. F. Williams, "Practical Guide to Laboratory Animals." Mosby, St. Louis, Missouri, 1976. T. K. Basu and C. J. Schorah, "Vitamin C in Health and Disease." AVI, Westport, Connecticut, 1982.
[76]
USE OF EXOGENOUSRADIOPROTECTORS
693
degradedin the tissues and rapidly excreted in the urine; hence, most of it is lost within 2 hr. It is toxic to humans in the doses required for radioprotection, but its beneficial effects in animals and in cultures of animal cells are well documented. 6,~6,22,25 Our injection of 6 rag/mouse is consistent with the previous usage of this radioprotector, tS,t6 In our work, s each mouse receives the desired dose in 1 ml of fluid, injected into the peritoneal sac through the belly wall, which had been shaved and cleansed with alcohol. The control group of mice is injected in the same way a n d at the same time as the treated groups but with the saline carrier only. The time of administration of the radioprotectors can be optimized by changing the time interval between the administration of the protector and irradiation, keeping all other factors unchanged. Timing must allow a balance between the attainment of uniform distribution and elimination through normal metabolic processes. Where information is not available from previous studies in the literature, simple tests on the time profile of the level of the radioprotector in the body fluids and organs of mice, subsequent to its administration, may allow the optimum time for the administration of the protector to be determined with economy of time and mice. The concentration of a radioprotector can be optimized by changing its concentration and monitoring the effect, keeping all other factors unchanged. Again, it may be useful to establish first how the in vivo concentration increases with increased amounts of the radioprotector being administered. Many radioprotectors may be toxic to mice at very high concentrations, so their levels should be kept low enough to avoid toxicity but high enough to get the maximum possible benefit. Once the concentrations of various radioprotectors have been individually optimized, their optimization in mixtures can be accomplished by variations in the levels of each one. Irradiation
Mice can be irradiated with X-rays, y-rays (6°C0), electron beams from an electron accelerator, or heavier energetic particles such as protons, neutrons, helium ions, and other heavier ions. 35,36 The use of the first three is most prevalent. It is important that there be a zone for uniform irradiation of 10 mice at a time, housed in a suitable, ventilated holder.
35 I. G. Draganic and Z. D. Draganic, "The Radiation Chemistry of Water." Academic Press, New York, 1971. W. M. Saunders, Radiat. Phys. Chem. 24, 365 0984).
694
ORGAN, TISSUE,
AND CELL DAMAGE
[76]
They should not be unduly warm; however, the holder should not be large enough to permit them too much movement. The mouse holder should preferably be of organic material, e.g., polycarbamate, polycarbonate, or polystyrene, so that the absorption of radiation by it is similar to that of the mice. A metal holder may introduce complex dose contours and shadows owing to its higher density. The uniformity of irradiation and the dose rate in the mouse holder, at the precise site for irradiation, can be determined with many dosimeter systems, 37 the Fricke dosimeter being the most common. We used Theraton F (Manitoba Cancer Foundation) for the y-irradiation of mice. Its output is frequently checked and calibrated for therapeutic purposes. The 6°Co source of the Theratron emits ~/-rays as a collimated beam for precise periods by the carefully timed opening of its port. The rate at which the power of the ~/-emission decays is well known ( - 1 % per month). From the dosimetry data, the known decay rate for 6°Co, and the exposure time, the dose delivered to the mice is calculated) For irradiation the mice are loaded in groups of 10 into a 10-celled polycarbamate holder, at room temperature. Each cell of the holder is 56 x 28 × 43 mm and is ventilated with three 7-mm holes in the lid and two in the floor. The cells are arranged in two parallel banks of five so that all are encompassed within a cross section of 17.5 × 16.0 cm. The mice receive the required dose, uniformly, within 5 min. Since all the irradiations are done in one evening, the dose rate is constant, for all practical purposes. The entire holder is collared by a 31-mm thickness of solid polycarbamate. This keeps scatter from the incident radiation uniform throughout the holder. Four identical holders are used, one at a time, to enable a large number of mice to be irradiated in a continuous session.
Generation of Survival Curves The curves obtained by plotting the animal survival data at a given dose against time are called survival curves.~ Our work is done in the range of 700 to 1300 rads (7 to 13 Gy). 8 For the purpose of discussion here, our detailed data at 1000 rads is given in Fig. 2. These data show that (1) both DF and MEA protect but AA sensitizes mice under the conditions used; (2) the protection offered by a mixture of MEA and AA is less than that offered by MEA alone; and (3) the protection offered by MEA and DF is much greater than that offered by MEA alone. In the case of results such as shown for saline and AA in Fig. 2, it is advisable to obtain a 37 N. W. Holm and R. G. Berry, "Manual on Radiation Dosimetry." Dekker, New York, 1970.
[76]
USE OF EXOGENOUSRADIOPROTECTORS Lz
I00
/1
i
AA
80
Q\
I
SALINE ~
I
MEA 4-
0........MEA o ~,... -
DF
\,E,.A,
4o
0
695
0
// zr
I
8
~/
12
16
J
28
32
DAYS AFTER IRRADIATION
FIG. 2. Survival of BALB/c mice treated with exogenous radioprotectors, on irradiation to 1000rads (10 Gy): AA, ascorbic acid; DF, Desferai; MEA, mercaptoethylamine, x, ©, e , l , Data from A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. yon Sonntag, eds.), p. 587. Plenum, New York, 1989. [3, A, Unpublished data (A. Singh, H. Singh, J. S. Henderson, R. D. Migliore, J. Rousseau, and J. E. Van Lier, 1985).
greater number of points for statistical analysis) 8to increase the degree of certainty of the conclusions being drawn. In Fig. 2, the radioprotection seen with MEA is similar to that reported by others.15,2YThe radiosensitization seen with AA is consistent with that reported by Tewfik et al. 26 and Forsberg e t ai. 25 However, the data in Fig. 2 suggest radiosensitization at a dose of I000 rads, whereas Tewfik et al. 26 reported radiosensitization at doses below 700 rads. This difference could be due to the different strains of mice used in the two studies and due to the different modes of administration of AA (injection8 versus orally in drinking water26). The drastic reduction of the protection offered by MEA by the simultaneous administration of AA is also consistent with the previous work by Forsberg e t al.25; however, the effect of AA seems to be greater according to the data in Fig. 2, again perhaps owing to the different strains of mice in the two studies. The radioprotection offered by DF (Fig. 2) supports the view4,28that reactions (I) and (2) play important roles in radiobiological damage. The much larger protection offered by DF and MEA administered together as compared to MEA alone is very promising and warrants extension of this work. The survival curves shown in Fig. 2 can be reproduced, or similar curves obtained with other mice and other radioprotectors, by following 3s p. Armitage, "Statistical Methods in Medical Research." Blackwell, Oxford, 1971.
696
ORGAN, TISSUE, AND CELL DAMAGE
[77]
the details given above. Our investigation of three radioprotectors and their two mixtures were conducted simultaneously on a nearly homogeneous population of mice irradiated during one session. Therefore one control group satisfactorily served all the protective protocols tested. From the data on mortality of mice in 30 days as a function of dose, LDs0 values for the mice chosen can be obtained. 1 Published LDs0 values should not be used as a benchmark reference for such work, since many factors can influence them. They should only be used as a guide in the choice of the doses to be used.
Concluding Remarks The mouse provides a good model animal system to investigate radioprotection by chemical and biochemical agents. A promising approach is to investigate radioprotection by mixtures of protectors, each agent aimed against a different mechanism of radiobiological damage. A good example of this approach is the result obtained with a mixture of MEA and DF, where chelation of ferric ions by DF seems to enhance the protection offered by MEA at a dose of 1000 rads.
[77] I n f l u e n c e of Thiols on T h e r m o s e n s i t i v i t y of M a m m a l i a n Cells in Vitro
By ROLF D. ISSELS and ARr~ONAGELE Introduction The cytotoxic effects of a number of chemotherapeutic agents have been reported to be enhanced by heat. 1,2 The exact mechanisms underlying thermosensitization are not understood in detail and might differ with the various compounds. We recently demonstrated that the enhanced toxicity at 37 and 44° of aminothiols like cysteamine is due to the generation of activated oxygen species during the autoxidation of these compounds and is significantly reduced in the presence of c a t a l a s e 3 (see Fig. 1). When studying the thermosensitizing capability of thiols, the following observations have to be taken into account. As demonstrated for Chinese G. M. H a h n , Cancer Res, 39, 2264 (1979). 2 G. M. H a h n and G. C. Li, Natl. Cancer Inst. Monogr. 61, 467 (1982). 3 R. D. Issels, J. E. Biaglow, L. Epstein, a n d L. Gerweek, Cancer Res. 44, 3911 (1984).
METHODS IN ENZYMOLOGY, VOL. 186
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