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Effect of varying concentrations of caffeine and ascorbic acid on the radiosensitivity of barley seed irradiated in oxygenated or oxygen-free hydration medium at 25 and 37°C
Environmental and Experimental Botany, 1977, Vol. 17, pp. 129 to 133. Pergamon Press. Printed in Great Britain.
EFFECT OF V A R Y I N G C O N C E N T R A T I O N S OF CAFFEINE A N D ASCORBIC ACID ON THE R A D I O S E N S I T I V I T Y OF BARLEY SEED I R R A D I A T E D IN O X Y G E N A T E D OR O X Y G E N - F R E E H Y D R A T I O N M E D I U M AT 25 A N D 37°C S. M. JAVED AFZKL and P. C. NIgSAV/~N
School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110057. India (Received 22 June 1976 and in Revised Form 28 April 1977) AFZALS. M.J. and KESAVANP. C. Effect of varying concentrations of caffeine and ascorbic acid on the radiosensitivity of barley seed irradiated in oxrgenated or oxrgen-free hydration medium at 25 and 37°C. ENVIRONMENTALANDEXPERIMENTALBOTA.XY17, 129--133, 1977. The moditication ofradiosensitivity of barley seed with 1.75 x 10 -3 M and 3.8 x 10 -3 M concentrations of caffeine and ascorbic acid during irradiation in oxygenated and oxygen-free hydration medium was studied at 25 and 37C, respectively. Both concentrations of caffeine and ascorbic acid afford protection against oxic radiation damage which is maximal at 25°C. Caffeine effectively potentiates the anoxic component of damage but ascorbic acid has no influence at all. At 25°C there is no concentration-dependent effect of caffeine or ascorbic acid. At 37°C, there is no effect, whatsoever, of either concentration of ascorbic acid, whereas caffeine dramatically potentiates the radiation damage under both oxygenated and oxygen-free conditions, and the magnitude of potentiation is concentrationdependent. The possible reactivity of caffeine and aseorbic acid towards the precursors of oxygendependent and -independent components of damage in determining the mode and magnitudes of modification is discussed briefly.
INTRODUCTION EXTENSIVE studies of KESAVAN and coworkers~l, v-ls~ have established that caffeine (1,3,7trimethyl xanthine) acts as a radioprotector or radiosensitizer depending upon the relative predominance of the radiation-induced oxygendependent and oxygen-independent components of damage. Caffeine partially protects against oxic radiation damage in dry as well as metabolizing barley seeds yet potentiates and oxygenindependent component of damage. (v - 15) Further investigations on dry and metabolizing barley seeds have shown that once the radiation-induced, oxygen-sensitive sites are lost (either by reaction with oxygen or by recombination) caffeine dramatically potentiates an oxygen-independent component of damage. (v' 8.10.15) T h e radioprotective action of caffeine has been interpreted on the basis of a mutually annihilatory reaction of caffeine
molecules with the precursors of oxic damage (oxygen-sensitive sites)F' 8, 14) KESAVAN and AFZAL(s) observed that the magnitude of the oxygen efl'ect is significantly lowered at 37 °C, due possibly to an accelerated rate of decay of the radiation-induced, oxygen-sensitive sites. O u r postulate was that the reactivity of the test chemicals towards the radiation-induced precursors ofoxic damage may be the first step towards radioprotection, even in the metabolizing systems. O u r data (s) did show that with a significant reduction in the magnitude ofoxic damage in the metabolizing barley seeds irradiated at 37°C, cysteine and potassium p e r m a n g a n a t e lose their protective action, whereas caffeine acts as a sensitizer. The concentration of caffeine is a significant factor in influencing the mode and magnitude of modification of radiation damage in d r y irradiated seeds. (11) The present study was therefore undertaken in order to assess the 129
130
S.M. JAVED and P. C. KESAVAN
influence of two different concentrations (1.75 x 1 0 - 3 M and 3.8x 1 0 - 3 M ) of caffeine on the radiation damage induced in metabolizing seeds at 25°C and 37°C. Parallel studies were also undertaken with ascorbic acid, a well-known radioprotector of plant seeds, (2 - 4~ to investigate its reactivity towards the radiation-induced precursors ofoxic and anoxic components of damage at 25 and 37°C.
MATERIAL AND M E T H O D S
Pure-line seeds (caryopses) of a hull-less barley strain (IB 65) were used. For each treatment, 30 seeds were put into three 30ml glass vials (replicates) and were pre-soaked in either oxygenated or oxygen-fi'ee water (prepared as reported earlier c8)) for 4h at 25°C. These seeds were then transferred to fresh hydration media, which consisted of oxygenated or oxygen-free water, and oxic and anoxic solutions of 3.8 x 10- 3 M and 1.75 x l 0 - 3 M of caffeine (Sigma, U.S.A.) and ascorbic acid (British Drug House, India). A constant temperature of 25°C for one set of experiments, and 37°C for another was maintained; hydration was for an additional 4 h. In the middle of the second 4-h treatment, the vials containing the seeds for irradiation were exposed to 10krad of gamma-rays, using a 5500 Ci6°Co gamma-chamber 4000 (B.A.R.C., India) at a dose-rate of 89R/sec. The seeds were thus irradiated while immersed in either water or treatment solution. After the 4 h of treatment, the seeds were washed and planted on moist filter paper in Petri dishes and cultured in a growth chamber at 25°C and under constant illumination. The growth of 8-day seedlings (i.e., length of the first leaf) was measured and the data analysed statistically. The levels of significance between various treatments were calculated using the standard errors of the mean. Seedling injury, i.e. the percentage reduction in the mean seedling height as compare d with the control was determined for each treatment from an average height of three replicates of 25 seedlings each. ~8) Seedling injury data were used to compute the magnitude of protection or potentiation caused by the test chemicals under different treatment conditions.
RESULTS
Eight-day seedling growth of unirradiated seeds was not appreciably influenced either by the nature of the gas or the chemical dissolved in the hydration media. There was no effect of increasing the temperature on the response of non-irradiated seeds to caffeine and ascorbic acid. Therefore, only the average control values are provided in Table 1. Data presented in Table 1 show the influence of two concentrations of caffeine and ascorbic acid on the oxic and anoxic components of radiation damage in terms of seedling injury at 25 and 37°C, respectively. The seedling growth of unirradiated controls treated at 37°C was significantly (p <0.01) lower than those treated at 25°C. Both concentrations of caffeine and ascorbic acid afford significant (p N 0.01 ) radioprotection against the oxic damage developing at 25°C. Under anoxic irradiation conditions at 25°C, application of caffeine during irradiation potentiates the anoxic component of damage significantly (p<0.01), whereas ascorbic acid exerts no influence on this component of damage. At this temperature there is no concentration-dependent effect of caffeine and ascorbic acid on the oxic and anoxic components of damage. Both concentrations (3.8 x 10 -3 M and 1.75 x 10 .3 M) of ascorbic acid and caffeine protect equally against oxic radiation damage, 2.9 fold and 1.4 fold, respectively. The magnitude of anoxic sensitization is also the same for both concentrations of caffeine ( ~ 2 fold) at 25°C. With a substantial reduction of the oxic damage at 37°C, the protective effect of ascorbic acid and caffeine was lost. Ascorbic acid had no influence on the anoxic component of irradiation damage; hence, one would not expect a concentration effect. At 37°C, caffeine begins to act as a radiosensitizer even under oxygenated conditions and its potentiation of radiation damage is concentration-dependent. The seedling injury data indicate that caffeine concentrations of 3.8mM and 1.75mM potentiate (p_<0.01) the radiation damage by 3.2- and 2.7-folds, respectively, over the oxic and 7.9- and 6.3-folds, respectively, over the anoxic levels of damage. DISCUSSION
Extensive studies of KESAVAN and coworkers ¢l'v-ls'aT) have shown that a wide range of
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chemical compounds (caffeine and analogues of caffeine, cysteine, N-ethylmaleimide, hydroxyurea, H2S, KMnO4, KI, KNO3 and K4Fe(CN)6) afford significant protection against the radiation-induced oxic damage in dry as well as metabolizing barley seeds. Recent studies of KESAVANand AFZAL(s' and KESAVANand DODD(12) have suggested that the so-called "immediate" oxygen effect in the metabolizing seeds is indeed initiated by A nspecies of DoDD and EBERX(5) which in dried seeds and spores of O s m u n d a postirradiatively react with oxygen and results in the socalled post-irradiation oxygen effect. The characteristic feature of this class of precursors is that they decay at 310 K whereas the precursors of true class II damage do not. (16) The oxygen effect mediated by A n species is considerably reduced at 37°C as compared at 25°C (s) and this supports the contention that A n species are responsible for the oxygen effect observed in dry as well as metabolizing barley seeds. Caffeine-mediated radioprotection against oxic damage has been ascribed to the mutually" annihilatory reaction of caffeine molecules with radiation-induced A n species. (s' 11-13) Data obtained from kinetic studies (1 a~have also shown that radioprotection occurs only' when catteine is introduced into tile system betore the decay of A, species. The presence of caffeine in seeds which could develop only the oxygen-independent component of damage (either because the seeds have high moisture content or because the A , species are experimentally removed {lm) causes potentiation of the radiation damage. The mechanisms of potentiation of an oxygen-independent component of damage by caffeine are yet to be elucidated. It is assumed that the radioprotective action of caffeine against oxic damage is due not only to annihilation of the radiation-induced precursors ofoxic damage butalso to a reciprocal removal of caffeine molecules which are presumed to react with the precursors of an oxygenindependent component of damage (or alternatively with the enzymatic repair processes acting preferentiaily under anoxic conditions ~lm) to cause potentiation of anoxic damage. The hypothesis of a mutually annihilatory reaction of caffeine molecules with the precursors of oxic damage tbr the manifestation of radioprotection has been further strengthened by the de-
monstration of KESAVAN and AHMAD(11} that for optimal radioprotection there is a delicate equilibrium between the caffeine concentration and the yield and stability of the radiation-induced, oxygen-sensitive sites. I f the caffeine concentration is much higher than that required for the mutually annihilatory reaction (s) with the precursors ofoxic damage then the remaining molecules even under oxic conditions would be able to potentiate the oxygen-independent component of damage. Under these circumstances the partial protection against the oxic damage is not discernible owing to a much higher degree of potentiation of the anoxic damage than the degree of protection against oxic damage. In the present studies a two-fold variation in caffeine concentration does not result in discernibh, diflierences in the magnitude of protection agai~;,t oxic damage at 25°C; a significant reduction in the oxic damage occurs at 37°C, with 3.8 x 10- 3 M caffeine causing more potentiation than 1.75 x 1 0 - 3 M caffeine. The lack of observable differences in the magnitude of protection against oxic damage is possibly due to the fact that a 2-fold change in the concentration of caffeine is not enough to cause measurable differences and/or that seedling injury is not a sensitive enough parameter. At 37°C the An precursors decay even before they react with caffeine molecules; thus, a 2-fold difference in the concentration of caffeine molecules is able to produce measurable differences in the magnitude ofradiosensitization. It should also be pointed out that the radiation-induced, oxygensensitive sites are heterogenous as evidenced from the rates of decay and response to thermal annealment.(6, s - 10) Furthermore, caffeine reacts with that fraction of oxygen-sensitive sites which is also harmlessly removed by heat. (1°) Therefore, notwithstanding the presence of a small amount of precursors of oxic damage, caffeine causes only potentiation. Cysteine, a well known radioprotector, affords radioprotection only against oxic damage but, unlike caffeine, shows no effect on the anoxic component of damage. (7-9) The results of the present studies with ascorbic acid suggest that it too affords protection only against oxic (Table 1) and not the anoxic component of damage. The magnitude of radioprotection afforded by both concentrations of ascorbic acid at 25°C is not as much as oxygen-free hydration, thereby suggest-
EFFECT OF CONCENTRATIONS OF CAFFEINE AND ASCORBIC ACID
9. KESAVANP. C. and AHMADA. (1974) Influence of caffeine and cysteine on post-irradiation oxygendependent and independent components of damage in Hordeum vulgare. Experientia 30, 942. 10. KESAVANP. C. and AHMAD A. (1974) Modification of genetic damage by post-treatment with caffeine applied during oxygenated and oxygen-free hydration in Hordeum vulgate. Mutat, Res. 23, 337-346. 1 l, KESAVANP. C. and AHMAD A. (1976) Modification of the radiosensitivity of barley seed by posttreatment with caffeine III. Influence of dose of irradiation and concentration of caffeine. Int. J. Radiat. Biol. 29, 395-398. 12. KESAVANP. C. and Door) N. J. F. (1976) Modification of radiation-induced oxic and anoxic damage REFERENCES by caffeine and potassium permanganate in barley BALACHANDRANR. and KESAVANP. C. (1974) A seeds. Int. J. Radiat. Biol. 30, 171-178. sequential caffeine-cysteine treatment and en- 13. KESAVANP. C., SHARMAG. J. and AFZALS. M . J . hanced radioprotection of Viciafaba chromosomes. (1977) Differential modification ofoxic and anoxic Experientia 30, 91. radiation damage by chemicals I. Simulation of the BISWAS S. and MATSUO T. (1966) Protective and action of caffeine by certain inorganic radical recovery effects of chemicals on plant growth, scavengers. Radiat. Res. (in press). chromosome aberration and mutation in irradiated 14. KESAVAN P. C., TRAS1 S. and AHMAD A. (1973) seed of crop plants. Radiat. Bot. 6, 575 587. Modification of barley seed radiosensitivity by postCONGERB. V. (1973) The etfizcts of ascorbic acid treatment with caffeine I. Effect of post-irradiation and sodium azide on seedling growth of irradiated heat-shock and nature of hydration. Int. ,7. Radiat. and nonirradiated barley seeds. Radiat. Bot. 13, Biol. 24, 581-587. 375 379. 15. NADKARNI S. and KESAVAN P. C. (1975) ModificCON(;}:R B. V. (1975) Radioprotective effects of ation of the radiosensitivity of barley seed by postascorbic acid in barley seeds. Radiat. Bot. 15, 3948. treatment with caffeine II. Kinetics of decay of 1)ore) N, .1. F. and EBERT M. (1970) Effects of caffeine-reactive oxygen-sensitive sites, hit. J. ionizing radiation on dried spores ofOsmunda regalis. Radiat. Biol. 27, 569-576. I 1, Modification of survival and its correlation with 16. POWERSE. L. (1965) Experimental biological basis the ti-ee radical changes. Int. J. Radiat. Biol. 18, 463of oxygen effects. Proc. Xlth Intern. Conq. Radiol., 473. Excerpta Medi. Int. Conq. Ser. 105, 1451-1457. Dotm N . J . F . and EIsERxM. (1972)Post-irradiation 17. SHARMAG. J, and KESAVANP. C. (1975) Use of temperature effects in dried spores of Osmunda hydrogen sulphide and N-ethylmaleimide in the regalis. Radiat. Bot. 12, 151-158. post-irradiation modification of oxic and anoxic KESAVANP. C. (1973) Effect of caffeine and cysteine components of damage in Hordeum vulgare. Radiat. applied during post-irradiation hydration on an Bot. 15, 261-266. oxygen-independent component of damage in 18. TowN C. D., SMIxn K. C. and KAPLANH. S. (1972) barley seeds. Radiat. Bot. 13, 355 359. Influence ofultrafast repair processes (Independent KESAVAN P. C. and AFZAL S. M. J. (1975) of DNA polymerase I) on the yield of DNA-single Dependence of chemical radioprotection on the strand breaks in Escherichin coil K- 12 X-irradiated in stability of radiation-induced oxygen-sensitive sites. presence or absence ofox)gen. Radial. Res. 52, 99Int. J. Radiot. Biol. 28, 495~-99. 114.
ing that only a tew |i'actions of a heterogenous p o p u l a t i o n of oxygen-sensitive sites are removed by ascorbic acid. This i n t e r p r e t a t i o n gains additional support by the fact that with the reduction of the oxygen effect at 37°C, ascorbic acid loses its radioprotective action; this indicates that ascorbic acid does not exhibit reactivity towards all possible categories of oxygen-sensitive sites. T h e fact that ascorbic acid does not exert any effect on the anoxic c o m p o n e n t of d a m a g e (both at 25 as well as 37°C) is indicative of its lack of reactivity towards the precursors of o x y g e n - i n d e p e n d e n t c o m p o n e n t of d a m a g e . 1.
2.
3.
4. 5.
6.
7.
8.
133
EFFECT OF V A R Y I N G C O N C E N T R A T I O N S OF CAFFEINE A N D ASCORBIC ACID ON THE R A D I O S E N S I T I V I T Y OF BARLEY SEED I R R A D I A T E D IN O X Y G E N A T E D OR O X Y G E N - F R E E H Y D R A T I O N M E D I U M AT 25 A N D 37°C S. M. JAVED AFZKL and P. C. NIgSAV/~N
School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110057. India (Received 22 June 1976 and in Revised Form 28 April 1977) AFZALS. M.J. and KESAVANP. C. Effect of varying concentrations of caffeine and ascorbic acid on the radiosensitivity of barley seed irradiated in oxrgenated or oxrgen-free hydration medium at 25 and 37°C. ENVIRONMENTALANDEXPERIMENTALBOTA.XY17, 129--133, 1977. The moditication ofradiosensitivity of barley seed with 1.75 x 10 -3 M and 3.8 x 10 -3 M concentrations of caffeine and ascorbic acid during irradiation in oxygenated and oxygen-free hydration medium was studied at 25 and 37C, respectively. Both concentrations of caffeine and ascorbic acid afford protection against oxic radiation damage which is maximal at 25°C. Caffeine effectively potentiates the anoxic component of damage but ascorbic acid has no influence at all. At 25°C there is no concentration-dependent effect of caffeine or ascorbic acid. At 37°C, there is no effect, whatsoever, of either concentration of ascorbic acid, whereas caffeine dramatically potentiates the radiation damage under both oxygenated and oxygen-free conditions, and the magnitude of potentiation is concentrationdependent. The possible reactivity of caffeine and aseorbic acid towards the precursors of oxygendependent and -independent components of damage in determining the mode and magnitudes of modification is discussed briefly.
INTRODUCTION EXTENSIVE studies of KESAVAN and coworkers~l, v-ls~ have established that caffeine (1,3,7trimethyl xanthine) acts as a radioprotector or radiosensitizer depending upon the relative predominance of the radiation-induced oxygendependent and oxygen-independent components of damage. Caffeine partially protects against oxic radiation damage in dry as well as metabolizing barley seeds yet potentiates and oxygenindependent component of damage. (v - 15) Further investigations on dry and metabolizing barley seeds have shown that once the radiation-induced, oxygen-sensitive sites are lost (either by reaction with oxygen or by recombination) caffeine dramatically potentiates an oxygen-independent component of damage. (v' 8.10.15) T h e radioprotective action of caffeine has been interpreted on the basis of a mutually annihilatory reaction of caffeine
molecules with the precursors of oxic damage (oxygen-sensitive sites)F' 8, 14) KESAVAN and AFZAL(s) observed that the magnitude of the oxygen efl'ect is significantly lowered at 37 °C, due possibly to an accelerated rate of decay of the radiation-induced, oxygen-sensitive sites. O u r postulate was that the reactivity of the test chemicals towards the radiation-induced precursors ofoxic damage may be the first step towards radioprotection, even in the metabolizing systems. O u r data (s) did show that with a significant reduction in the magnitude ofoxic damage in the metabolizing barley seeds irradiated at 37°C, cysteine and potassium p e r m a n g a n a t e lose their protective action, whereas caffeine acts as a sensitizer. The concentration of caffeine is a significant factor in influencing the mode and magnitude of modification of radiation damage in d r y irradiated seeds. (11) The present study was therefore undertaken in order to assess the 129
130
S.M. JAVED and P. C. KESAVAN
influence of two different concentrations (1.75 x 1 0 - 3 M and 3.8x 1 0 - 3 M ) of caffeine on the radiation damage induced in metabolizing seeds at 25°C and 37°C. Parallel studies were also undertaken with ascorbic acid, a well-known radioprotector of plant seeds, (2 - 4~ to investigate its reactivity towards the radiation-induced precursors ofoxic and anoxic components of damage at 25 and 37°C.
MATERIAL AND M E T H O D S
Pure-line seeds (caryopses) of a hull-less barley strain (IB 65) were used. For each treatment, 30 seeds were put into three 30ml glass vials (replicates) and were pre-soaked in either oxygenated or oxygen-fi'ee water (prepared as reported earlier c8)) for 4h at 25°C. These seeds were then transferred to fresh hydration media, which consisted of oxygenated or oxygen-free water, and oxic and anoxic solutions of 3.8 x 10- 3 M and 1.75 x l 0 - 3 M of caffeine (Sigma, U.S.A.) and ascorbic acid (British Drug House, India). A constant temperature of 25°C for one set of experiments, and 37°C for another was maintained; hydration was for an additional 4 h. In the middle of the second 4-h treatment, the vials containing the seeds for irradiation were exposed to 10krad of gamma-rays, using a 5500 Ci6°Co gamma-chamber 4000 (B.A.R.C., India) at a dose-rate of 89R/sec. The seeds were thus irradiated while immersed in either water or treatment solution. After the 4 h of treatment, the seeds were washed and planted on moist filter paper in Petri dishes and cultured in a growth chamber at 25°C and under constant illumination. The growth of 8-day seedlings (i.e., length of the first leaf) was measured and the data analysed statistically. The levels of significance between various treatments were calculated using the standard errors of the mean. Seedling injury, i.e. the percentage reduction in the mean seedling height as compare d with the control was determined for each treatment from an average height of three replicates of 25 seedlings each. ~8) Seedling injury data were used to compute the magnitude of protection or potentiation caused by the test chemicals under different treatment conditions.
RESULTS
Eight-day seedling growth of unirradiated seeds was not appreciably influenced either by the nature of the gas or the chemical dissolved in the hydration media. There was no effect of increasing the temperature on the response of non-irradiated seeds to caffeine and ascorbic acid. Therefore, only the average control values are provided in Table 1. Data presented in Table 1 show the influence of two concentrations of caffeine and ascorbic acid on the oxic and anoxic components of radiation damage in terms of seedling injury at 25 and 37°C, respectively. The seedling growth of unirradiated controls treated at 37°C was significantly (p <0.01) lower than those treated at 25°C. Both concentrations of caffeine and ascorbic acid afford significant (p N 0.01 ) radioprotection against the oxic damage developing at 25°C. Under anoxic irradiation conditions at 25°C, application of caffeine during irradiation potentiates the anoxic component of damage significantly (p<0.01), whereas ascorbic acid exerts no influence on this component of damage. At this temperature there is no concentration-dependent effect of caffeine and ascorbic acid on the oxic and anoxic components of damage. Both concentrations (3.8 x 10 -3 M and 1.75 x 10 .3 M) of ascorbic acid and caffeine protect equally against oxic radiation damage, 2.9 fold and 1.4 fold, respectively. The magnitude of anoxic sensitization is also the same for both concentrations of caffeine ( ~ 2 fold) at 25°C. With a substantial reduction of the oxic damage at 37°C, the protective effect of ascorbic acid and caffeine was lost. Ascorbic acid had no influence on the anoxic component of irradiation damage; hence, one would not expect a concentration effect. At 37°C, caffeine begins to act as a radiosensitizer even under oxygenated conditions and its potentiation of radiation damage is concentration-dependent. The seedling injury data indicate that caffeine concentrations of 3.8mM and 1.75mM potentiate (p_<0.01) the radiation damage by 3.2- and 2.7-folds, respectively, over the oxic and 7.9- and 6.3-folds, respectively, over the anoxic levels of damage. DISCUSSION
Extensive studies of KESAVAN and coworkers ¢l'v-ls'aT) have shown that a wide range of
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chemical compounds (caffeine and analogues of caffeine, cysteine, N-ethylmaleimide, hydroxyurea, H2S, KMnO4, KI, KNO3 and K4Fe(CN)6) afford significant protection against the radiation-induced oxic damage in dry as well as metabolizing barley seeds. Recent studies of KESAVANand AFZAL(s' and KESAVANand DODD(12) have suggested that the so-called "immediate" oxygen effect in the metabolizing seeds is indeed initiated by A nspecies of DoDD and EBERX(5) which in dried seeds and spores of O s m u n d a postirradiatively react with oxygen and results in the socalled post-irradiation oxygen effect. The characteristic feature of this class of precursors is that they decay at 310 K whereas the precursors of true class II damage do not. (16) The oxygen effect mediated by A n species is considerably reduced at 37°C as compared at 25°C (s) and this supports the contention that A n species are responsible for the oxygen effect observed in dry as well as metabolizing barley seeds. Caffeine-mediated radioprotection against oxic damage has been ascribed to the mutually" annihilatory reaction of caffeine molecules with radiation-induced A n species. (s' 11-13) Data obtained from kinetic studies (1 a~have also shown that radioprotection occurs only' when catteine is introduced into tile system betore the decay of A, species. The presence of caffeine in seeds which could develop only the oxygen-independent component of damage (either because the seeds have high moisture content or because the A , species are experimentally removed {lm) causes potentiation of the radiation damage. The mechanisms of potentiation of an oxygen-independent component of damage by caffeine are yet to be elucidated. It is assumed that the radioprotective action of caffeine against oxic damage is due not only to annihilation of the radiation-induced precursors ofoxic damage butalso to a reciprocal removal of caffeine molecules which are presumed to react with the precursors of an oxygenindependent component of damage (or alternatively with the enzymatic repair processes acting preferentiaily under anoxic conditions ~lm) to cause potentiation of anoxic damage. The hypothesis of a mutually annihilatory reaction of caffeine molecules with the precursors of oxic damage tbr the manifestation of radioprotection has been further strengthened by the de-
monstration of KESAVAN and AHMAD(11} that for optimal radioprotection there is a delicate equilibrium between the caffeine concentration and the yield and stability of the radiation-induced, oxygen-sensitive sites. I f the caffeine concentration is much higher than that required for the mutually annihilatory reaction (s) with the precursors ofoxic damage then the remaining molecules even under oxic conditions would be able to potentiate the oxygen-independent component of damage. Under these circumstances the partial protection against the oxic damage is not discernible owing to a much higher degree of potentiation of the anoxic damage than the degree of protection against oxic damage. In the present studies a two-fold variation in caffeine concentration does not result in discernibh, diflierences in the magnitude of protection agai~;,t oxic damage at 25°C; a significant reduction in the oxic damage occurs at 37°C, with 3.8 x 10- 3 M caffeine causing more potentiation than 1.75 x 1 0 - 3 M caffeine. The lack of observable differences in the magnitude of protection against oxic damage is possibly due to the fact that a 2-fold change in the concentration of caffeine is not enough to cause measurable differences and/or that seedling injury is not a sensitive enough parameter. At 37°C the An precursors decay even before they react with caffeine molecules; thus, a 2-fold difference in the concentration of caffeine molecules is able to produce measurable differences in the magnitude ofradiosensitization. It should also be pointed out that the radiation-induced, oxygensensitive sites are heterogenous as evidenced from the rates of decay and response to thermal annealment.(6, s - 10) Furthermore, caffeine reacts with that fraction of oxygen-sensitive sites which is also harmlessly removed by heat. (1°) Therefore, notwithstanding the presence of a small amount of precursors of oxic damage, caffeine causes only potentiation. Cysteine, a well known radioprotector, affords radioprotection only against oxic damage but, unlike caffeine, shows no effect on the anoxic component of damage. (7-9) The results of the present studies with ascorbic acid suggest that it too affords protection only against oxic (Table 1) and not the anoxic component of damage. The magnitude of radioprotection afforded by both concentrations of ascorbic acid at 25°C is not as much as oxygen-free hydration, thereby suggest-
EFFECT OF CONCENTRATIONS OF CAFFEINE AND ASCORBIC ACID
9. KESAVANP. C. and AHMADA. (1974) Influence of caffeine and cysteine on post-irradiation oxygendependent and independent components of damage in Hordeum vulgare. Experientia 30, 942. 10. KESAVANP. C. and AHMAD A. (1974) Modification of genetic damage by post-treatment with caffeine applied during oxygenated and oxygen-free hydration in Hordeum vulgate. Mutat, Res. 23, 337-346. 1 l, KESAVANP. C. and AHMAD A. (1976) Modification of the radiosensitivity of barley seed by posttreatment with caffeine III. Influence of dose of irradiation and concentration of caffeine. Int. J. Radiat. Biol. 29, 395-398. 12. KESAVANP. C. and Door) N. J. F. (1976) Modification of radiation-induced oxic and anoxic damage REFERENCES by caffeine and potassium permanganate in barley BALACHANDRANR. and KESAVANP. C. (1974) A seeds. Int. J. Radiat. Biol. 30, 171-178. sequential caffeine-cysteine treatment and en- 13. KESAVANP. C., SHARMAG. J. and AFZALS. M . J . hanced radioprotection of Viciafaba chromosomes. (1977) Differential modification ofoxic and anoxic Experientia 30, 91. radiation damage by chemicals I. Simulation of the BISWAS S. and MATSUO T. (1966) Protective and action of caffeine by certain inorganic radical recovery effects of chemicals on plant growth, scavengers. Radiat. Res. (in press). chromosome aberration and mutation in irradiated 14. KESAVAN P. C., TRAS1 S. and AHMAD A. (1973) seed of crop plants. Radiat. Bot. 6, 575 587. Modification of barley seed radiosensitivity by postCONGERB. V. (1973) The etfizcts of ascorbic acid treatment with caffeine I. Effect of post-irradiation and sodium azide on seedling growth of irradiated heat-shock and nature of hydration. Int. ,7. Radiat. and nonirradiated barley seeds. Radiat. Bot. 13, Biol. 24, 581-587. 375 379. 15. NADKARNI S. and KESAVAN P. C. (1975) ModificCON(;}:R B. V. (1975) Radioprotective effects of ation of the radiosensitivity of barley seed by postascorbic acid in barley seeds. Radiat. Bot. 15, 3948. treatment with caffeine II. Kinetics of decay of 1)ore) N, .1. F. and EBERT M. (1970) Effects of caffeine-reactive oxygen-sensitive sites, hit. J. ionizing radiation on dried spores ofOsmunda regalis. Radiat. Biol. 27, 569-576. I 1, Modification of survival and its correlation with 16. POWERSE. L. (1965) Experimental biological basis the ti-ee radical changes. Int. J. Radiat. Biol. 18, 463of oxygen effects. Proc. Xlth Intern. Conq. Radiol., 473. Excerpta Medi. Int. Conq. Ser. 105, 1451-1457. Dotm N . J . F . and EIsERxM. (1972)Post-irradiation 17. SHARMAG. J, and KESAVANP. C. (1975) Use of temperature effects in dried spores of Osmunda hydrogen sulphide and N-ethylmaleimide in the regalis. Radiat. Bot. 12, 151-158. post-irradiation modification of oxic and anoxic KESAVANP. C. (1973) Effect of caffeine and cysteine components of damage in Hordeum vulgare. Radiat. applied during post-irradiation hydration on an Bot. 15, 261-266. oxygen-independent component of damage in 18. TowN C. D., SMIxn K. C. and KAPLANH. S. (1972) barley seeds. Radiat. Bot. 13, 355 359. Influence ofultrafast repair processes (Independent KESAVAN P. C. and AFZAL S. M. J. (1975) of DNA polymerase I) on the yield of DNA-single Dependence of chemical radioprotection on the strand breaks in Escherichin coil K- 12 X-irradiated in stability of radiation-induced oxygen-sensitive sites. presence or absence ofox)gen. Radial. Res. 52, 99Int. J. Radiot. Biol. 28, 495~-99. 114.
ing that only a tew |i'actions of a heterogenous p o p u l a t i o n of oxygen-sensitive sites are removed by ascorbic acid. This i n t e r p r e t a t i o n gains additional support by the fact that with the reduction of the oxygen effect at 37°C, ascorbic acid loses its radioprotective action; this indicates that ascorbic acid does not exhibit reactivity towards all possible categories of oxygen-sensitive sites. T h e fact that ascorbic acid does not exert any effect on the anoxic c o m p o n e n t of d a m a g e (both at 25 as well as 37°C) is indicative of its lack of reactivity towards the precursors of o x y g e n - i n d e p e n d e n t c o m p o n e n t of d a m a g e . 1.
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