INT . J . RADIAT . BIOL .,
1978,
VOL .
33 .
NO .
1, 69-73
CORRESPONDENCE The reduction of radiation-induced mitotic delay by caffeine :
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a test of the cyclic AMP hypothesise N . L . OLEINICK$, E . N . BREWER and R . C . RUSTAD§ Division of Radiation Biology, Department of Radiology, Case Western Reserve University, School of Medicine,- Cleveland, Ohio 44106, U .S .A . (Received 19 September 1977 ; accepted 6 October 1977)
1.
Introduction One of the well-known biochemical effects of caffeine is the inhibition of cyclic nucleotide phosphodiesterases (Butcher and Sutherland 1962) . Thus, it seemed plausible to propose that the reduction in X-radiation-induced mitotic delay by caffeine may be the result of an elevated cyclic AMP content (Scaife 1971, Boynton, Evans and Crouse 1974, Walters, Gurley and Tobey 1974) . To test this hypothesis, we have characterized the caffeine effect on radiationinduced mitotic delay in the naturally-synchronous plasmodial slime mould Physarum polycephalum during late G 2 and early prophase, and have compared the action of that compound with others of similar structure and/or physiological function . Our results indicate that the reduction of mitotic delay is not a result of altered cyclic AMP levels . 2.
Materials and methods Macroplasmodia containing synchronously-dividing nuclei were prepared (Daniel and Baldwin 1964) from stock cultures of Physarum polycephalum, strain M3 C, using the modified medium of Brewer and Prior (1976) . Methodologies for gamma-irradiation, drug treatment, and determination of mitotic stages in plasmodial sectors were essentially as described previously (Rustad, Oleinick and Brewer 1975) .
3.
Results The caffeine concentration dependence for the reduction of radiation-induced mitotic delay in Physarum is shown in figure 1 . Sectors of a plasmodium were irradiated with 920 rad of 60Co gamma-rays 27 min before metaphase, and mitosis was delayed for 29 min in an irradiated plasmodial sector compared with a control sector (figure 1 (a)) . When other sectors were placed into medium containing 0 . 1-10 mM caffeine within 1 min after irradiation, less mitotic delay was observed . With 10 mM caffeine there was no mitotic delay (over that due to the drug alone) . When plasmodial sectors were irradiated earlier in the mitotic cycle (74 min before metaphase, figure 1 (b)), a somewhat smaller mitotic delay was observed fi Copyright U .S . Government . I Also affiliated with the Department of Biochemistry and the School of Dentistry . § Also affiliated with the Departments of Anatomy and Biology .
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70
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0 2
Figure 1 . The reduction of radiation-induced mitotic delay by various concentrations of caffeine . Sectors (twelfths of a plasmodium) were exposed to 0 or 920 rad 60Co gamma-radiation and immediately transferred to fresh growth medium containing 0-10 mM caffeine . Mitotic delay is defined as the difference between the time of appearance of metaphase in treated and in untreated sectors . Irradiation was at 27 min (a) or 74 min (b) before control metaphase . •, unirradiated ; •, 920 rad gamma-radiation .
in the absence of caffeine, in agreement with earlier results (Nygaard, Brewer, Evans and Wolpaw 1973), and the efficacy of each caffeine concentration was greater (with the exception of 10 mM which was somewhat toxic (Trevithick and Braun 1977)) . Essentially complete reversal of the delay was seen with 0 . 3 mM caffeine . In another experiment the mitotic delay resulting from a dose as high as 10 krad (261 min) was reduced by 84 per cent in the presence of 3 mM caffeine . Maximal reduction of radiation-induced mitotic delay resulted when caffeine (1 mM) was present throughout the post-irradiation period (figure 2) . Plasmodial sectors were incubated continuously in caffeine beginning at various times after irradiation . The protective effect of the drug decreased dramatically as a function of the length of time prior to the addition of caffeine (figure 2 (a)) . In the experiments described by figure 2 (b), exposure to caffeine was begun within 1 min after irradiation, and the plasmodial sectors were returned to normal growth medium at various intervals thereafter . The protection against radiation induced mitotic delay by the drug increased with the duration of the exposure . In contrast to post-irradiation exposure, treatment with caffeine and subsequent removal of the plasmodia from the drug prior to irradiation did not modify the radiation response (data not shown) . Two other methylxanthines, theophylline and theobromine, which differ from caffeine only by a single methyl group each, appeared to mimic caffeine
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Correspondence
71
Figure 2 . Variation of mitotic delay with interval of exposure to caffeine . Plasmodial sectors were transferred to fresh growth medium containing none or 1 mM caffeine following 3 . 4 krad gamma-radiation . (a) Sectors were placed in caffeine-free medium for various periods before exposure to caffeine . Irradiation occurred at 62 min ( .) or 36 min (A) before control metaphase . ` oo ' on the abscissa indicates no exposure to caffeine . (b) Sectors were exposed to caffeine immediately after irradiation, then transferred to caffeine-free medium at various times thereafter . Irradiation occurred at 62 min ( .) or 45 min (A) before control metaphase . All values for mitotic delay are relative to the time of mitosis observed in an unirradiated control sector.
Substance None Caffeine Theobromine Theophylline Dibutyryl cyclic AMP Cholera toxin Methylisobutyl xanthine Papaverine Ro 20-1724
Per cent mitotic delay relative to irradiation alone (100) 37 ± 6 (5) 34 ± 5 (4) 51 ± 9 (3) 93 ± 13 (4) 116 ± 10 (4) 126 ± 15 (3) 139 ±10 (7) 195 ± 22 (4)
Effect on radiation-induced mitotic delay of substances which elevate cyclic AMP levels . Plasmodial sectors were irradiated with 3 .4 krad in late G2 -early prophase (between 30 and 90 min before control metaphase), then transferred to fresh growth medium or to medium containing one of the above substances at 2 mM, with the exception of cholera toxin which was present at 1 µg/ml . Data are corrected for any small mitotic delays resulting from drug treatment alone and are expressed as mean ± standard error . Numbers in parentheses refer to the number of plasmodia tested with a given substance .
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72
Correspondence
in reducing mitotic delay (table) . All three of these compounds are known to be inhibitors of cyclic AMP phosphodiesterase . If their action on radiationinduced mitotic delay were due to inhibition of that enzyme, other such inhibitors, even though structurally dissimilar, should likewise reduce the mitotic delay . Methylisobutylxanthine (Beavo, Rogers, Crofford, Hardman, Sutherland and Newman 1970), Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] (Sheppard and Wiggan 1971), and papaverine [6,7-dimethoxy-1-veratrylisoquinoline] (Triner, Vulliemoz, Schwartz and Nahas 1970) are all reported inhibitors of the phosphodiesterase . These compounds increased rather than decreased mitotic delay at concentrations which have little or no effect on mitosis in unirradiated plasmodia . The addition of cholera toxin, a stimulator of adenyl cyclase activity in every system tested (Finkelstein 1973), also led to extended mitotic delays . Dibutyryl cyclic AMP itself had little effect on radiationinduced mitotic delay (table) . It appears that mere elevation of cyclic AMP within the plasmodium is insufficient to produce the reduction of mitotic inhibition shown by caffeine .
4.
Conclusions Several conclusions can be derived from this study . (1) The reduction of radiation-induced mitotic delay is related to increasing concentrations of caffeine over at least two orders of magnitude . (2) Pre-irradiation treatment with caffeine has no detectable effect . (3) For maximal effect, caffeine must be present for most, if not all, of the post-irradiation pre-mitotic period . (4) Chemicals which are reported to inhibit cyclic AMP phosphodiesterase may either reduce or increase radiation-induced mitotic delay . Thus, it seems unlikely that cyclic AMP metabolism per se is the basis for the ' healing ' effect of caffeine on radiation-induced mitotic delay . On the contrary, the present data suggest that elevation of cyclic AMP levels may actually prolong this delay, and that caffeine (theophylline, theobromine) may reduce mitotic delay by a mechanism(s) other than through the inhibition of cyclic AMP phosphodiesterase . Caffeine also engages in several other biochemical actions, e .g . alterations in Ca++_ transport (Huddart and Syson 1975, Rebhun, Jemiolo, Ivy, Mellon and Nath 1975), binding to DNA (Ts'o, Helmkamp and Sander 1962, Ts'o and Lu 1964), stimulation of cell-free protein synthesis (Fernandez-Puentes, Carrasco and Vazquez 1974), as well as other actions less well described . Thus the role of caffeine in reducing radiation-induced mitotic delay may be a complex one .
ACKNOWLEDGMENTS
This work was supported by the U .S . Energy Research and Development Administration . We thank Ms . Peggy Busacca for capable technical assistance, Dr . Michael E . Maguire, Department of Pharmacology, Case Western Reserve University for gifts of methylisobutylxanthine and Ro 20-1724, Dr . Herbert Sheppard, Hoffman-LaRoche, Inc ., for additional quantities of Ro 20 -1724, Dr . Charles Carpenter, Department of Medicine, for a gift of cholera toxin, Mr . K . Pillai for the dosimetry, and Dr . Helen H . Evans for reviewing the manuscript .
Correspondence
73
REFERENCES
J . A ., ROGERS, N . L ., CROFFORD, O . B ., HARDMAN, J . G ., SUTHERLAND, E . W ., and NEWMAN, E . V ., 1970, Molec . Pharmac ., 6, 597 . BOYNTON, A . L ., EVANS, T. C ., and CROUSE, D . A ., 1974, Radiat . Res ., 60, 89 . BREWER, E . N ., and PRIOR, A ., 1976, Physarum Newsl ., 8, 45 . BUTCHER, R . W ., and SUTHERLAND, E . W ., 1962, J . biol . Chem., 237, 1244 . DANIEL, J . W ., and BALDWIN, H . H ., 1964, Methods in Cell Physiology, Vol . 1 (New York : Academic Press) ; p . 9 . FERNANDEZ-PUENTES, C ., CARRASCO, L ., and VAZQUEZ, D ., 1974, FEBS Lett ., 45, 132 .
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BEAVO,
FINKELSTEIN, R . A ., 1973, CRC crit . Rev . Microbiol., 2, 553 . HUDDART, H ., and SYSON, A. J ., 1975, Y. exp . Biol ., 63, 131 . NYGAARD, O . F ., BREWER, E . N ., EVANS, T . E ., and WOLPAW, J . R ., 1973, Advances in Radiation Research, Vol . 2 (London : Gordon & Breach), p . 989 . REBHUN, L . I ., JEMIOLO, D ., Ivy, N ., MELLON, M ., and NATH, J ., 1975, Ann . N . Y. Acad . Sci ., 253, 362 . RuSTAD, R . C ., OLEINICK, N . L., and BREWER, E . N ., 1975, Expl Cell Res ., 93, 477 . SCAIFE, J . F ., 1971, Int . Y. Radiat . Biol., 19, 191 . SHEPPARD, H ., and WIGGAN, G ., 1971, Molec . Pharmac ., 7, 111 . TREVITHICK, J . R ., and BRAUN, R ., 1977, Experientia, 33, 28 . TRINER, L ., VULLIEMOZ, Y ., SCHWARTZ, I ., and NAHAS, G . G ., 1970, Biochem . biophys . Res . Commun ., 40, 64 . Ts'o, P .O .P ., HELMKAMP, G . K., and SANDER, C ., 1962, Proc . natn . Acad . Sci. U.S.A ., 48,686 . Ts'o, P . O . P ., and Lu, P ., 1964, Proc . natn . Acad . Sci. U.S . A ., 51, 17 . WALTERS, R . A ., GURLEY, L . R ., and ToBEY, R . A ., 1974, Biophys . Y., 14, 99 .
1978,
VOL .
33 .
NO .
1, 69-73
CORRESPONDENCE The reduction of radiation-induced mitotic delay by caffeine :
Int J Radiat Biol Downloaded from informahealthcare.com by Flinders University of South Australia on 01/11/15 For personal use only.
a test of the cyclic AMP hypothesise N . L . OLEINICK$, E . N . BREWER and R . C . RUSTAD§ Division of Radiation Biology, Department of Radiology, Case Western Reserve University, School of Medicine,- Cleveland, Ohio 44106, U .S .A . (Received 19 September 1977 ; accepted 6 October 1977)
1.
Introduction One of the well-known biochemical effects of caffeine is the inhibition of cyclic nucleotide phosphodiesterases (Butcher and Sutherland 1962) . Thus, it seemed plausible to propose that the reduction in X-radiation-induced mitotic delay by caffeine may be the result of an elevated cyclic AMP content (Scaife 1971, Boynton, Evans and Crouse 1974, Walters, Gurley and Tobey 1974) . To test this hypothesis, we have characterized the caffeine effect on radiationinduced mitotic delay in the naturally-synchronous plasmodial slime mould Physarum polycephalum during late G 2 and early prophase, and have compared the action of that compound with others of similar structure and/or physiological function . Our results indicate that the reduction of mitotic delay is not a result of altered cyclic AMP levels . 2.
Materials and methods Macroplasmodia containing synchronously-dividing nuclei were prepared (Daniel and Baldwin 1964) from stock cultures of Physarum polycephalum, strain M3 C, using the modified medium of Brewer and Prior (1976) . Methodologies for gamma-irradiation, drug treatment, and determination of mitotic stages in plasmodial sectors were essentially as described previously (Rustad, Oleinick and Brewer 1975) .
3.
Results The caffeine concentration dependence for the reduction of radiation-induced mitotic delay in Physarum is shown in figure 1 . Sectors of a plasmodium were irradiated with 920 rad of 60Co gamma-rays 27 min before metaphase, and mitosis was delayed for 29 min in an irradiated plasmodial sector compared with a control sector (figure 1 (a)) . When other sectors were placed into medium containing 0 . 1-10 mM caffeine within 1 min after irradiation, less mitotic delay was observed . With 10 mM caffeine there was no mitotic delay (over that due to the drug alone) . When plasmodial sectors were irradiated earlier in the mitotic cycle (74 min before metaphase, figure 1 (b)), a somewhat smaller mitotic delay was observed fi Copyright U .S . Government . I Also affiliated with the Department of Biochemistry and the School of Dentistry . § Also affiliated with the Departments of Anatomy and Biology .
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70
Correspondence
0 2
Figure 1 . The reduction of radiation-induced mitotic delay by various concentrations of caffeine . Sectors (twelfths of a plasmodium) were exposed to 0 or 920 rad 60Co gamma-radiation and immediately transferred to fresh growth medium containing 0-10 mM caffeine . Mitotic delay is defined as the difference between the time of appearance of metaphase in treated and in untreated sectors . Irradiation was at 27 min (a) or 74 min (b) before control metaphase . •, unirradiated ; •, 920 rad gamma-radiation .
in the absence of caffeine, in agreement with earlier results (Nygaard, Brewer, Evans and Wolpaw 1973), and the efficacy of each caffeine concentration was greater (with the exception of 10 mM which was somewhat toxic (Trevithick and Braun 1977)) . Essentially complete reversal of the delay was seen with 0 . 3 mM caffeine . In another experiment the mitotic delay resulting from a dose as high as 10 krad (261 min) was reduced by 84 per cent in the presence of 3 mM caffeine . Maximal reduction of radiation-induced mitotic delay resulted when caffeine (1 mM) was present throughout the post-irradiation period (figure 2) . Plasmodial sectors were incubated continuously in caffeine beginning at various times after irradiation . The protective effect of the drug decreased dramatically as a function of the length of time prior to the addition of caffeine (figure 2 (a)) . In the experiments described by figure 2 (b), exposure to caffeine was begun within 1 min after irradiation, and the plasmodial sectors were returned to normal growth medium at various intervals thereafter . The protection against radiation induced mitotic delay by the drug increased with the duration of the exposure . In contrast to post-irradiation exposure, treatment with caffeine and subsequent removal of the plasmodia from the drug prior to irradiation did not modify the radiation response (data not shown) . Two other methylxanthines, theophylline and theobromine, which differ from caffeine only by a single methyl group each, appeared to mimic caffeine
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Correspondence
71
Figure 2 . Variation of mitotic delay with interval of exposure to caffeine . Plasmodial sectors were transferred to fresh growth medium containing none or 1 mM caffeine following 3 . 4 krad gamma-radiation . (a) Sectors were placed in caffeine-free medium for various periods before exposure to caffeine . Irradiation occurred at 62 min ( .) or 36 min (A) before control metaphase . ` oo ' on the abscissa indicates no exposure to caffeine . (b) Sectors were exposed to caffeine immediately after irradiation, then transferred to caffeine-free medium at various times thereafter . Irradiation occurred at 62 min ( .) or 45 min (A) before control metaphase . All values for mitotic delay are relative to the time of mitosis observed in an unirradiated control sector.
Substance None Caffeine Theobromine Theophylline Dibutyryl cyclic AMP Cholera toxin Methylisobutyl xanthine Papaverine Ro 20-1724
Per cent mitotic delay relative to irradiation alone (100) 37 ± 6 (5) 34 ± 5 (4) 51 ± 9 (3) 93 ± 13 (4) 116 ± 10 (4) 126 ± 15 (3) 139 ±10 (7) 195 ± 22 (4)
Effect on radiation-induced mitotic delay of substances which elevate cyclic AMP levels . Plasmodial sectors were irradiated with 3 .4 krad in late G2 -early prophase (between 30 and 90 min before control metaphase), then transferred to fresh growth medium or to medium containing one of the above substances at 2 mM, with the exception of cholera toxin which was present at 1 µg/ml . Data are corrected for any small mitotic delays resulting from drug treatment alone and are expressed as mean ± standard error . Numbers in parentheses refer to the number of plasmodia tested with a given substance .
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72
Correspondence
in reducing mitotic delay (table) . All three of these compounds are known to be inhibitors of cyclic AMP phosphodiesterase . If their action on radiationinduced mitotic delay were due to inhibition of that enzyme, other such inhibitors, even though structurally dissimilar, should likewise reduce the mitotic delay . Methylisobutylxanthine (Beavo, Rogers, Crofford, Hardman, Sutherland and Newman 1970), Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] (Sheppard and Wiggan 1971), and papaverine [6,7-dimethoxy-1-veratrylisoquinoline] (Triner, Vulliemoz, Schwartz and Nahas 1970) are all reported inhibitors of the phosphodiesterase . These compounds increased rather than decreased mitotic delay at concentrations which have little or no effect on mitosis in unirradiated plasmodia . The addition of cholera toxin, a stimulator of adenyl cyclase activity in every system tested (Finkelstein 1973), also led to extended mitotic delays . Dibutyryl cyclic AMP itself had little effect on radiationinduced mitotic delay (table) . It appears that mere elevation of cyclic AMP within the plasmodium is insufficient to produce the reduction of mitotic inhibition shown by caffeine .
4.
Conclusions Several conclusions can be derived from this study . (1) The reduction of radiation-induced mitotic delay is related to increasing concentrations of caffeine over at least two orders of magnitude . (2) Pre-irradiation treatment with caffeine has no detectable effect . (3) For maximal effect, caffeine must be present for most, if not all, of the post-irradiation pre-mitotic period . (4) Chemicals which are reported to inhibit cyclic AMP phosphodiesterase may either reduce or increase radiation-induced mitotic delay . Thus, it seems unlikely that cyclic AMP metabolism per se is the basis for the ' healing ' effect of caffeine on radiation-induced mitotic delay . On the contrary, the present data suggest that elevation of cyclic AMP levels may actually prolong this delay, and that caffeine (theophylline, theobromine) may reduce mitotic delay by a mechanism(s) other than through the inhibition of cyclic AMP phosphodiesterase . Caffeine also engages in several other biochemical actions, e .g . alterations in Ca++_ transport (Huddart and Syson 1975, Rebhun, Jemiolo, Ivy, Mellon and Nath 1975), binding to DNA (Ts'o, Helmkamp and Sander 1962, Ts'o and Lu 1964), stimulation of cell-free protein synthesis (Fernandez-Puentes, Carrasco and Vazquez 1974), as well as other actions less well described . Thus the role of caffeine in reducing radiation-induced mitotic delay may be a complex one .
ACKNOWLEDGMENTS
This work was supported by the U .S . Energy Research and Development Administration . We thank Ms . Peggy Busacca for capable technical assistance, Dr . Michael E . Maguire, Department of Pharmacology, Case Western Reserve University for gifts of methylisobutylxanthine and Ro 20-1724, Dr . Herbert Sheppard, Hoffman-LaRoche, Inc ., for additional quantities of Ro 20 -1724, Dr . Charles Carpenter, Department of Medicine, for a gift of cholera toxin, Mr . K . Pillai for the dosimetry, and Dr . Helen H . Evans for reviewing the manuscript .
Correspondence
73
REFERENCES
J . A ., ROGERS, N . L ., CROFFORD, O . B ., HARDMAN, J . G ., SUTHERLAND, E . W ., and NEWMAN, E . V ., 1970, Molec . Pharmac ., 6, 597 . BOYNTON, A . L ., EVANS, T. C ., and CROUSE, D . A ., 1974, Radiat . Res ., 60, 89 . BREWER, E . N ., and PRIOR, A ., 1976, Physarum Newsl ., 8, 45 . BUTCHER, R . W ., and SUTHERLAND, E . W ., 1962, J . biol . Chem., 237, 1244 . DANIEL, J . W ., and BALDWIN, H . H ., 1964, Methods in Cell Physiology, Vol . 1 (New York : Academic Press) ; p . 9 . FERNANDEZ-PUENTES, C ., CARRASCO, L ., and VAZQUEZ, D ., 1974, FEBS Lett ., 45, 132 .
Int J Radiat Biol Downloaded from informahealthcare.com by Flinders University of South Australia on 01/11/15 For personal use only.
BEAVO,
FINKELSTEIN, R . A ., 1973, CRC crit . Rev . Microbiol., 2, 553 . HUDDART, H ., and SYSON, A. J ., 1975, Y. exp . Biol ., 63, 131 . NYGAARD, O . F ., BREWER, E . N ., EVANS, T . E ., and WOLPAW, J . R ., 1973, Advances in Radiation Research, Vol . 2 (London : Gordon & Breach), p . 989 . REBHUN, L . I ., JEMIOLO, D ., Ivy, N ., MELLON, M ., and NATH, J ., 1975, Ann . N . Y. Acad . Sci ., 253, 362 . RuSTAD, R . C ., OLEINICK, N . L., and BREWER, E . N ., 1975, Expl Cell Res ., 93, 477 . SCAIFE, J . F ., 1971, Int . Y. Radiat . Biol., 19, 191 . SHEPPARD, H ., and WIGGAN, G ., 1971, Molec . Pharmac ., 7, 111 . TREVITHICK, J . R ., and BRAUN, R ., 1977, Experientia, 33, 28 . TRINER, L ., VULLIEMOZ, Y ., SCHWARTZ, I ., and NAHAS, G . G ., 1970, Biochem . biophys . Res . Commun ., 40, 64 . Ts'o, P .O .P ., HELMKAMP, G . K., and SANDER, C ., 1962, Proc . natn . Acad . Sci. U.S.A ., 48,686 . Ts'o, P . O . P ., and Lu, P ., 1964, Proc . natn . Acad . Sci. U.S . A ., 51, 17 . WALTERS, R . A ., GURLEY, L . R ., and ToBEY, R . A ., 1974, Biophys . Y., 14, 99 .
