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Effects of caffeine and theophylline on DNA synthesis in untreated and UV-irradiated mammalian cells
192
3rd
ANNUAL MEETING, UPPSALA
duced in bean roots by MH and TT appears to be independent of the temperature during the post-treatment. Both in bean roots and in hamster cells, potentiation was obtained only when cells were exposed to caffeine during the S-phase. Abbreviations: MH, maleic hydrazide; TT, thiotepa.
14
KRATOCHVILOVA,J., Abteilung ftir Genetik, Gesellschaft ftir Strahlen- und Umweltforschung, Mtinchen-Neuherberg (W. Germany). Fertilization ability of male mice after t r e a t m e n t with t u m o r inhibitors Male mice were injected intraperitoneally with 3.5 mg/kg of MC or with IOO mg/kg of FU and mated with untreated females for IO consecutive weeks. Tubal ova were examined 3 5 ~ 5 h after copulation. Approx. i i ova were recovered per female. Two-cell-stage ova were considered to be fertilized, one-cell-stage ova to be unfertilized. Sperm in the ejaculate were counted 2 h after copulation. In the MC experiment the frequency of fertilized ova was normal until 20 days post-treatment. The number of fertilized ova decreased from day 21 to day 42, followed by 9 days of complete sterility. The fertilization ability increased from day 52 to day 60, when control values were reached again. In the FU experiment all ova were fertilized until 30 days post-treatment. From day 13 to day 34 the frequency of fertilized ova decreased and reached a minimum of 3 ° % from day 34 to day 38. Thereafter the number of fertilized ova increased, reaching the control level b y 43 days posttreatment. In both experiments the sperm counts were markedly lower during the periods of impaired fertilization ability. The frequency of fertilized ova compared with the results of the dominant lethal experiment (EHLING, Mutation Res., I I (1971)35) indicates that MC-induced dominant lethal mutations in spermatocytes manifested as loss before implantation. In addition MC affects the maturation of spermatocytes and spermatogonia. Both effects are partly due to the induction of chromatid aberrations in these germ cell stages (ADLER, this meeting). FU seems to affect only the maturation of spermatocytes. Abbreviations: FU, 5-fluorouracil; MC, mitomycin C.
15
LEHMANN, A. R., The University of Sussex, Brighton (Great Britain). Effects of caffeine and theophylline on D N A synthesis in untreated and UV-irradiated mammalian cells The effects of methylated xanthines on the rate of DNA synthesis, and on the size of newly synthesized DNA made in unirradiated and UVdrradiated rodent cell lines have been examined, with the following results:
EUROPEAN ENVIRONMENTAL
MUTAGEN SOCIETY
193
(z) In mouse L5178Y lymphoma cells caffeine did not introduce breaks into pre-existing DNA, nor did it inhibit the rejoining of X-ray-induced single-strand breaks in this DNA; (2) In L5178Y cells, LS929 mouse fibroblasts and Chinese hamster V79 cells, high doses of caffeine or theophylline (greater than 0.3 mg/ml) inhibited DNA synthesis, as measured by the uptake of E3Hlthymidine into DNA. In UV-irradiated cells this inhibition of the rate of DNA synthesis was slightly smaller than in unirradiated cells; (3) Alkaline sucrose gradient studies showed that the average size of DNA strands synthesized in the presence of caffeine or theophylline (o.3-1.5 mg/ml) was somewhat smaller than that in untreated cells. This effect varied between the cell lines studied and was quite small compared to the effects on UV-irradiated cells (see below). It probably indicates that the DNA was synthesized in smaller replicating units. On further incubation in the absence of the drug, the molecular weight of the new DNA rapidly reached values similar to those of untreated controls; (4) DNA synthesized after UV-irradiation of cells contains gaps, presumably opposite the UV-induced pyrimidine dimers in the parental strands. These gaps are subsequently sealed. This gap-filling process is strongly inhibited by relatively low doses of caffeine or theophylline (o.15-o. 3 mg/ml). As a consequence of this, in UV-irradiated cells low molecular weight newly synthesized DNA accumulates in the presence of caffeine or theophylline, whereas in the absence of the drugs, the new DNA eventually attains the size of that in unirradiated cells. The magnitude of all these effects varied between the cell lines studied. The results probably account for the well-documented sensitization of cells to UV light by caffeine and theophylline, and for the observations that caffeine only exerts many of its cytological effects if it is present during the DNA synthetic period.
16 LILLY, L. J., Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London (Great Britain).
An investigation of possible chromosome damage in disulfiramtreated alcoholics PILINSKAYA (Genetika, 6, No. 7, (197o) 157-163) showed that there was an increased incidence of chromosome aberrations in leucocytes from industrial workers who had been exposed to the fungicide ziram. Later (Genetika, 7, No. 6, (1971) 138-142 ) he showed that ziram also caused an increase in aberrations in chromosomes of leucocytes cultured in vitro. The fungicide ziram has a chemical structure which is similar but not identical to that of disulfiram ("antabuse") a drug given to alcoholics. Both compounds m a y be used as rubber accelerators or fungicides. Alcohol causes acute nausea, vasodilation, unconsciousness or even death if taken after disulfiram. Because of its possibly fatal reaction, disulfiram is no longer so widely prescribed. However, as it is still used for intractable cases it is important to establish whether or not it induces chromosome aberrations. To do this we are investigating the frequency of aberrations in leucocytes from patients treated with disulfiram and also examining the effects of disulfiram added to leucocyte cultures in vitro. In this paper we shall consider some preliminary results from patients treated
3rd
ANNUAL MEETING, UPPSALA
duced in bean roots by MH and TT appears to be independent of the temperature during the post-treatment. Both in bean roots and in hamster cells, potentiation was obtained only when cells were exposed to caffeine during the S-phase. Abbreviations: MH, maleic hydrazide; TT, thiotepa.
14
KRATOCHVILOVA,J., Abteilung ftir Genetik, Gesellschaft ftir Strahlen- und Umweltforschung, Mtinchen-Neuherberg (W. Germany). Fertilization ability of male mice after t r e a t m e n t with t u m o r inhibitors Male mice were injected intraperitoneally with 3.5 mg/kg of MC or with IOO mg/kg of FU and mated with untreated females for IO consecutive weeks. Tubal ova were examined 3 5 ~ 5 h after copulation. Approx. i i ova were recovered per female. Two-cell-stage ova were considered to be fertilized, one-cell-stage ova to be unfertilized. Sperm in the ejaculate were counted 2 h after copulation. In the MC experiment the frequency of fertilized ova was normal until 20 days post-treatment. The number of fertilized ova decreased from day 21 to day 42, followed by 9 days of complete sterility. The fertilization ability increased from day 52 to day 60, when control values were reached again. In the FU experiment all ova were fertilized until 30 days post-treatment. From day 13 to day 34 the frequency of fertilized ova decreased and reached a minimum of 3 ° % from day 34 to day 38. Thereafter the number of fertilized ova increased, reaching the control level b y 43 days posttreatment. In both experiments the sperm counts were markedly lower during the periods of impaired fertilization ability. The frequency of fertilized ova compared with the results of the dominant lethal experiment (EHLING, Mutation Res., I I (1971)35) indicates that MC-induced dominant lethal mutations in spermatocytes manifested as loss before implantation. In addition MC affects the maturation of spermatocytes and spermatogonia. Both effects are partly due to the induction of chromatid aberrations in these germ cell stages (ADLER, this meeting). FU seems to affect only the maturation of spermatocytes. Abbreviations: FU, 5-fluorouracil; MC, mitomycin C.
15
LEHMANN, A. R., The University of Sussex, Brighton (Great Britain). Effects of caffeine and theophylline on D N A synthesis in untreated and UV-irradiated mammalian cells The effects of methylated xanthines on the rate of DNA synthesis, and on the size of newly synthesized DNA made in unirradiated and UVdrradiated rodent cell lines have been examined, with the following results:
EUROPEAN ENVIRONMENTAL
MUTAGEN SOCIETY
193
(z) In mouse L5178Y lymphoma cells caffeine did not introduce breaks into pre-existing DNA, nor did it inhibit the rejoining of X-ray-induced single-strand breaks in this DNA; (2) In L5178Y cells, LS929 mouse fibroblasts and Chinese hamster V79 cells, high doses of caffeine or theophylline (greater than 0.3 mg/ml) inhibited DNA synthesis, as measured by the uptake of E3Hlthymidine into DNA. In UV-irradiated cells this inhibition of the rate of DNA synthesis was slightly smaller than in unirradiated cells; (3) Alkaline sucrose gradient studies showed that the average size of DNA strands synthesized in the presence of caffeine or theophylline (o.3-1.5 mg/ml) was somewhat smaller than that in untreated cells. This effect varied between the cell lines studied and was quite small compared to the effects on UV-irradiated cells (see below). It probably indicates that the DNA was synthesized in smaller replicating units. On further incubation in the absence of the drug, the molecular weight of the new DNA rapidly reached values similar to those of untreated controls; (4) DNA synthesized after UV-irradiation of cells contains gaps, presumably opposite the UV-induced pyrimidine dimers in the parental strands. These gaps are subsequently sealed. This gap-filling process is strongly inhibited by relatively low doses of caffeine or theophylline (o.15-o. 3 mg/ml). As a consequence of this, in UV-irradiated cells low molecular weight newly synthesized DNA accumulates in the presence of caffeine or theophylline, whereas in the absence of the drugs, the new DNA eventually attains the size of that in unirradiated cells. The magnitude of all these effects varied between the cell lines studied. The results probably account for the well-documented sensitization of cells to UV light by caffeine and theophylline, and for the observations that caffeine only exerts many of its cytological effects if it is present during the DNA synthetic period.
16 LILLY, L. J., Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London (Great Britain).
An investigation of possible chromosome damage in disulfiramtreated alcoholics PILINSKAYA (Genetika, 6, No. 7, (197o) 157-163) showed that there was an increased incidence of chromosome aberrations in leucocytes from industrial workers who had been exposed to the fungicide ziram. Later (Genetika, 7, No. 6, (1971) 138-142 ) he showed that ziram also caused an increase in aberrations in chromosomes of leucocytes cultured in vitro. The fungicide ziram has a chemical structure which is similar but not identical to that of disulfiram ("antabuse") a drug given to alcoholics. Both compounds m a y be used as rubber accelerators or fungicides. Alcohol causes acute nausea, vasodilation, unconsciousness or even death if taken after disulfiram. Because of its possibly fatal reaction, disulfiram is no longer so widely prescribed. However, as it is still used for intractable cases it is important to establish whether or not it induces chromosome aberrations. To do this we are investigating the frequency of aberrations in leucocytes from patients treated with disulfiram and also examining the effects of disulfiram added to leucocyte cultures in vitro. In this paper we shall consider some preliminary results from patients treated