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combined effects of chemicals on mammalian cells in culture. Effect of order and intervals of administration of methyl methanesulfonate and ethyl methanesulfonate
364 metabolic activation after extraction with boiling water and adsorption to blue cotton. The numbers of His + revertant colonies/5 g of the meat heatdried without charring at 2 2 0 ° C for 15 min were about 3000 for bonito, about 1000 for tunny, less than 500 for mackerel, salmon, swordfish, sardine, horse mackerel and cod, and 0 for cuttlefish. The mutagens in the heat-dried bonito meat were purified by thin-layer chromatography and highperformance liquid chromatography. They were identified as MelQx and 4,8-DiMelQx by comparison with the authentic specimen with respect to R f values, retention times, ultraviolet adsorption spectra and mass spectra. The contents of MelQx and 4,8-DiMelQx in the bonito meat were estimated to be 5.2 and 5.4 ng/g, respectively. The major mutagens produced in the bonito, tunny and mackerel meats heated without charring at 1 0 0 ° C for 48 h and at 2 2 0 ° C for 15 min were found to be MelQx and 4,8-DiMelQx. It is interesting to note that the bonito and sardine meats broiled with charring for 15 min contained MelQx and 4,8-DiMelQx but higher mutagenicity was observed in the fraction containing other mutagens such as IQ, MelQ a n d / o r Glu-P-2.
19 Kinouchi, T., K. Nishifuji and Y. Ohnishi, Department of Bacteriology, School of Medicine, University of Tokushima, Tokushima 770 (Japan) In vivo metabolism of l-nitropyrene: detection of glutathione conjugates of l-nitropyrene 4,5-oxide in the bile of rats administered 1-nitropyrene orally High-performance liquid chromatographic (HPLC) analysis of bile excreted from rats administered [3H]l-nitropyrene (NP), orally, indicated the presence of 8 radioactive peaks, A - H . Each peak was collected and treated with ]3-glucuronidase, aryl sulfatase and y-glutamtyl transpeptidase (yGTP). Peak G, which accounted for 5.3% of the radioactive biliary metabolites, contained glucuronide conjugates of hydroxy-NPs. Peak H contained both glucuronide (23.5%) and sulfate (2.7%) conjugates of hydroxy-NPs. Peaks C, D and F were substrates for y G T P and, thus, appeared to be glutathione (GSH) conjugates. These
peaks accounted for 9.1, 3.6 and 9.6% of the biliary radioactivity, respectively. Peak F had the same HPLC retention time as a conjugate obtained from reacting 1-NP 4,5-oxide with GSH, in vitro. Furthermore, both peak F and the synthetic 1-NP 4,5-oxide GSH conjugate were converted to the same product upon treatment with 7GTP. Additional evidence for the reaction of 1-NP 4,5-oxide and 1-NP 9,10-oxide with GSH was obtained by conducting mutagenesis assays with Salmonella. In the absence of GSH, the mutagenicity of 1-NP 4,5-oxide and 1-NP 9,10-oxide was ( r e v e r t a n t s / n m o l e / p l a t e ) : 413, 324 [TA98, ( - ) $ 9 ] ; 735, 22 [TA98, (+)$9]; 1060, 476 [TA100, ( - ) $ 9 ] ; 112, 13 [TA100, (+)$9]; 76, 95 [TA98NR, ( - ) $ 9 ] ; and 175, 362 [TA98/1,8-DNP6, ( - ) $ 9 ] , respectively. Addition of GSH to the preincubation mixture, which contained the 1-NP oxide and $9 mix, resulted in a decrease in mutations.
20 Kojima, H., H. Konishi and Y. Kuroda 1, Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., Ohgaki, Gifu 503, and 1 Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 411 (Japan) Combined effects of chemicals on mammalian cells in culture. Effect of order and intervals of administration of methyl methanesulfonate and ethyl methanesulfonate We previously reported on effects of simultaneous treatments with methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on 6thioguanine (6TG)-resistant mutation in Chinese hamster V79 cells. In these experiments, we found that the mutation frequency induced by simultaneous treatments with the above two chemicals was higher than the sum of the frequencies obtained by single treatment with each chemical. In the present work, to investigate the combined effects further, cells were treated with MMS and EMS successively for each 3 h, or incubated in normal medium for 3 h between the two treatments. In successive treatments with MMS and EMS, the induced mutation frequency was higher than that obtained in simultaneous treatment, but
365 a reversal of the sequence of treatments gave no detectable changes in the frequency. By inserting an incubation in normal medium between the MMS and EMS treatments the induced mutation frequency decreased to a value almost equal to that observed in the simultaneous treatment. These results suggest that the mutation frequency induced by a combination of MMS and EMS varies due to the difference of treatment conditions such as order and intervals of their administration.
21 Koshi, K., F. Serita, K. Sawatari and Y. Suzuki, National Institute of Industrial Health, Tama-ku, Kawasaki 214 (Japan) Cytogenetic analysis of bone marrow cells and peripheral blood lymphocytes from rats exposed to chromium fumes by inhalation Male Sprague-Dawley rats were exposed to chromium fumes generated from powders of chromium metal by a plasma flame sprayer at 1.84 + 0.55 m g / m 3 or 0.55 + 0.07 m g / m 3 in fume concentration, for 5 h a day, 5 days a week, during a period of 1 week or 2 months, respectively. The cytogenetic analysis was carried out 20 h, 3 days, 7 days and 1 month after the end of exposure. No significant increase in chromosomal aberrations in the bone marrow cells was caused by the 1-week and 2-month exposures. In the peripheral blood lymphocytes, higher frequencies of sister-chromatid exchanges were observed in each exposure group compared with the controls. The incidence of structural chromosomal aberrations in the peripheral blood lymphocytes in both exposure groups was higher than that in the controls with respect to aberrant metaphases, chromatid and chromosome gaps and chromatid breaks at each period of the examination. No changes in the number of chromosomes in the bone marrow cells and peripheral blood lymphocytes were found.
22 Kuroda, Y., Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 411 (Japan)
Antimutagenic mechanism of vitamin C and its derivatives in mammalian cells in culture The effect of vitamin C and its derivatives, dehydro-vitamin C and iso-vitamin C on the cytotoxicity and mutagenicity of ethyl methanesulfonate (EMS) in Chinese hamster V79 cells was investigated. EMS alone had a cytotoxic effect on the cells, showing an LDs0 of 541 /~g/ml. In the presence of vitamin C or its derivatives at concentrations of 50 or 100/~g/ml, the LDs0 of EMS was reduced to more than 1 0 0 0 / t g / m l for vitamin C, and 639 / t g / m l for iso-vitamin C. Dehydrovitamin C enhanced the cytotoxicity of EMS, giving an LDs0 of 234 /~g/ml. EMS alone had a strong activity in inducing 6-thioguanine-resistant (6TG r) mutations in these cells. At a concentration of 1000 ~ g / m l , EMS induced 6TG r mutations at a frequency of 88/105 survivors. Vitamin C decreased the EMS-induced mutations to 1 / 3 - 1 / 4 . Dehydro-vitamin C and iso-vitamin C also decreased the EMS-induced mutations to 1/2-1/3. When cells were treated with vitamin C after treatment with EMS, the induced mutation frequency was not affected. The pretreatment with vitamin C was effective in reducing the EMS-induced mutations. Vitamin C mixed with EMS and incubated for 2 h had a strong effect on reducing the mutations. This suggests that vitamin C was desmutagenic rather than antimutagenic for induction of mutations by EMS.
23 Masubuchi, Y., S. Fujita and T. Suzuki, Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoicho, Chiba 260 (Japan) A possible mechanism for Sudan Ill-induced prevention of DMBA carcinogenesis 7,12-Dimethylbenzanthracene ( D M B A ) - i n duced carcinogenesis is prevented by Sudan III (Huggins et al. (1978) Proc. Natl. Acad. Sci. (U.S.A.)). We have shown that Sudan III induces cytochrome P-448 and UDP-glucuronyltransferase ( U D P G T ) (Fujita et al. (1982) Chem.-Biol. Inter-
19 Kinouchi, T., K. Nishifuji and Y. Ohnishi, Department of Bacteriology, School of Medicine, University of Tokushima, Tokushima 770 (Japan) In vivo metabolism of l-nitropyrene: detection of glutathione conjugates of l-nitropyrene 4,5-oxide in the bile of rats administered 1-nitropyrene orally High-performance liquid chromatographic (HPLC) analysis of bile excreted from rats administered [3H]l-nitropyrene (NP), orally, indicated the presence of 8 radioactive peaks, A - H . Each peak was collected and treated with ]3-glucuronidase, aryl sulfatase and y-glutamtyl transpeptidase (yGTP). Peak G, which accounted for 5.3% of the radioactive biliary metabolites, contained glucuronide conjugates of hydroxy-NPs. Peak H contained both glucuronide (23.5%) and sulfate (2.7%) conjugates of hydroxy-NPs. Peaks C, D and F were substrates for y G T P and, thus, appeared to be glutathione (GSH) conjugates. These
peaks accounted for 9.1, 3.6 and 9.6% of the biliary radioactivity, respectively. Peak F had the same HPLC retention time as a conjugate obtained from reacting 1-NP 4,5-oxide with GSH, in vitro. Furthermore, both peak F and the synthetic 1-NP 4,5-oxide GSH conjugate were converted to the same product upon treatment with 7GTP. Additional evidence for the reaction of 1-NP 4,5-oxide and 1-NP 9,10-oxide with GSH was obtained by conducting mutagenesis assays with Salmonella. In the absence of GSH, the mutagenicity of 1-NP 4,5-oxide and 1-NP 9,10-oxide was ( r e v e r t a n t s / n m o l e / p l a t e ) : 413, 324 [TA98, ( - ) $ 9 ] ; 735, 22 [TA98, (+)$9]; 1060, 476 [TA100, ( - ) $ 9 ] ; 112, 13 [TA100, (+)$9]; 76, 95 [TA98NR, ( - ) $ 9 ] ; and 175, 362 [TA98/1,8-DNP6, ( - ) $ 9 ] , respectively. Addition of GSH to the preincubation mixture, which contained the 1-NP oxide and $9 mix, resulted in a decrease in mutations.
20 Kojima, H., H. Konishi and Y. Kuroda 1, Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., Ohgaki, Gifu 503, and 1 Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 411 (Japan) Combined effects of chemicals on mammalian cells in culture. Effect of order and intervals of administration of methyl methanesulfonate and ethyl methanesulfonate We previously reported on effects of simultaneous treatments with methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on 6thioguanine (6TG)-resistant mutation in Chinese hamster V79 cells. In these experiments, we found that the mutation frequency induced by simultaneous treatments with the above two chemicals was higher than the sum of the frequencies obtained by single treatment with each chemical. In the present work, to investigate the combined effects further, cells were treated with MMS and EMS successively for each 3 h, or incubated in normal medium for 3 h between the two treatments. In successive treatments with MMS and EMS, the induced mutation frequency was higher than that obtained in simultaneous treatment, but
365 a reversal of the sequence of treatments gave no detectable changes in the frequency. By inserting an incubation in normal medium between the MMS and EMS treatments the induced mutation frequency decreased to a value almost equal to that observed in the simultaneous treatment. These results suggest that the mutation frequency induced by a combination of MMS and EMS varies due to the difference of treatment conditions such as order and intervals of their administration.
21 Koshi, K., F. Serita, K. Sawatari and Y. Suzuki, National Institute of Industrial Health, Tama-ku, Kawasaki 214 (Japan) Cytogenetic analysis of bone marrow cells and peripheral blood lymphocytes from rats exposed to chromium fumes by inhalation Male Sprague-Dawley rats were exposed to chromium fumes generated from powders of chromium metal by a plasma flame sprayer at 1.84 + 0.55 m g / m 3 or 0.55 + 0.07 m g / m 3 in fume concentration, for 5 h a day, 5 days a week, during a period of 1 week or 2 months, respectively. The cytogenetic analysis was carried out 20 h, 3 days, 7 days and 1 month after the end of exposure. No significant increase in chromosomal aberrations in the bone marrow cells was caused by the 1-week and 2-month exposures. In the peripheral blood lymphocytes, higher frequencies of sister-chromatid exchanges were observed in each exposure group compared with the controls. The incidence of structural chromosomal aberrations in the peripheral blood lymphocytes in both exposure groups was higher than that in the controls with respect to aberrant metaphases, chromatid and chromosome gaps and chromatid breaks at each period of the examination. No changes in the number of chromosomes in the bone marrow cells and peripheral blood lymphocytes were found.
22 Kuroda, Y., Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 411 (Japan)
Antimutagenic mechanism of vitamin C and its derivatives in mammalian cells in culture The effect of vitamin C and its derivatives, dehydro-vitamin C and iso-vitamin C on the cytotoxicity and mutagenicity of ethyl methanesulfonate (EMS) in Chinese hamster V79 cells was investigated. EMS alone had a cytotoxic effect on the cells, showing an LDs0 of 541 /~g/ml. In the presence of vitamin C or its derivatives at concentrations of 50 or 100/~g/ml, the LDs0 of EMS was reduced to more than 1 0 0 0 / t g / m l for vitamin C, and 639 / t g / m l for iso-vitamin C. Dehydrovitamin C enhanced the cytotoxicity of EMS, giving an LDs0 of 234 /~g/ml. EMS alone had a strong activity in inducing 6-thioguanine-resistant (6TG r) mutations in these cells. At a concentration of 1000 ~ g / m l , EMS induced 6TG r mutations at a frequency of 88/105 survivors. Vitamin C decreased the EMS-induced mutations to 1 / 3 - 1 / 4 . Dehydro-vitamin C and iso-vitamin C also decreased the EMS-induced mutations to 1/2-1/3. When cells were treated with vitamin C after treatment with EMS, the induced mutation frequency was not affected. The pretreatment with vitamin C was effective in reducing the EMS-induced mutations. Vitamin C mixed with EMS and incubated for 2 h had a strong effect on reducing the mutations. This suggests that vitamin C was desmutagenic rather than antimutagenic for induction of mutations by EMS.
23 Masubuchi, Y., S. Fujita and T. Suzuki, Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoicho, Chiba 260 (Japan) A possible mechanism for Sudan Ill-induced prevention of DMBA carcinogenesis 7,12-Dimethylbenzanthracene ( D M B A ) - i n duced carcinogenesis is prevented by Sudan III (Huggins et al. (1978) Proc. Natl. Acad. Sci. (U.S.A.)). We have shown that Sudan III induces cytochrome P-448 and UDP-glucuronyltransferase ( U D P G T ) (Fujita et al. (1982) Chem.-Biol. Inter-