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DNA damage and its repair in germ cells of male mice treated with ethyl nitrosourea
396
JEMS Abstracts/Mutation Research 334 (1995) 385-427
histidine to selectively grow revertants only. The bacterial DNA is extracted at 100°C for 5 min, followed by amplification of the hisG gene by PCR using two primers which give 343-bp DNA. Then the double-stranded DNA is directly cyclesequenced by AmpliTaq DNA polymerase with fluorescent labelled primer and is analyzed in the automatic DNA sequencer. This method was applied to N-methyl-N'nitro-N-nitrosoguanidine as a mutagen on TA1535. The analyzed data pattern of the hisG gene showed the revertant base sequence, -TCC-, on the mutant base sequence, -CCC-. This result suggests that this new method can be useful in estimating the genetic level of mutation, and thus it can provide an effective way to clarify the mechanism of mutation. 24 Itoh, S. and H. Shimada, Drug Safety Research Center, Developmental Research Laboratories, Daiichi Pharmaceutical Co., Ltd., 16-13 Kita-kasai 1-Chome, Edogawa-ku, Tokyo 134, Japan In vivo mutagenicity of hexavalent chromium We investigated the mutagenicity of hexavalent chromium (K2CrO 4) using the MutaMouse positive selection system in bone marrow and liver. K2CrO 4 was injected intraperitoneally to male MutaMouse, and bone marrow and liver were removed after 1 or 7 days expression. DNA was isolated from the tissue and the mutational target gene, lacZ, was rescued by packaging into lambda phage preheads. Galactose-sensitive E. coli C ( G a l E - ) were infected with the phages and cultured to obtain l a c Z - mutant plaques on a phenyl galactoside selection plate. In bone marrow, mutant frequency (MF) was increased on day 1 after treatment with K2CrO 4 and then decreased to the spontaneous level on day 7. In the liver, the increase in MF was observed on day 7 after treatment only. The difference in response between bone marrow and liver may be caused by the difference in their cell turnover rate. 25 Inoue, H., H. Baba, K. Awano and K. Yoshikawa, Toxicology Laborarory, Life Science Research
Sector, Research Center, Mitsubishi Kasei Corporation, 1000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan Use of high-bioactivation strain in Drosophila somatic cell mutagenicity testing A simple procedure was developed for screening strains having a putative high-bioactivation capacity which confers high potency to procarcinogens in the wing spot test a n d / o r the DNA repair test. The procedure was applied to three insecticide-resistant strains, Oregon R(R), Haag79 and Hikone-R, and two insecticide-sensitive strains, Canton-S and w. Hybrid larvae produced by mating FM7 / mei-9 a mei-41°5; mwh / mwh females and males from each strain were fed on a procarcinogen, and the lethality of DNA repair defective, mei-9 m e i - 4 1 / Y hybrid males was compared among the strains. Five procarcinogens tested showed 3-30-fold differences in LC70 concentration among the hybrid males. The relative sensitivities to procarcinogens were Oregon R(R) = Canton-S = w > Hikone R > Haag-79 for aflatoxin B1, benzo[a]pyrene and 7,12-dimethylbenz [a]anthracene, Oregon R(R) = Canton-S = w = Haag-79 > Hikone-R for 2-acetylaminofluorene and Oregon R ( R ) = Haag-79 > Hikone-R > Canton-S = w for urethane. Of the five strains tested, Oregon R(R) was found to have a superior transforming capacity for a variety of procarcinogens to active forms being detected in both the wing spot test and the DNA repair test. The wing/repair test, an assay for concomitant performance of both the wing spot test and the DNA repair test, was performed using the Oregon R(R) flies. In this assay, 58% (11/19) of Ames-negative carcinogens listed by IARC (1987) were detected as positive. From these findings, we conclude that the wing/repair test with high-bioactivation capacity is useful to complement the Ames test for detection of genotoxic carcinogens. 26 Inoue, M. and M. Miyagoshi, Medical Research Institute, Kanazawa Medical University, Daigaku 1-1, Uchinada-machi, Kahoku-gun, Ishikawa 92002, Japan
JEMS Abstracts/Mutation Research 334 (1995) 385-427
DNA damage and its repair in germ cells of male mice treated with ethyl nitrosourea
Male mice i.p. treated with ethyl nitrosourea (ENU) and unscheduled DNA synthesis (UDS) in their germ cells were studied after testicular injection of 3H-thymidine. The UDS was induced in spermatogonia to early spermatid stages, but not in late spermatid to spermatozoa. Levels of DNA base damage and their repair in the germ cells were analyzed in 3H-ENU treated mice. About half of the base damage (N7-ethyl guanine, O6-ethyl guanine and N3-ethyl adenine) detected was repaired within 24 h after ENU treatment. Replicative DNA synthesis (RDS) in spermatogonia was enhanced by ENU treatment in a dose dependent manner. The DNA synthesis in mice treated with 250 mg/kg of ENU increased to about 4 times that of the control. However, the RDS in spermatozoa of treated mice was lower than that of the control. Number of sperm and weight of testis in ENU-treated groups were low when compared with the control. It is suggested that spermatogonia with DNA synthesis induced by ENU degenerated during differentiation into sperm. 27 Inose, H., H. Okuda and T. Watabe, Laboratory of Drug Metabolism and Toxicology, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachiojishi, Tokyo 192-03, Japan 32p-Postlabeling analysis of DNA adducted by the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene
Hepatic DNA of rats injected i.p. with the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene (5-HCR), as well as calf thymus DNA incubated with 5-HCR sulfate in vitro, was analyzed with 32p-postlabeling techniques. In the calf thymus DNA, four adduct spots were detected, and the most potent radioactive adduct spot (adduct 1) was chromatographed on HPLC after being eluted from the thin-layer chromatogram with 2-propanol-ammonium water.
397
Adduct 1 was identified as 2'-deoxy-N6-(chrysen5-yl)methyladenosine (5-HCR-dA) 3',5'-bisphosphate using the synthetic specimen, which was synthesized by the reaction of 5-aminomethylchrysene with 6-chloropurine 2'-deoxyriboside followed by phosphorylation with tetrachloropyrophosphate. Two other adduct spots were characterized as oligonucleotides containing the 5HCR-dA residue resulting from digestion of the adducts with nuclease P1 and phosphodiesterase I produced 5-HCR-dA 5'-monophosphate, which was identified with the authentic specimen. A TLC map of the 32p-postlabeled DNA adduct obtained from rat liver was very similar to that from calf thymus DNA, though an extra minor adduct was observed in rat liver DNA. The adduct level in rat liver DNA treated with 5-HCR sulfate (0.25 /zmol/g body weight) was 77/106 nucleotides, and the adenine adduct was estimated to comprise 80% of the total adducts. 28 Iwase, Y., S. Tanaka, Y. Takeda and K. Yoshikawa, Toxicology Laboratory, Life Science Sector, Research Center, Mitsubishi Kasei Co., 1000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan Effects of 10 chemicals on gap junctional intercellular communication (GJIC) in Chinese hamster V79 cells assessed with a dye transfer assay
The metabolic cooperation assay using 6-thioguanine is not an appropriate method when detection after relatively long-term exposure of ceils to chemicals is required. In this study, a dye transfer assay was applied to measure GJIC. Eight nongenotoxic carcinogens, which had shown negative responses in the metabolic cooperation assay, and two chemicals which had shown positive responses, were examined. Wild-type Chinese hamster V79 cells (2 x 105 cells for 24 h exposure and 2 x 104 cells for 72 h exposure) were cultivated for 24 h in 35 mm dishes and treated with a single test chemical for 24 and 72 h. Effects on GJIC were measured by counting dye-coupled cells after microinjection of Lucifer yellow CH solution into individual cells. Four nongenotoxic
JEMS Abstracts/Mutation Research 334 (1995) 385-427
histidine to selectively grow revertants only. The bacterial DNA is extracted at 100°C for 5 min, followed by amplification of the hisG gene by PCR using two primers which give 343-bp DNA. Then the double-stranded DNA is directly cyclesequenced by AmpliTaq DNA polymerase with fluorescent labelled primer and is analyzed in the automatic DNA sequencer. This method was applied to N-methyl-N'nitro-N-nitrosoguanidine as a mutagen on TA1535. The analyzed data pattern of the hisG gene showed the revertant base sequence, -TCC-, on the mutant base sequence, -CCC-. This result suggests that this new method can be useful in estimating the genetic level of mutation, and thus it can provide an effective way to clarify the mechanism of mutation. 24 Itoh, S. and H. Shimada, Drug Safety Research Center, Developmental Research Laboratories, Daiichi Pharmaceutical Co., Ltd., 16-13 Kita-kasai 1-Chome, Edogawa-ku, Tokyo 134, Japan In vivo mutagenicity of hexavalent chromium We investigated the mutagenicity of hexavalent chromium (K2CrO 4) using the MutaMouse positive selection system in bone marrow and liver. K2CrO 4 was injected intraperitoneally to male MutaMouse, and bone marrow and liver were removed after 1 or 7 days expression. DNA was isolated from the tissue and the mutational target gene, lacZ, was rescued by packaging into lambda phage preheads. Galactose-sensitive E. coli C ( G a l E - ) were infected with the phages and cultured to obtain l a c Z - mutant plaques on a phenyl galactoside selection plate. In bone marrow, mutant frequency (MF) was increased on day 1 after treatment with K2CrO 4 and then decreased to the spontaneous level on day 7. In the liver, the increase in MF was observed on day 7 after treatment only. The difference in response between bone marrow and liver may be caused by the difference in their cell turnover rate. 25 Inoue, H., H. Baba, K. Awano and K. Yoshikawa, Toxicology Laborarory, Life Science Research
Sector, Research Center, Mitsubishi Kasei Corporation, 1000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan Use of high-bioactivation strain in Drosophila somatic cell mutagenicity testing A simple procedure was developed for screening strains having a putative high-bioactivation capacity which confers high potency to procarcinogens in the wing spot test a n d / o r the DNA repair test. The procedure was applied to three insecticide-resistant strains, Oregon R(R), Haag79 and Hikone-R, and two insecticide-sensitive strains, Canton-S and w. Hybrid larvae produced by mating FM7 / mei-9 a mei-41°5; mwh / mwh females and males from each strain were fed on a procarcinogen, and the lethality of DNA repair defective, mei-9 m e i - 4 1 / Y hybrid males was compared among the strains. Five procarcinogens tested showed 3-30-fold differences in LC70 concentration among the hybrid males. The relative sensitivities to procarcinogens were Oregon R(R) = Canton-S = w > Hikone R > Haag-79 for aflatoxin B1, benzo[a]pyrene and 7,12-dimethylbenz [a]anthracene, Oregon R(R) = Canton-S = w = Haag-79 > Hikone-R for 2-acetylaminofluorene and Oregon R ( R ) = Haag-79 > Hikone-R > Canton-S = w for urethane. Of the five strains tested, Oregon R(R) was found to have a superior transforming capacity for a variety of procarcinogens to active forms being detected in both the wing spot test and the DNA repair test. The wing/repair test, an assay for concomitant performance of both the wing spot test and the DNA repair test, was performed using the Oregon R(R) flies. In this assay, 58% (11/19) of Ames-negative carcinogens listed by IARC (1987) were detected as positive. From these findings, we conclude that the wing/repair test with high-bioactivation capacity is useful to complement the Ames test for detection of genotoxic carcinogens. 26 Inoue, M. and M. Miyagoshi, Medical Research Institute, Kanazawa Medical University, Daigaku 1-1, Uchinada-machi, Kahoku-gun, Ishikawa 92002, Japan
JEMS Abstracts/Mutation Research 334 (1995) 385-427
DNA damage and its repair in germ cells of male mice treated with ethyl nitrosourea
Male mice i.p. treated with ethyl nitrosourea (ENU) and unscheduled DNA synthesis (UDS) in their germ cells were studied after testicular injection of 3H-thymidine. The UDS was induced in spermatogonia to early spermatid stages, but not in late spermatid to spermatozoa. Levels of DNA base damage and their repair in the germ cells were analyzed in 3H-ENU treated mice. About half of the base damage (N7-ethyl guanine, O6-ethyl guanine and N3-ethyl adenine) detected was repaired within 24 h after ENU treatment. Replicative DNA synthesis (RDS) in spermatogonia was enhanced by ENU treatment in a dose dependent manner. The DNA synthesis in mice treated with 250 mg/kg of ENU increased to about 4 times that of the control. However, the RDS in spermatozoa of treated mice was lower than that of the control. Number of sperm and weight of testis in ENU-treated groups were low when compared with the control. It is suggested that spermatogonia with DNA synthesis induced by ENU degenerated during differentiation into sperm. 27 Inose, H., H. Okuda and T. Watabe, Laboratory of Drug Metabolism and Toxicology, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachiojishi, Tokyo 192-03, Japan 32p-Postlabeling analysis of DNA adducted by the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene
Hepatic DNA of rats injected i.p. with the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene (5-HCR), as well as calf thymus DNA incubated with 5-HCR sulfate in vitro, was analyzed with 32p-postlabeling techniques. In the calf thymus DNA, four adduct spots were detected, and the most potent radioactive adduct spot (adduct 1) was chromatographed on HPLC after being eluted from the thin-layer chromatogram with 2-propanol-ammonium water.
397
Adduct 1 was identified as 2'-deoxy-N6-(chrysen5-yl)methyladenosine (5-HCR-dA) 3',5'-bisphosphate using the synthetic specimen, which was synthesized by the reaction of 5-aminomethylchrysene with 6-chloropurine 2'-deoxyriboside followed by phosphorylation with tetrachloropyrophosphate. Two other adduct spots were characterized as oligonucleotides containing the 5HCR-dA residue resulting from digestion of the adducts with nuclease P1 and phosphodiesterase I produced 5-HCR-dA 5'-monophosphate, which was identified with the authentic specimen. A TLC map of the 32p-postlabeled DNA adduct obtained from rat liver was very similar to that from calf thymus DNA, though an extra minor adduct was observed in rat liver DNA. The adduct level in rat liver DNA treated with 5-HCR sulfate (0.25 /zmol/g body weight) was 77/106 nucleotides, and the adenine adduct was estimated to comprise 80% of the total adducts. 28 Iwase, Y., S. Tanaka, Y. Takeda and K. Yoshikawa, Toxicology Laboratory, Life Science Sector, Research Center, Mitsubishi Kasei Co., 1000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan Effects of 10 chemicals on gap junctional intercellular communication (GJIC) in Chinese hamster V79 cells assessed with a dye transfer assay
The metabolic cooperation assay using 6-thioguanine is not an appropriate method when detection after relatively long-term exposure of ceils to chemicals is required. In this study, a dye transfer assay was applied to measure GJIC. Eight nongenotoxic carcinogens, which had shown negative responses in the metabolic cooperation assay, and two chemicals which had shown positive responses, were examined. Wild-type Chinese hamster V79 cells (2 x 105 cells for 24 h exposure and 2 x 104 cells for 72 h exposure) were cultivated for 24 h in 35 mm dishes and treated with a single test chemical for 24 and 72 h. Effects on GJIC were measured by counting dye-coupled cells after microinjection of Lucifer yellow CH solution into individual cells. Four nongenotoxic