336 novo gap-filling repair may not take place in the cells. Further, NHSF cells were pre-labeled with B U d R for 2 h, irradiated with 0 or 20 J m -2, and then incubated in [3H]BUdR for 1 to 2 h. The neutral CsC1 profile of the extracted DNA s h o w ed that the DNA replicating in irradiated cells was composed of a large portion of intermediate-density DNA (1.730 g m1-1 for hybrid 1.752 g m1-1 and light band 1.700 g ml-'), hybrid (usually seen in unirradiated cells) and a small amount of heavy-heavy DNA (1.805 g ml-1). The intermediate and heavyheavy DNAs were chased into a single hybrid peak after an additional 2-h chase in BUdR. Thus, replication of NHSF cells seems blocked at a UV lesion without forming a gap, and perhaps a newly synthesized strand may be utilized as a template to replicate a damaged site in the parental DNA, thereby restoring the proper genetic information. A Grant-in-Aid from the Ministry of Education is acknowledged. A b b r e v i a t i o n s : TdR, thymidine; BUdR, 5-bromodeoxyuridine.
3 S. Hitotsumachi and Y. Kikuchi, Drug Safety Research Centre, Takeda Chemical Industries Ltd., Osaka {Japan) Mitomycin-C-induced dominant lethality in mice The dose effect of mitomycin C (MC) in the induction of dominant lethality was studied in CF1 mice. Adult males were given a single i.p. injection of 1, 2 or 3 mg of MC per kg. The treated male was mated with untreated virgin females during 7 weeks after treatment. On the 13th day of pregnancy the female was killed and examined for corpora lutea as well as living and dead implants. The treatment at 3 mg/kg induced marked pre- and post-implantation dominant lethality J=l
number of living embryos per female in experiment number of living embryos per female in control
The sensitive stage was from spermatogonia to early spermatids. At 2 mg/kg, only spermatocytes were sensitive in regard to increase of J value. No increase of J value was induced at any stage of spermatogenesis with MC at 1 mg/kg. Increase of dominant lethality in the present study was always associated with high pre-implantation egg loss, which could be due to decrease of sperm in the ejaculate [Kratochvilova, Mutation Res., 21 (1973) 192]. To examine this possibility, post-implantation dominant lethality J ' = 1 -- number of living embryos/all implants in experiment number of living embryos/all implants in control was also calculated. According to this J ' index, 3 mg of MC per kg seemed to have induced the mutation only in late spermatocytes and early spermatids. However, the J ' index was increased, though slightly, even with MC at 1 mg/kg at the late spermatocyte stage.
337 4 M. Horikawa, F. Suzuki and S. Ban *, Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920, and * Department of Experimental Radiology, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan) Mammalian cell mutagenesis: comparison of the sensitivity in assay systems of mutations induced by radiations and chemicals Establishment of sensitive assay systems for mutations of mammalian cells in vitro is urgent for detection of various potential mutagens present in the human environment as well as for elucidation of the mechanisms of somatic mutation and phenotypic expression in cultured mammalian cells. We have isolated several kinds of mutant [azg-resistant cells, azg-sensitive cells, prototrophic cells (Ala÷, Asn ÷, Pro ÷, Asp ÷, Hyp ÷, Glu ÷) and auxotrophic cells (TdR-)] from a single cultured Chinese hamster hai cell line and compared the induced forward- and reverse-mutation frequencies by radiations (X-rays and UV) and typical chemical mutagens (ethyl methanesulfonate, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine) using these mutants. The forward mutation system using prototrophic cells was most sensitive for assay of mutations induced by radiations and chemicals. The reverse mutation system using azg-resistant cells and auxotrophic cells was also useful. The forward system using azg-sensitive cells was not sensitive as an assay system, when compared with other systems reported by many workers to date. These results point out the complexity of studies on mutagenesis with cultured mammalian cells, especially since the induced mutation frequency in cultured cells is affected by mutation expression time, size of cell inoculum, concentration of selective agent, etc. Additionally, we found that AF-2 (or furylfuramide), a nitrofuran antimicrobial food additive, had a powerful mutagenic activity in our forward mutation system using prototrophic cells.
5 M. Inouye and U. Murakami, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai (Japan) Teratogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine in mice The teratogenic effect of a potent mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), on the mouse fetus was studied. Eighteen groups of pregnant JCL-CF1 mice were injected intraperitoneally with a single dose of MNNG at 40, 60 or 80 mg/kg on day 7, 8, 9, 10, 11 or 12 of pregnancy. Fetuses were examined on day 18. Many malformed fetuses were found. The malformations were exencephalia (groups treated on day 7 or 8), hydrocephalus (day 8, 9 or 10), hydromicrocephalus (days 9, 10 or 11), microcephalia (day 11 or 12), cleft palate (day 9, 10 or 11), phocomelia (day 9 or 10), dysmelia (day 11 or 12), polydactylia (day 9 or 10) and ectrodactylia (day 9, 10 or 11). The incidence of such mal-
3 S. Hitotsumachi and Y. Kikuchi, Drug Safety Research Centre, Takeda Chemical Industries Ltd., Osaka {Japan) Mitomycin-C-induced dominant lethality in mice The dose effect of mitomycin C (MC) in the induction of dominant lethality was studied in CF1 mice. Adult males were given a single i.p. injection of 1, 2 or 3 mg of MC per kg. The treated male was mated with untreated virgin females during 7 weeks after treatment. On the 13th day of pregnancy the female was killed and examined for corpora lutea as well as living and dead implants. The treatment at 3 mg/kg induced marked pre- and post-implantation dominant lethality J=l
number of living embryos per female in experiment number of living embryos per female in control
The sensitive stage was from spermatogonia to early spermatids. At 2 mg/kg, only spermatocytes were sensitive in regard to increase of J value. No increase of J value was induced at any stage of spermatogenesis with MC at 1 mg/kg. Increase of dominant lethality in the present study was always associated with high pre-implantation egg loss, which could be due to decrease of sperm in the ejaculate [Kratochvilova, Mutation Res., 21 (1973) 192]. To examine this possibility, post-implantation dominant lethality J ' = 1 -- number of living embryos/all implants in experiment number of living embryos/all implants in control was also calculated. According to this J ' index, 3 mg of MC per kg seemed to have induced the mutation only in late spermatocytes and early spermatids. However, the J ' index was increased, though slightly, even with MC at 1 mg/kg at the late spermatocyte stage.
337 4 M. Horikawa, F. Suzuki and S. Ban *, Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920, and * Department of Experimental Radiology, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan) Mammalian cell mutagenesis: comparison of the sensitivity in assay systems of mutations induced by radiations and chemicals Establishment of sensitive assay systems for mutations of mammalian cells in vitro is urgent for detection of various potential mutagens present in the human environment as well as for elucidation of the mechanisms of somatic mutation and phenotypic expression in cultured mammalian cells. We have isolated several kinds of mutant [azg-resistant cells, azg-sensitive cells, prototrophic cells (Ala÷, Asn ÷, Pro ÷, Asp ÷, Hyp ÷, Glu ÷) and auxotrophic cells (TdR-)] from a single cultured Chinese hamster hai cell line and compared the induced forward- and reverse-mutation frequencies by radiations (X-rays and UV) and typical chemical mutagens (ethyl methanesulfonate, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine) using these mutants. The forward mutation system using prototrophic cells was most sensitive for assay of mutations induced by radiations and chemicals. The reverse mutation system using azg-resistant cells and auxotrophic cells was also useful. The forward system using azg-sensitive cells was not sensitive as an assay system, when compared with other systems reported by many workers to date. These results point out the complexity of studies on mutagenesis with cultured mammalian cells, especially since the induced mutation frequency in cultured cells is affected by mutation expression time, size of cell inoculum, concentration of selective agent, etc. Additionally, we found that AF-2 (or furylfuramide), a nitrofuran antimicrobial food additive, had a powerful mutagenic activity in our forward mutation system using prototrophic cells.
5 M. Inouye and U. Murakami, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai (Japan) Teratogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine in mice The teratogenic effect of a potent mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), on the mouse fetus was studied. Eighteen groups of pregnant JCL-CF1 mice were injected intraperitoneally with a single dose of MNNG at 40, 60 or 80 mg/kg on day 7, 8, 9, 10, 11 or 12 of pregnancy. Fetuses were examined on day 18. Many malformed fetuses were found. The malformations were exencephalia (groups treated on day 7 or 8), hydrocephalus (day 8, 9 or 10), hydromicrocephalus (days 9, 10 or 11), microcephalia (day 11 or 12), cleft palate (day 9, 10 or 11), phocomelia (day 9 or 10), dysmelia (day 11 or 12), polydactylia (day 9 or 10) and ectrodactylia (day 9, 10 or 11). The incidence of such mal-
