Detection of clastogens in vitro using rat hepatocyte primary culture

412

JEMS Abstracts/'Mutation Research 334 (1995) 385-427

School of Natural Science and Technology and c Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan Characteristics of menadione-induced mutations in the lacI gene of Escherichia coli

Using the lacI + to l a c I - or lacO c forward mutation system in the wild-type and m u t M strains of Escherichia coli, we investigated the characteristics of menadione-induced mutations. With treatment of the wild-type strain with 0.4 mM menadione at 37°C for 45 min, the number of l a d - or lacO c mutant colonies per plate increased from 477 (per 2.7 × 108 surviving cells) for a control without menadione to 622 (per 4.7 × 107 surviving cells; surviving fraction, 18%) for those treated with menadione. This corresponded to a 7-fold increase of the mutation frequency. The mutation frequency increase was 6-fold with strain m u t M . Thus, the menadione-induced mutation frequencies were similar in the wild-type and the m u t M strains. The m u t M strain is deficient in the MutM protein which is capable of removing 8-ohG lesions in DNA produced by reactive oxygen species. It appears, therefore, that the MutM protein does not play a role in the menadione-mediated mutagenesis. These results show that it is unlikely that 8-ohG formation in DNA is involved in these menadione-induced mutations. 64 Ono, Y. a, I. Somiya a, T. Kawaguchi b and Y. Oda c, a Department of Environmental & Sanitary Engineering, Kyoto University, YoshidaHonmachi, Sakyo-ku, Kyoto 606-01, b Kurita Industry Co. and c Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan Detection of genotoxicity on aromatic amines and nitroarenes in municipal sewage

Genotoxicity was evaluated in the performance of nightsoil treatment plants, because the human feces and urine were expected to produce some genotoxic effects. Strong genotoxic activity was

detected in the influents of nightsoil treatment plants and also effluents from the plants after biological treatment using a bacterial assay, the u m u test. A newly developed assay using a Salmonella typhimurium NM series, which has high sensitivity due to high copy numbers of nitroreductase and O-acetyltransferase, was applied to the samples to detect activities induced by nitroarenes and aromatic amines that might come from cooked foods and metabolites consumed by humans. Genotoxic potencies of the samples were revealed with dividing by a value detected in a positive control. Genotoxicity induced by aromatic amines and nitroarenes could not be removed by biological process or ultrafiltration, but was completely removed by a coagulation-sedimentation process. Advanced treatment such as ozonation was proposed to obtain safe effluent conditions. 65 Ota, M., M. Hara and I. Nakatsuka, Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohanaku, Osaka 554, Japan Detection of clastogens in vitro using rat hepatocyte primary culture

Liver plays an important role in metabolizing exogenous chemicals and is a target organ for many mutagens/carcinogens. We have developed an easy method to detect the clastogenicity of chemicals in liver using rat hepatocyte primary culture, which has drug metabolizing activity. Hepatocytes were isolated from Sprague-Dawley male rats by two-step collagenase perfusion, and cultured in William's medium E supplemented with 5% fetal calf serum and insulin (10 -7 M). Epidermal growth factor (EGF, 20 ng/ml) was used as a mitogen for growth stimulation. Hepatocytes showed a maximal mitotic index at 43 h after growth stimulation. The clastogenic activity of 2-acetylaminofluorene (2-AAF), cyclophosphamide (CP) and mitomycin C (MMC) was evaluated in this test. We conducted two treatment procedures to find the optimum test condition: (1) ceils were treated

JEMS Abstracts / Mutation Research 334 (1995) 385-427

with chemicals under growth stimulation conditions (treatment A), or (2) cells were stimulated to grow after chemical treatments (treatment B). All three chemicals induced dose-dependent increases in chromosomal aberration in rat hepatocytes. There was not much difference in the induction of chromosomal aberration between treatments A and B, but treatment B showed lower mitotic indices than treatment A, indicating that treatment A is more suitable for this test. In conclusion, this in vitro chromosomal aberration test using rat hepatocytes with EGF growth stimulation is useful and convenient for detecting clastogens in liver. 66 Otsuka, C., K.F. Miura, T. Saito and M. Ishidate, Jr., Chromosome Research Center, Olympus Optical Co., Ltd., 2-3 Kuboyama-cho, Hachioji, Tokyo 192, Japan Possible metabolic pathway of PhlP in the induction of chromosomal aberrations in cultured mammalian cells

We have reported that PhIP and its metabolite, N-OH PhIP, induced chromosomal aberrations in C H L / I U cells and in human fibroblast cell line TIG7 cells, with and without rat $9 for metabolic activation, respectively. In this study, a variant subline, YG10003, which was newly established from C H L / I U by introducing an O-acetyltransferase gene of S. typhimurium was used. The YG10003 cells were several times more sensitive than C H L / I U , when the cells were treated with PhIP (10/zg/ml) for 6 h in the presence of rat microsome fraction (Ms) and sampled at 24 h. Pentachlorophenol (20-40/zg/ml), an arylsulfotransferase inhibitor, showed no effect on the induction of aberrations by PhIP either in C H L / I U or in YG10003 cells, while it apparently inhibited the induction of aberrations by another carcinogen, 7.12-dimethylbenz[a]anthracene in the presence of Ms. These results indicate that the effect of PhIP on the induction of chromosomal aberrations in mammalian cells is mainly through the metabolic

413

activation with acetyltransferase occurring within the cells. 67 Provost, G.S., M.J. Dycaico, P.L. Kretz, B.J. Rogers, D. Wyborsky and J.M. Short, Stratagene, 11011 M. Torrey Pines Rd., La Jolla, CA 92037, USA LacI mutation assays using the Big Blue ® mouse and rat

Mutation frequency data and parameters for statistical evaluation will be presented for analysis of the standardized Big Blue ® (BB) color mutation assay. These mutation data will include comparisons of spontaneous germ and somatic mutation in BB transgenic mice and rats. Also analysis of existing positive selection assays and continuing advancement of new methodologies will be discussed in terms of assay standardization, reproducibility, and the effects of 'directed mutation' in systems under selective pressure. 68 Sasaki, Y.F. a, M. Sakaguchi a and H. Yamada b, a Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Hachinohe, Aomori 039-11 and b Faculty of Pharmaceutical Science, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama, Toyama 930-01, Japan Antagonizing effect of triphenyltin chloride on cytosine-l-15-D-arabinofuranoside potentiation of chromosome aberrations induced by mitomycin C

We previously reported that the organotin triphenyltin chloride (TPTC), which has been widely used as an anti-fouling coating for fishing nets and ship bottoms, potentiated clastogen-induced chromosome aberrations during the G2 phase of the cell cycle. In this study, CHO ceils treated with mitomycin C (MMC) were posttreated with TPTC in the presence and absence of other agents having a similar G2 effect cytosine-1-/3-D-arabinofuranoside (araC), hydroxyurea, or caffeine - during the G2 phase of