Mutagenicity, clastogenicity and cytotoxicity of procarcinogens in a rat hepatoma cell line competent for xenobiotic metabolism

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Abstract
The present studies were aimed at evaluating the suitability of the differentiated Reuber hepatoma cells H4DEC3/G− for monitoring permanent damage to the DNA caused by hepato-trophic chemicals. First we determined the profile of xenobiotic metabolizing enzymes. The cells expressed various cytochrome P-450-dependent monooxygenases, UDP-glucu-ronosyl-, phenol sulpho- and glutathione S-transferase, cytochrome c (P-450) reductase and carboxylesterases. We then established the conditions for genotoxicity testing in H4IIEC/G− cells. Induction of resistance against 6-thio-guanine and appearance of micronuclei served as indicators for mutagenicity and clastogenicity, respectively. 6-Thio-guanine-resistant H4HEC3/G− cells were phenotypically stable for at least 30 cell cycles; recovery of 6-thioguanine-resistant cells was not significantly affected by the number of cells seeded for mutant selection up to at least 106 cells/100-mm dish; expression time of chemically induced mutants was 12–15 days; a period of 24 h after treatment appeared to be sufficient to allow for the formation of micro-nuclei. Finally we tested the genotoxic effects of promutagens which are typically activated or inactivated in liver. Aflatoxin B1, N-nitrosodiethylamine and cyclophosphamide were genotoxic to H4IIEC3/G− cells at concentrations of 10–30 nM, 2–20 mM and 1 mM, respectively. N-Nitroso-dimethylamine and benzo[a]pyrene were not or only weakly cytotoxic and genotoxic to the cells, but this appears most likely to be due to protective mechanisms rather than to lack of metabolic activation. The results indicate that differentiated hepatoma cells such as H4HEC3/G− offer a means of studying the potential of chemicals for inducing permanent DNA damage in liver cells.