Geno- and cytotoxicity of nitrosamines, aflatoxin B1, and benzo[a]-pyrene in continuous cultures of rat hepatoma cells

Two closely related hepatoma cell lines were examined for theirresponse to carcinogens requiring metabolic activation: H₅,a dedifferentiated line expressing cytochrome P-448-dependentmonooxygenase(s); and HF_1–4, a differentiated line whichalso expresses cytochrome P-450-dependent monooxygenase(s).The hepatocarcinogens dimethyl- and diethylnitrosamine and aflatoxinB₁, preferred substrates for cytochrome P-450-dependent monooxygenase(s),and the non-hepatocarcinogen benzoa]pyrene, which is preferentiallymetabolized by cytochrome P-448-dependent monooxygenase forms,were used as test agents. Their effects were compared to thoseof the directly alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and N-ethyl-N-nitrosourea (ENU). The cytotoxicity wasevaluated by plating efficiency, the genotoxicity by the appearanceof alkaline labile DNA sites. The nitrosamines had a cytotoxicand genotoxic effect on the differentiated HF_1–4 cells,but had no effect on H₅ cells. Aflatoxin B₁ affected both celllines, but was 10-times more potent in the HF_1–4 thanin the H₅ cells. In contrast to the nitrosamines and the mycotoxin,benzoa]-pyrene exerted a stronger effect on the dedifferentiatedcell line. Pretreatment of cultures with dexamethasone increasedboth the cytotoxicity and genotoxicity of the hepatotoxic agents.MNNG and ENU induced a similar degree of DNA-damage after short-term(2 h) exposure in the two cell lines. When cells were allowedto recover for 16 h HF_1–4 cells, but not H₅ cells, regainedtheir full growth potential suggesting a marked capacity forthe repair of MNNG- and ENU-induced lesions in the HF_1–4cells. The results indicate that continuous lines of mammaliancells may retain a considerable degree of organ-specific responseto chemical carcinogens. Hepatoma cells of the type describedabove may be useful for screening the wide spectrum of chemicalswhich are potentially genotoxic in liver and in extrahepatictissues and for analyzing their metabolic activation and mechanismof action.