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

Abstract

Two closely related hepatoma cell lines were examined for their response to carcinogens requiring metabolic activation: H₅, a dedifferentiated line expressing cytochrome P-448-dependent monooxygenase(s); and HF_1–4, a differentiated line which also expresses cytochrome P-450-dependent monooxygenase(s). The hepatocarcinogens dimethyl- and diethylnitrosamine and aflatoxin B₁, preferred substrates for cytochrome P-450-dependent monooxygenase(s), and the non-hepatocarcinogen benzo[a]pyrene, which is preferentially metabolized by cytochrome P-448-dependent monooxygenase forms, were used as test agents. Their effects were compared to those of the directly alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N-nitrosourea (ENU). The cytotoxicity was evaluated by plating efficiency, the genotoxicity by the appearance of alkaline labile DNA sites. The nitrosamines had a cytotoxic and genotoxic effect on the differentiated HF_1–4 cells, but had no effect on H₅ cells. Aflatoxin B₁ affected both cell lines, but was ∼10-times more potent in the HF_1–4 than in the H₅ cells. In contrast to the nitrosamines and the mycotoxin, benzo[a]-pyrene exerted a stronger effect on the dedifferentiated cell line. Pretreatment of cultures with dexamethasone increased both 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 allowed to recover for 16 h HF_1–4 cells, but not H₅ cells, regained their full growth potential suggesting a marked capacity for the repair of MNNG- and ENU-induced lesions in the HF_1–4 cells. The results indicate that continuous lines of mammalian cells may retain a considerable degree of organ-specific response to chemical carcinogens. Hepatoma cells of the type described above may be useful for screening the wide spectrum of chemicals which are potentially genotoxic in liver and in extrahepatic tissues and for analyzing their metabolic activation and mechanism of action.