Biochemical properties of carcinogen-metabolizing enzymes in cultured hepatoma cells

We have previously demonstrated the inducibility of both cytochrome P-448- and P-450-dependent monooxygenases in the differentiated rat hepatoma cell line MH1C1. Further experiments with these cells on the expression of different forms of cytochrome P-450, inducible not only by phenobarbital (PB) and 3-methylcholanthrene (MC), but also by metyrapone (MP), ethanol (E), and beta-naphthoflavone (BNF) are reported here. The effects of the in vitro addition of the inhibitors alpha-naphthoflavone and beta-naphthoflavone on the aryl hydroxylase activity (AHH) and the influence of protein synthesis on the induction of cytochrome P-450 were also assessed. Cultures were exposed to the inducers PB, MC, BNF, and MP during the last 6 days of culture and to E for 10 days. The inhibition of protein synthesis was obtained by adding cycloheximide (CY) to the cultured cells during the last 24 hr. The exposure of MH1C1 cells to various concentrations of MP resulted in a dose-dependent increase in AHH activity. The treatment of MH1C1 cells with different concentrations of ethanol produced a significant dose-dependent increase of monooxygenases. AHH activity, induced by the various treatments, was inhibited in a dose-dependent way by alpha-naphthoflavone and beta-naphthoflavone. Cy reduced the concentration of cytochrome P-450 and the AHH activity induced by the various treatments, thus indicating an implication of the protein synthesis in the mechanism(s) of induction.