Induction of cytochrome P450IA1 and its recombinant construct in H4IIE rat hepatoma cells

Abstract

1. 1. Our previous studies have shown that benzo(a)pyrene (BP), 3-methylcholanthrene (3MC) and tetrachlorodibenzofuran (TCDBF) can induce the expression of the cytochrome P450IA1 mRNA in the rat hepatoma cell line, H4IIE, although the kinetics of induction differed. 2. 2. In the present study, by using biochemical, immunochemical and recombinant DNA approaches, the effects of these inducers have been examined on the steady state level of endogenous cytochrome P450IA1 protein and on induction of chloramphenicol acetyltransferase activity (CAT) in the H4IIE cells transfected with pMC 1CAT (a recombinant construct consisting of CAT linked to 5' upstream DNA sequence of the rat cytochrome P450IA1 gene). 3. 3. From 7-ethoxyresorufin O-deethylase activity (EROD) and immunochemical analysis of cytochrome P450IA1, the optimal concentrations of BP, 3MC and TCDBF for induction in the H4IIE cells were determined as 1, 0.1-1 and 0.1 μ M, respectively. 4. 4. The elevated expression of the protein was more sustained in the TCDBF-exposed cells than in the BP or 3MC-treated cells. 5. 5. After 1.5 hr of treatment, little if any detectable P450IA1 protein was observed in the H4IIE cells although a considerable amount of mRNA was present. 6. 6. In addition, no cytochrome P450IA2 protein was detected in the control or induced H4IIE cells. 7. 7. H4IIE cells were transfected by pMC 1CAT, and the induction ratio of CAT expression in the transfected H4IIE cells after BP, 3MC or TCDBF treatment was 10-, 17- and 40-fold, respectively. 8. 8. These results indicate that the rat H4IIE cell line offers a valid homologous system for studies of the regulation of the rat cytochrome P450IA1 gene.