Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes

The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepat ocytes. Hepatocytes were suspended and cultured in diluted Matrigel(R) in a basal serum-free Dulbecco's modified Eagle medium and exposed to the proto typical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydroco rtisone, and clofibrate for 48 h. Total RNA and microsomes were isolated an d prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1 , CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotr ansferase was determined at the mRNA level with reverse transcriptase polym erase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1 CYP2C11, CY P2E1, and CYP4A1 was also measured at the apoprotein level by Western immun oblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can he maintained in c ulture for up to 7 d at the mRNA and apoprotein levels. In addition, hepato cytes were found to respond to chemical enzyme inducers with marked increas es in enzyme expression at either the mRNA or protein level and in a concen tration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigatio n indicate that the presence of diluted Matrigel(R) (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 mu M), hydrocortis one (0.1 mu M), and serum-free culture medium can maintain the differentiat ed phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzy me induction in adult male rat hepatocytes shows close agreement with enzym e induction observed in the livers of rats exposed to these or similar prot otypical enzyme inducers. Rat hepatocytes cultured in the presence of dilut ed Matrigel(R) coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to a ssess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enymes.