INDUCTION OF CYP3A AND ASSOCIATED TERFENADINE N-DEALKYLATION IN RAT HEPATOCYTES COCULTURED WITH 3T3 CELLS

Long-term culture of hepatocytes has been challenged by the loss of di fferentiated functions. In particular, there is a rapid decline in cyt ochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morpho logy, and expression of CYP3A. Terfenadine was incubated with the cult ures, and its biotransformation was quantitatively analyzed by HPLC. T erfenadine is metabolized by two major pathways: C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, an d N-dealkylation to azacyclonol. In rat liver, only the N-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubat ions of terfenadine with liver microsomes. Freshly isolated rat hepato cytes were seeded on top of confluent 3T3 cells. Cultures were maintai ned in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mu mol/L or 5 mu mol/L dexamethasone. In pure hepatocyte c ultures, viability, as determined by lactate dehydrogenase (LDH) activ ity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial level s on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mu mol/L dexamethasone, and wa s associated with the increased formation of azacyclonol. On day 5, ne arly 50% of added 5 mu mol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and R NA-slot blot analyses confirmed that treatment with 5 mu mol/L dexamet hasone induced CYP3A mRNA expression and CYP3A protein expression. Thi s coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.