Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1. which were expressed in both cell types during the coculture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.