REGULATION OF THE MAJOR DETOXICATION FUNCTIONS BY PHENOBARBITAL AND 3-METHYLCHOLANTHRENE IN COCULTURES OF RAT HEPATOCYTES AND LIVER EPITHELIAL-CELLS

In the present study, we analysed the expression of monooxygenase acti vities and mRNAs associated with cytochrome P-450 (CYP), including CYP 1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alp ha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cul tures of primary rat hepatocytes and rat liver epithelial cells. We ob served that pentoxyresorufin O-deethylation activity was well maintain ed and ethoxyresorufin O-deethylation activity gradually decreased dur ing co-culture time. In addition, we showed that phenobarbital and 3-m ethylcholanthrene treatments resulted in a significant increase of the se 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. I n addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated c o-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3 A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treate d co-cultures. Furthermore, we demonstrated that liver-specific gene e xpression 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 hepa tocytes and rat liver epithelial cells. Finally, to investigate whethe r co-cultures could be used to study the molecular mechanisms regulati ng CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CY P2B2 (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 bas al 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 metaboli sm, and shows promise as a new tool for analysing transcriptional regu lation under the influence of xenobiotics within primary hepatocytes.