Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Abstract

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CUP), including CYPlAU2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase a (GSTa), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin 0-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. CYPl A1 /2, CYP2B1/2, CYP3All2, GSTa, 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 CYPl A1/2 (13.6-fold) and GSTu (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (1 1 .Zfold), GSTu (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 co-culture, 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 CYP2B 1 (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.

Nandrolone Decanoate Enhances the Activities of Cholanthrene Induced Glutathione-s-Transferase in Liver Tissue of Albino Mice

“…This enhancement effect of 3- methylcholanthrene on glutathione-s-transferase activity is supported by various investigators [22,23]. This finding suggests the role of glutathione-s-transferase in detoxification of polycyclic aromatic hydrocarbons.…”

Selective control of liver and kidney cells migration during organotypic cocultures inside fibronectin-coated rectangular silicone microchannels

“…For that purpose, liver cells cocultures (hepatocytes with non-parenchymal cell [17,18] and hepatocytes with biliary cells [19]) have been developed, and these models have demonstrated that cell-cell interactions preserved hepatocytes, functionality [20]. Even if various studies are done on an artificial 3D matrix [21,22] and in coculture [23,24], the reconstructed physiology does not completely mimic in vivo 3D tissue.…”

Liver cell models in in vitro toxicology.

“…A considerable step forward in long-term maintenance of differentiated hepatocytes was made by adding another liver epithelial cell type (126). When cocultured with rat liver epithelial cells probably derived from primitive biliary cells, hepatocytes from various species including humans survive for several weeks and retain various liverspecific functions; these include production of plasma proteins, expression of both phase I and phase II drug metabolizing enzymes, and taurocholate uptake (52,71,(127)(128)(129)(130)(131)(132)(133)(134)(135) (71,126,128) and with preservation of communication through gap junctions (136). These two features could be related because in pure hepatocyte cultures the addition of proteoglycans improves gap junction activity (137).…”