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 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 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.
Lerche
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INSERM U456, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes I, France.
Fautrel
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Shaw
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PM
Glaise
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Ballet
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F
Guillouzo
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Corcos
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D018920:Coculture Techniques; D019363:Cytochrome P-450 CYP1A1 / Q000187:drug effects / Q000235:genetics; D019362:Cytochrome P-450 CYP2B1 / Q000187:drug effects / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D008658:Inactivation, Metabolic; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology*; D010634:Phenobarbital / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000187:drug effects / Q000235:genetics
D012333:RNA, Messenger; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; C025619:steroid 16-beta-hydroxylase; D010634:Phenobarbital