Akrawi, May Ghanim; (1995) Long-term preservation and induction of biotransformation enzymes in co-cultured rat hepatocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

When hepatocytes are cultured under conventional conditions they undergo a rapid decline in the expression of many of the enzymes responsible for xenobiotic metabolism, most markedly members of the cytochrome P450 (CYP) superfamily. Members of the subfamily CYP2B are no longer inducible by the anti-epileptic drug phenobarbital. However, when rat hepatocytes are co-cultured with rat liver epithelial cells they survive for up to two months in culture and maintain the expression of CYP2B1/2 for up to 14 days in culture. Both CYP2B1/2 mRNA and protein amounts are elevated by treatment with phenobarbital. Using RNase protection assays with antisense RNA probes designed to distinguish between CYP2B1 and CYP2B2 mRNAs, the ratio of abundance and inducibility of the two mRNA species was found to be similar to that observed in vivo. Another anti-epileptic drug, valproic acid, was shown to be a stronger inducer of both CYP2B1 and 2 and their proteins in vitro but a poor inducer in vivo. Two other components of the mixed function mono-oxygenase, namely P450 reductase and cytochrome b5are also maintained in co-culture and their levels of expression elevated by both drugs in vivo and in vitro. In contrast to phenobarbital, valproate is found to be a strong inducer of the CYP4A family and in this case the induction is observed in vivo as well as in the cultured hepatocytes. The induction of CYP4A1, CYP4A2 and CYP4A3 mRNA species by valproate, was also observed in vivo and in vitro using an RNase protection assay and antisense probes that were designed to discriminate between them. Valproate was also found to decrease the expression of GST 3/4 subunits in vitro. The expression of HNF-4 was increased by phenobarbital, in vivo and in vitro, and by valproate, only in vitro.

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