In Vitro Induction of Rat Hepatic Cytochrome P450 Utilizing a High-Throughput In Situ mRNA Hybridization Assay
Rinehart, Jesse J.
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Cytochrome P450 enzymes are a superfamily of hemoproteins highly expressed in the liver. Through hydroxylation, hepatic P450 enzymes detoxify pharmacological agents and a multitude of endogenous substances such as steroids. Investigating the increase of hepatic cytochrome P450 concentrations, or induction, by drugs is important in metabolism research. In this study, a novel in situ hybridization method was used to measure P450 induction with 33p labeled antisense mRNA probes hybridized in situ. The novel in situ hybridization assay successfully measured changes in specific cytochrome P450, CYPIA2 and CYP3A, mRNA levels in primary cultures of rat hepatocytes exposed to the prototypic P450 inducers pregnenolone-6α-carbonitrile, dexamethasone, and 3-methylcholanthrene. Parallel experiments with hepatocytes cultured on 100 mm plates were performed under identical conditions. The experimental conditions, cell culture and drug treatment, used in the study were based on previous studies. Northern blots for specific mRNAs, Western blots for proteins, and enzyme activity correlate with the in situ hybridization data. The combined experimental evidence suggests that the use of a high-throughput mRNA assay for analysis of P450 expression in vitro might be of utility in research. With further development, this method could be used to perform studies measuring the induction of multiple P450 enzymes by a number of compounds. This assay could be most applicable to drug discovery and research since P450 induction is important to the understanding of drug metabolism.With honors.