Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes - Analysis with a novel reverse transcriptase-polymerase chain reaction assay

The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzym es for drug metabolism, can be elevated by chemical inducers. In the presen t study, we investigated in cultured rat hepatocytes the induction of all f our currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vi vo. To this end, CYP3A mRNA levels were quantified with a never, radioactiv e reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT-PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-ph osphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucl eotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT-PCR assay, it was found that the expression of all CYP3A isoforms in rat hepato cytes, cultured on a collagen matrix, was decreased by 80-90% within one da y of cultivation. After addition of dexamethasone, at one day after isolati on, CYP3A1 mRNA levels were elevated to levels comparable to those in fresh ly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and C YP3A18 mRNA levels were not affected by dexamethasone treatment, and were h ardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addit ion of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after d examethasone administration. However, in contrast to the situation in cultu red hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser e xtent (4- and I-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interfer ence by any of the other CYP3A isoenzymes. (C) 2000 Elsevier Science Inc.