title: Glucocorticoid receptor-independent transcriptional induction of cytochrome P450 3A1 by metyrapone and its potentiation by glucocorticoid.
abstract: Metyrapone administration to 21- and 90-day-old male rats causes a transcriptional induction of the hepatic glucocorticoid-inducible CYP3A1 gene within an hour as determined by nuclear run-on experiments. Analyses performed 24 hr after metyrapone administration in both ages of rat demonstrate that the transcriptional induction of CYP3A1 gene expression is followed by significant increases in CYP3A1 mRNA, CYP3A-immunoreactive microsomal protein and total microsomal cytochrome P450 (CYP). In 21-day-old rats, there is a significant increase in microsomal CYP3A dependent steroid 6 beta-hydroxylase activity but not in 90-day-old rats, possibly because of a slower clearance of this drug, which inhibits CYP activities. In hepatocytes cultured in serum- and glucocorticoid hormone-free medium, metyrapone alone induces CYP3A1 mRNA expression, which demonstrates that metyrapone transcriptionally induces hepatic CYP3A1 by a direct interaction with the liver. Metyrapone does not compete with the binding of the synthetic glucocorticoid and potent transcriptional CYP3A1 inducer dexamethasone to the glucocorticoid receptor (GR) in soluble fractions from liver. This suggests that metyrapone is not a ligand for the GR and induces CYP3A1 by a mechanism independent of the GR. Addition of glucocorticoid to cultured hepatocytes at levels that induce GR-dependent genes potentiate CYP3A1 mRNA induction by metyrapone without inducing CYP3A1 mRNA alone. A GR-dependent mechanism may therefore mediate the potentiation of CYP3A1 transcriptional induction by metyrapone. The CYP3A1 transcriptional inducer and glucocorticoid antagonist pregnenolone 16 alpha-carbonitrile at 100 microM blocks dexamethasone binding to the GR in 21-day-old rat liver soluble fractions but is less effective in 90-day-old rat liver soluble fractions in contrast with 10 microM glucocorticoid antagonist RU486, which is equally effective at blocking dexamethasone binding to the GR. The inability of pregnenolone 16 alpha-carbonitrile to fully compete with dexamethasone for cytosolic binding in adult animals suggests that there may exist variant receptors with different affinities for dexamethasone and pregnenolone 16 alpha-carbonitrile and may explain the mechanism by which low concentrations of dexamethasone potentiate the transcriptional induction of CYP3A1 mediated by high concentrations of pregnenolone 16 alpha-carbonitrile [J. Biol, Chem. 270:28917-28923 (1995)]. Examination of membrane-bound dexamethasone binding activity, with which other steroidal and nonsteroidal CYP3A inducers have been shown to compete, indicates that binding activity is detectable in 90- but not 21-day-old rat liver microsomes. The absence of membrane-bound glucocorticoid binding site activity and the presence of a functional CYP3A1 transcriptional response in 21-day-old rats suggest that membrane-bound glucocorticoid binding site activity is not involved in the transcriptional activation of CYP3A1 expression. These data suggest that both glucocorticoids and nonsteroidal compounds may trigger the transcriptional induction of CYP3A1 by a GR-independent mechanism that may be potentiated by a GR-dependent mechanism.
authors.affiliation: DH Department of Toxicology, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK. m.c.wright@mds.qmw.ac.uk
authors.forename: M C
authors.initials: MC
authors.lastname: Wright
authors.forename: X J
authors.initials: XJ
authors.lastname: Wang
authors.forename: M
authors.initials: M
authors.lastname: Pimenta
authors.forename: V
authors.initials: V
authors.lastname: Ribeiro
authors.forename: A J
authors.initials: AJ
authors.lastname: Paine
authors.forename: M C
authors.initials: MC
authors.lastname: Lechner
mesh_terms: D000375:Aging / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology*; D004357:Drug Synergism; D004790:Enzyme Induction; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008797:Metyrapone / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011965:Receptors, Glucocorticoid / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
chemical_list: D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D008797:Metyrapone