ABSTRACT:The effects of treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors lovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin on the contents of cytochrome P450 mRNAs were examined in primary cultures of human hepatocytes prepared from three different livers. Treatment of 2-to 3-day-old human hepatocyte cultures with 3 ؋ 10 ؊5 M lovastatin, simvastatin, fluvastatin, or atorvastatin for 24 h increased the amounts of CYP2B6 and CYP3A mRNA by an average of 3.8-to 9.2-fold and 24-to 36-fold, respectively. In contrast, pravastatin treatment had no effect on the mRNA level of either CYP2B6 or CYP3A, although treatment with pravastatin did produce the expected compensatory increase in HMG-CoA reductase mRNA content, indicating effective inhibition of cholesterol biosynthesis. Although treatment with the active (؉), but not the inactive (؊), enantiomer of atorvastatin increased the amount of HMG-CoA reductase mRNA, treatment with each enantiomer significantly induced both CYP2B6 and CYP3A mRNA levels. Treatment of primary cultured rat hepatocytes with the atorvastatin enantiomers effectively increased the amount of CYP3A mRNA, but had no effect on CYP2B or CYP4A mRNA levels, in contrast to fluvastatin, which increased both. Findings for P450 proteins by Western blotting were consistent with the mRNA results. These findings indicate that the ability of a drug to inhibit HMG-CoA reductase activity does not predict its ability to produce P450 induction in primary cultured human hepatocytes, and demonstrate that some, but not all, of the effects of these drugs that occur in primary cultured rat hepatocytes are conserved in human hepatocyte cultures.Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase 2 ), also known as "statins", have achieved an important place in the arsenal of therapeutic agents available for the treatment of hypercholesterolemia, a major risk factor for the development of coronary artery disease, the leading cause of death in the United States. Five drugs of this class are currently approved for use in the United States, namely, lovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin (Food and Drug Adminstration Orange Book; http://www.fda.gov/cder/ob/default.htm). In general, these drugs have proven to be safe and effective when taken over a period of years (Davidson, 2001), and additional therapeutic uses of these drugs are under investigation (White, 1999;Puddu et al., 2001;Whitfield, 2001).However, one precaution in the use of these drugs is that most of them have been shown to interact, in one manner or another, with the cytochrome P450 system of drug-metabolizing enzymes, thereby leading to the possibility for pharmacokinetic interactions with coadministered drugs. Thus, abundant information indicates that lovastatin, simvastatin, and atorvastatin interact with human CYP3A, both as substrates and inhibitors (Prueksaritanont et al., 1997;Beaird, 2000;Cohen et al., 2000;Farmer and Torre-Amione, 20...
This article is available online at http://dmd.aspetjournals.org A common theme in cell physiology is that endogenous metabolites regulate their own levels, through both feed-back and feed-forward mechanisms. Many of these molecules exert their effects by activating transcription factors of the nuclear receptor superfamily. A prominent class of such endogenous metabolites is the "oxysterols
Because our previous studies indicated that squalestatin 1 treatment induces CYP2B expression in primary cultures of rat hepatocytes as a direct consequence of squalene synthase inhibition, we investigated possible underlying mechanisms. Cotransfection of cultured Sprague-Dawley male rat hepatocytes with each of the three sterol regulatory element binding protein (SREBP) transcription factors failed to induce luciferase expression from a squalestatin 1-responsive CYP2B1 reporter plasmid. Squalestatin 1 treatment of primary hepatocyte cultures from male Wistar-Kyoto rats produced a greater induction of CYP2B mRNA than occurred in cultures from female rats, consistent with the previously demonstrated response dimorphism that has been attributed to differences in constitutive androstane receptor (CAR) levels. Cotransfection of female Wistar-Kyoto rat hepatocyte cultures with plasmid expressing either mouse or rat CAR restored squalestatin 1-inducible CYP2B1-reporter expression. Cotransfection of Sprague-Dawley rat hepatocyte cultures with plasmid expressing rat CAR lacking the C-terminal AF-2 subdomain inhibited squalestatin 1-inducible CYP2B1-reporter expression. Squalestatin 1-mediated CYP2B mRNA induction in rat hepatocyte cultures was completely abolished by pretreatment with the 3-hydroxymethyl-3-glutaryl CoA reductase inhibitor pravastatin and was rescued by mevalonate supplementation, whereas phenobarbitalmediated induction was unaffected by these treatments. Finally, direct addition of trans,trans-farnesol to the culture medium caused the rapid induction of CYP2B mRNA. These results indicate that squalestatin 1 treatment induces CYP2B expression, not by inhibiting sterol synthesis and activating SREBPs, but by evoking the accumulation of an endogenous isoprenoid and activating CAR.
The effects of inhibitors of 2,3-oxidosqualene:lanosterol cyclase (cyclase) on cytochrome P450 expression were investigated in primary cultures of rat hepatocytes. Treatment of hepatocyte cultures for 24 h with either of the inhibitors [4Ј-(6-allyl-methyl-amino-hexyloxy)-2Ј-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071) or trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79) selectively increased CYP3A mRNA and immunoreactive protein contents, with maximal accumulations occurring at 3 ϫ 10 Ϫ5 M Ro 48-8071 and 10 Ϫ4 M BIBX 79. The abilities of Ro 48-8071, BIBX 79, and 3-(2-diethylaminoethoxy)androst-5-en-17-one⅐HCl (U18666A) to induce murine CYP3A were abolished in hepatocyte cultures prepared from pregnane X receptor (PXR)-null mice, and cotransfection of primary cultured rat hepatocytes with a dominant-negative PXR prevented cyclase inhibitorinducible luciferase expression from a PXR-responsive reporter plasmid. Cyclase inhibitor-mediated CYP3A mRNA induction was eliminated when primary cultured rat hepatocytes were cotreated with any of the following agents that inhibit steps upstream of cyclase in the cholesterol biosynthetic pathway: squalestatin 1 (squalene synthase inhibitor), (E)Nethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3Ј-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598, squalene monooxygenase inhibitor), or pravastatin (HMG-CoA reductase inhibitor). Ro 48-8071-inducible CYP3A mRNA expression was restored when pravastatin-treated cultures were incubated with medium containing mevalonate. The concentration-dependence of Ro 48-8071-mediated CYP3A mRNA induction corresponded to the cellular contents of metabolically labeled squalene 2,3-oxide and squalene 2,3:22,23-dioxide, but not 24(S),25-epoxycholesterol. These results indicate that cyclase inhibitors are capable of inducing CYP3A expression in primary cultured rat and mouse hepatocytes and that the effect is mediated as a consequence of cyclase blockade through the evoked accumulation of one or more squalene metabolites that activate the PXR.
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