Abstract
The effects of [4′-(6-allyl-methyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 μM Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor (PXR)-responsive transactivation assay in HepG2 cells was used. Ro 48-8071, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79), and 3β-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC50s of 0.113, 0.916, and 0.294 μM, respectively. Treatment of the HepG2 system with (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3′-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598), an inhibitor of squalene monooxygenase, at concentrations sufficient to achieve cholesterol biosynthesis inhibition significantly inhibited cyclase inhibitor-mediated, but not rifampicin-mediated, reporter induction. Direct treatment of the HepG2 system with 1 to 10 μM squalene 2,3:22,23-dioxide, but not squalene 2,3-oxide, significantly activated PXR-responsive reporter expression. Also, squalene 2,3:22,23-dioxide bound to human PXR in vitro with an IC50 of 3.35 μM. These data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.
Footnotes
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This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL50710]; the National Institutes of Health National Institute of Environmental Health Sciences [Grant ES05823]; the National Institutes of Health National Institute of Environmental Health Sciences Center [Grant P30-ES06639] (Cell Culture and Gene Transfer Technologies, Imaging and Cytometry, and Microarray and Bioinformatics Facility Cores); and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Contract N01-DK70004 HHSN267200700004C].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.025130.
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ABBREVIATIONS: PXR, pregnane X receptor; CAR, constitutive androstane receptor; cyclase, 2,3-oxidosqualene:lanosterol cyclase; Ro 48-8071, [4′-(6-allyl-methyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate; NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3′-bithiophen-5-yl)methoxy]benzenemethanamine; U18666A, 3β-(2-diethylaminoethoxy)androst-5-en-17-one HCl; BIBX 79, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine; DMSO, dimethyl sulfoxide; TBST, Tris-buffered saline/Tween 20; MVA, mevalonate; TR-FRET, time-resolved fluorescence resonance energy transfer; SXR, steroid and xenobiotic receptor; T0901317, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide.
- Received October 13, 2008.
- Accepted January 14, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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