title: Human pregnane X receptor activation and CYP3A4/CYP2B6 induction by 2,3-oxidosqualene:lanosterol cyclase inhibition.
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 microM 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 3beta-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC(50)s of 0.113, 0.916, and 0.294 microM, 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 microM 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 IC(50) of 3.35 microM. 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.
authors.affiliation: Institute of Environmental Health Sciences, 2727 Second Avenue, Room 4000, Detroit, MI 48201, USA.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Duniec-Dmuchowski
authors.forename: Hai-Lin
authors.initials: HL
authors.lastname: Fang
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Strom
authors.forename: Ewa
authors.initials: E
authors.lastname: Ellis
authors.forename: Melissa
authors.initials: M
authors.lastname: Runge-Morris
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kocarek
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D015153:Blotting, Western; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors*; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis*; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004791:Enzyme Inhibitors; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D019751:Intramolecular Transferases; C021924:lanosterol synthase