Induction of CYP3A by 2,3-oxidosqualene:lanosterol cyclase inhibitors is mediated by an endogenous squalene metabolite in primary cultured rat hepatocytes.
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 [4'-(6-allyl-methyl-amino-hexyloxy)-2'-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 x 10(-5) M Ro 48-8071 and 10(-4) M BIBX 79. The abilities of Ro 48-8071, BIBX 79, and 3beta-(2-diethylaminoethoxy)androst-5-en-17-one.HCl (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 inhibitor-inducible 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)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-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.
Shenoy
Sarita D
SD
Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 48201, USA.
Spencer
Thomas A
TA
Mercer-Haines
Nancy A
NA
Abdolalipour
Masumeh
M
Wurster
William L
WL
Runge-Morris
Melissa
M
Kocarek
Thomas A
TA
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001577:Benzophenones / Q000494:pharmacology*; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D004790:Enzyme Induction / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors*; D008297:Male; D051379:Mice; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013185:Squalene / Q000378:metabolism*; D014162:Transfection
D001577:Benzophenones; D012333:RNA, Messenger; C105726:Ro 48-8071; D013185:Squalene; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D019751:Intramolecular Transferases; C021924:lanosterol synthase