Metabolism of an oxysterol, 7-ketocholesterol, by sterol 27-hydroxylase in HepG2 cells.
7-Ketocholesterol (7K) is a quantitatively important oxysterol in both atherosclerotic lesions and macrophage foam cells. We reported recently that radiolabeled 7K delivered to rodents in a modified lipoprotein or chylomicron remnant-like emulsion, both cleared predominantly by the liver, was rapidly excreted into the intestine as water-soluble products, presumably bile acids. Herein, we aimed to elucidate the early or initial reactions in 7K metabolism. The hypothesis was tested that sterol 27-hydroxylase, a mitochondrial cytochrome P450 and the first enzyme of the acidic bile acid pathway, is responsible for the initial metabolism of 7K by HepG2 cells, a human hepatoblastoma cell-line. The 27-hydroxylated product of 7K (27OH-7K) was shown to be the initial, lipid-soluble product of 7K metabolism. It was produced in mitochondrial incubations and whole cells and was readily released into the media from cells. Intact cells generated metabolites of 7K that had undergone conversion from lipid-soluble precursors to water-soluble products rapidly and extensively. Their production was ablated with cyclosporin A, a sterol 27-hydroxylase inhibitor. Furthermore, we demonstrated the effectiveness of two novel selective inhibitors of this enzyme, GW273297X and GI268267X. These inhibitors also ablated the production of water-soluble products by cells; and the inhibitor of choice, GW273297X, decreased the production of 27OH-7K in mitochondrial preparations. This is the first study to demonstrate that sterol 27-hydroxylase plays an important role in the metabolism of oxysterols such as 7K in liver cells.
Lyons
M A
MA
Cell Biology Group, Heart Research Institute, Sydney, New South Wales, Australia.
Brown
A J
AJ
D053493:Cholestanetriol 26-Monooxygenase; D002784:Cholesterol / Q000031:analogs & derivatives / Q000494:pharmacology; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018197:Hepatoblastoma / Q000201:enzymology*; D006801:Humans; D007653:Ketocholesterols / Q000378:metabolism* / Q000494:pharmacology; D008113:Liver Neoplasms / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; C445140:G1268267X; C445139:GW27329TX; D007653:Ketocholesterols; D016572:Cyclosporine; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; C003001:7-ketocholesterol