Biochemical characterization of coumarin 7-hydroxylase activity in chick embryo liver microsomes.
Coumarin occurs naturally in the diet and can induce and inhibit cytochrome P450 enzymes. Hepatic coumarin 7-hydroxylase activity is the major pathway for coumarin metabolism in humans but not in rats, most strains of mice, or other laboratory animals. Coumarin 7-hydroxylase activity and the effects of chemical inhibitors and inducers on this activity were studied in 19-day-old chick embryo liver microsomes. Activity was between 35 and 75 nmol/mg protein/hr which is approximately 2-fold higher than reported for human liver microsomes. The pH optimum was 7.8 and the Km determined by both an ether extraction and a high performance liquid chromatography method was 7.3 +/- 0.9 (+/- SD) microM. Substrate inhibition was evident at coumarin concentrations above 250 microM (activities at 1000 and 4000 microM coumarin were 84 and 40% of Vmax, respectively). The Ki values (+/- SD) for inhibitors of microsomal coumarin 7-hydroxylase activity in vitro were: alpha-naphthoflavone, 46.9 +/- 19.8 nM; metyrapone, 0.8 +/- 0.9 microM; aniline, 12.3 +/- 8.2 microM; cimetidine, 70.9 +/- 27.9 microM; N-nitrosodimethylamine, 0.7 +/- 0.9 mM; and dimethyl sulfoxide, 7.9 +/- 1.9 mM. Treatment of chick embryos with pyrazole (40 mumol) increased coumarin 7-hydroxylase by 50% at 24 hr, but this activity was unaffected by treatment of embryos with 3-methylcholanthrene (2 mumol) or glutethimide (8 mumol). Thus, hepatic coumarin 7-hydroxylase activity in 19-day-old chick embryos is higher than in most laboratory animals and has similar biochemical properties as the enzyme in humans and mice. The chick embryo liver may be a useful system for studies on the biochemical effects of coumarin and the regulation of cytochrome P450-dependent coumarin 7-hydroxylase.
Goeger
D E
DE
Department of Preventive Medicine and Community Health, University of Texas medical Branch, Galveston 77550.
Anderson
K E
KE
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002642:Chick Embryo; D006224:Cricetinae; D065637:Cytochrome P-450 CYP2A6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D005984:Glutethimide / Q000494:pharmacology; D006168:Guinea Pigs; D008297:Male; D008647:Mesocricetus; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014468:Umbelliferones / Q000032:analysis
D065607:Cytochrome P-450 Enzyme Inhibitors; D011720:Pyrazoles; D014468:Umbelliferones; C031280:pyrazole; D008748:Methylcholanthrene; C031477:7-hydroxycoumarin; D003577:Cytochrome P-450 Enzyme System; D005984:Glutethimide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6