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.