Coumarin metabolism by rat esophageal microsomes and cytochrome P450 2A3

The rat esophagus is strikingly sensitive to tumor induction by nitrosamine s, and it has been hypothesized that this tissue contains cytochrome P450 e nzymes (P450s) which catalyze the metabolic activation of these carcinogens . The metabolic capacity of the esophagus is not well characterized. In the study described here, the products of C-14-coumarin metabolism by rat esop hageal microsomes were identified and quantified. Metabolite characterizati on was by LC/MS/MS and GUMS and comparison to standards, quantification was by radioflow HPLC. The coumarin metabolites formed by rat esophageal micro somes were compared to those formed by P450 2A3. The major metabolites form ed by esophageal microsomes were 8-hydroxycoumarin, o-hydroxyphenylacetalde hyde (o-HPA), and o-hydroxyphenylacetic acid (o-HPAA). A smaller amount of 5-hydroxycoumarin, about one-third the 8-hydroxycoumarin, was also formed. o-HPA and o-HPAA are products of coumarin 3,4-epoxidation. The relative rat es of coumarin 8-hydroxylation and 3,4-epoxidation were similar. Coumarin 8 -hydroxylation has not previously been reported as a major pathway in any t issue, and no P450s have yet been reported to catalyze this reaction. P450 2A3 catalyzed both the 7-hydroxylation and 3,4-epoxidation of coumarin. P45 0 2A3 was previously characterized as a coumarin 7-hydroxylase, however, in this study, we report that it catalyzes the formation of o-HPA more effici ently. The K-m and V-max were 1.3 +/- 0.35 muM and 0.65 +/- 0.06 nmol/min/n mol P450 for coumarin 7-hydroxylation and 1.4 +/- 0.58 muM and 3.1 +/- 0.46 nmol/min/nmol P450 for o-HPA formation.