Microsomal Metabolism of Furosemide Evidence for the Nature of the Reactive Intermediate Involved in Covalent Binding

Results indicate that the covalent binding of furosemide to microsomal protein is mediated by an arene oxide intermediate.

Abstract

Pathways for the cytochrome P-450 monooxygenase-mediated covalent binding of furosemide to microsomal protein were examined. Two pathways were considered: one leading to a highly electrophilic imine by C- or N-hydroxylation followed by dehydration, the other an epoxidation pathway leading to formation of an arene oxide. Microsomal covalent binding of [α-3H]furosemide, [35S]furosemide, [α-2H]furosemide, and [α,α9-H]furosemide was the same, indicating that formation of an imine intermediate is unlikely and that the α-carbon is not a site of metabolic activation. Covalent binding to microsomal protein was enhanced in the presence of an epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane, and did not occur when tetrahydro[35S]furosemide was used as substrate. The results indicate that the covalent binding is mediated by an arene oxide intermediate. The potential significance of a subsequent rearrangement of the arene oxide to other electrophilic species cannot presently be evaluated. Furosemide covalent binding assay results using microsomes from the inbred C57BL/6N (B6) and DBA/2N (D2) strains of mice which had been treated with 3-methylcholanthrene and phenobarbital were compared. Phenobarbital treatment increased covalent binding (p