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,α'-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 < 0.05) and 3-methylcholanthrene treatment had no effect, suggesting that a cytochrome P-450, rather than P-448, pathway is predominantly involved in mediating the covalent binding of furosemide.