CYTOCHROME P4502C11 IS A TARGET OF DICLOFENAC COVALENT BINDING IN RATS

Diclofenac antiserum was previously developed and used to detect prote in adducts of metabolites of dichlofenac in livers of mice and rats. I n this study, the antibody has been used to facilitate the purificatio n of a major 51 kDa microsomal adduct of diclofenac from the liver mic rosomes of male rats that were treated with diclofenac. The adduct was identified as male-specific cytochrome P4502C11 based on its N-termin al amino acid sequence, reaction with a cytochrome P4502C11 antibody, and by its absence from liver microsomes of diclofenac-treated female rats. When diclofenac was incubated with liver microsomes of control r ats in the presence of NADPH, only the 51 kDa adduct was produced. The formation of the adduct was inhibited by a cytochrome P4502C11 monocl onal antibody, but not by reduced glutathione or N-alpha-acety-L-lysin e. No adduct was detected when diclofenac was incubated with liver mic rosomes from female rats. Moreover, adduct formation in vivo appeared to lead to a 72% decrease in the activity of cytochrome P4502C11. The results indicate that cytochrome P4502C11 metabolizes diclofenac into a highly reactive product that covalently binds to this enzyme before it can diffuse away and react with other proteins.