METABOLIC-ACTIVATION AND IMMUNOCHEMICAL LOCALIZATION OF LIVER PROTEINADDUCTS OF THE NONSTEROIDAL ANTIINFLAMMATORY DRUG DICLOFENAC

Diclofenac is a nonsteroidal anti-inflammatory agent that is reported to cause serious hepatic injury in some patients. To investigate the p ossibility that protein adducts derived from reactive intermediates of diclofenac might be responsible for the hepatotoxicity produced by th is drug, we recently developed polyclonal antisera that recognized pro tein adducts of diclofenac. In the present study, we have characterize d further the diclofenac adducts in rat liver. Immunoblotting studies showed that diclofenac-labeled hepatic proteins were formed in a dose- and time-dependent manner in rats given diclofenac. Subcellular fract ionation of liver homogenates from diclofenac-treated rats showed that a 50-kDa microsomal protein and 110-, 140-, and 200-kDa plasma membra ne proteins were labeled preferentially. Immunofluorescence studies of isolated hepatocytes and immunohistochemical analysis of liver slices from diclofenac-treated mice and rats confirmed that plasma membrane proteins were labeled by diclofenac metabolites and showed that the bi le canalicular domain of the plasma membrane was a major site of diclo fenac adduct formation. Additionally, we found that cytochrome P-450 a nd UDP-glucuronosyltransferase, but not acyl-CoA synthase, catalyzed t he formation of reactive intermediates of diclofenac that were bound c ovalently to proteins in vitro. The metabolites catalyzed by cytochrom e P-450 in vitro were bound exclusively to a 60-kDa microsomal protein , even in the presence of albumin. In contrast, the 110-, 140-, and 20 0-kDa plasma membrane proteins as well as others appeared to be labele d when diclofenac was activated by UDP-glucuronosyltransferase. Chemic al stability studies of the adducts formed in vivo showed that they we re unstable under basic conditions and suggested that the metabolites were bound to cellular proteins as ester linkages. These results demon strated that plasma membrane proteins are major targets of diclofenac metabolites in vivo, and the presence of these adducts may be importan t in the mechanism of diclofenac hepatotoxicity.