IMMUNOCHEMICAL IDENTIFICATION OF MOUSE HEPATIC PROTEIN ADDUCTS DERIVED FROM THE NONSTEROIDAL ANTIINFLAMMATORY DRUGS DICLOFENAC, SULINDAC, AND IBUPROFEN

Reactive metabolite-modified hepatic protein adducts have been propose d to play important roles in the mechanism(s) of hepatotoxicity of non steroidal anti-inflammatory drugs (NSAIDs). In the present study, immu nochemical techniques have been used to compare the patterns of drug-p rotein adducts expressed in livers of mice given single doses of one o r other of three different NSAIDs. These were diclofenac and sulindac, which are widely used but potentially hepatotoxic drugs, and ibuprofe n, which is considered to be nonhepatotoxic. Specific polyclonal antis era were produced by immunization of rabbits with conjugates prepared by coupling each of the NSAIDs to the carrier protein keyhole limpet h emocyanin. Immunoblotting studies revealed dose-dependent formation of major 110 kDa polypeptide adducts in livers from mice sacrificed 6 h after administration of single doses of either diclofenac (0-300 mg/kg ) or sulindac (0-100 mg/kg). Lower levels of several other adducts, of 140 and 200 kDa, were also expressed in livers from these animals. In contrast, livers from mice treated with ibuprofen (0-200 mg/kg) predo minantly expressed a 60 kDa adduct and only relatively low levels of a 110 kDa adduct. The various adducts were shown by differential centri fugation to be concentrated in the nuclear fraction of liver homogenat es. Those derived from diclofenac and sulindac were further localized, by Percoll density gradient centrifugation, to a subfraction which co ntained a high activity of the bile canalicular marker enzyme alkaline phosphatase. This suggests that they are concentrated in the bile can alicular domain of hepatocytes. The different patterns of adduct forma tion raise the possibility that formation of certain NSAID protein add ucts, particularly 110 kDa adducts, has toxicological significance.