CHEMICAL AND IMMUNOCHEMICAL COMPARISON OF PROTEIN ADDUCT FORMATION OF4 CARBOXYLATE DRUGS IN RAT-LIVER AND PLASMA

Carboxylate drugs usually form acyl glucuronide conjugates as major me tabolites. These electrophilic metabolites are reactive, capable of un dergoing hydrolysis, rearrangement, and covalent binding reactions to proteins. The last-mentioned property has the potential to initiate im mune and other toxic responses in vivo. In this study, we compared the extent and pattern of covalent adduct formation in plasma and livers of rats dosed with the nonsteroidal antiinflammatory drugs (NSAIDs) zo mepirac (ZP) and diflunisal (DF), the hypolipidemic agent clofibric ac id (CA), and the anti-epileptic agent valproic acid (VPA). These drugs form acyl glucuronides with diverse intrinsic reactivities (apparent first order degradation t(1/2) values of 0.5, 0.6, 3, and 60 h, respec tively). Rats were dosed iv twice daily for 2 days (50 mg/kg for ZP, D F, and CA, 150 mg/kg for VPA). Chemical analysis of tissues obtained 6 h after the last dose revealed adduct concentrations of 0.31, 0.44, 0 .28, and 0.05 mu g of drug equivalents/ml of plasma and 2.21, 2.31, 0. 96, and 0.96 mu g of drug equivalents/g of liver for ZP, DF, CA and VP A treatments, respectively. For both plasma and liver, the higher conc entrations of adducts were found with ZP and DF, which have the more r eactive glucuronides. The low concentrations of VPA adducts found in p lasma were in keeping with the very low reactivity of its glucuronide. In liver, however, VPA adducts achieved concentrations of the same or der of magnitude as the other drugs and were accompanied by adducts of the (E)-2-en metabolite of VPA at 0.38 mu g of VPA equivalents/g of l iver. The liver data for VPA can be explained by an acyl CoA/beta-oxid ation pathway of adduct formation in addition to that from acyl glucur onidation. Immunoblotting using rabbit polyclonal antisera raised agai nst synthetic drug-protein adducts revealed major bands at 110, 140, a nd similar to 200 kDa in livers of ZP- and DF-treated rats. A fourth m ajor band at 70 kDa in ZP-treated liver had the same apparent molecula r weight as the only major band detected in CA-treated liver. A 140 kD a band was detected in liver tissue from VPA-treated rats, as well as several lower molecular weight bands. In plasma, the antisera specific ally detected drug-modified serum albumin in samples from rats treated with ZP, DF, and CA, but not VPA. The results with this small series of carboxylate drugs suggested that (a) adduct concentrations in plasm a but not liver could be related to acyl glucuronide reactivity, (b) w hile some modified proteins detected were common, the pattern of modif ication varied from drug to drug, and (c) caution should be exercised in attributing adduct formation exclusively to the acyl glucuronidatio n pathway.