Abstract
The nonsteroidal antiinflammatory drug diclofenac can bind irreversibly to hepatocellular proteins via its acyl glucuronide metabolite. In view of a possible involvement of these protein adducts in the pathogenesis of diclofenac-associated liver damage, we investigated the mechanism of adduct formation in rat hepatic microsomes. [14C]Diclofenac covalently bound to hepatic microsomal proteins as a function of exposure time and the concentration of the cofactor, UDP-glucuronic acid (UDPGA). The rate and extent of protein binding were significantly increased in the presence of the imine-trapping agent, sodium cyanide. Moreover, hepatic microsomes incubated with [14C]UDPGA and nonradiolabeled diclofenac resulted in similar covalent binding of the radiolabeled compound to microsomal proteins. Covalent binding of [14C]UDPGA was significantly decreased in the presence of 7,7,7-triphenylheptyl-UDP, a specific inhibitor of UDP-glucuronosyltransferase. Finally, the protein adducts formed after incubation with both the radiolabeled aglycone and radiolabeled glucuronic acid were resolved by sodium dodecyl sulfate gel electrophoresis. Under both conditions, a radiolabeled protein band of apparent M(r) 60 kDa was found by fluorographic analysis. These results indicate that diclofenac acyl glucuronide covalently binds to hepatic microsomal proteins by two apparent mechanisms. Besides nucleophilic displacement of the glucuronic acid, the open-chain glucuronic acid can form an imine bond with a nucleophilic site of the target protein and is thus retained within the adduct.
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