Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat canalicular conjugate export pump (Mrp2)

Previous work demonstrates that the reactive acyl glucuronide of the nonsteroidal antiinflammatory drug diclofenac forms selective protein adducts in the liver, which may play a causal role in the pathogenesis of diclofenac-associated liver toxicity. Because glucuronide conjugates can be exported into the bile, we explored the role of diclofenac glucuronide hepatobiliary transport in the formation of site-specific protein adducts. Specifically, to analyze intracellular (hepatocytes) versus extracellular (biliary tree) targeting of proteins, we have compared the pattern of diclofenac binding in normal Wistar rats with that in mutant transport-deficient (TR-) rats which lack the functional canalicular isoform of the conjugate export pump, Mrp2. In bile duct-cannulated normal rats, >50% of an iv injected dose of [C-14]diclofenac appeared in the bile over a 90-min period. In contrast, in TR- rats virtually no hepatobiliary excretion of diclofenac glucuronide was found. After administration of diclofenac (30 mg/kg/day, ip for 3 days) to rats of both genotypes, a major protein adduct of an apparent molecular mass of 118 kDa was selectively detected by immunoblotting in isolated canalicular, but not in basolateral, membrane subfractions of wild-type rats, whereas no plasma membrane adducts could be found in the livers of TR- rats. Furthermore, immunohistochemical analysis using an anti-diclofenac antibody revealed the presence of distinct diclofenac-modified proteins on canalicular membranes of liver sections from diclofenac-treated normal rats, whereas no adducts could be identified in livers of TR- rats. In Western blots, the major diclofenac-modified canalicular membrane protein did not comigrate with Mrp2, indicating that the glucuronide carrier itself was unlikely to be a target. Collectively, the results demonstrate that the reactive diclofenac glucuronide is selectively transported into bile via Mrp2 and that hepatobiliary transport is critical for diclofenac covalent binding to proteins in the biliary tree.