Bioactivation of diclofenac in human hepatocytes and the proposed human hepatic proteins modified by reactive metabolites.

To reveal putative bioactivation pathways of diclofenac, in vitro human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by 35S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by 14C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.In the 35S-cysteine trapping assay, three and two adducts with 35S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using 14C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of 14C-diclofenac to human liver microsomal proteins.Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.