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.
Keywords: Bioactivation; Diclofenac; covalent binding; cytochrome P450; human hepatocytes; protein identification; trapping; uridine diphosphate-glucuronosyltransferase.