Mechanistic Studies on the Bioactivation of Diclofenac:  Identification of Diclofenac-<i>S</i>-acyl-glutathione in Vitro in Incubations with Rat and Human Hepatocytes

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to diclofenac-1-O-acyl glucuronide (D-1-O-G), a chemically reactive conjugate that has been implicated as playing a role in the idiosyncratic hepatoxicity associated with its use. The present studies investigated the ability of diclofenac to be metabolized to diclofenac-S-acyl-glutathione thioester (D-SG) in vitro in incubations with rat and human hepatocytes and whether its formation is dependent on a transacylation-type reaction between D-1-O-G and glutathione. When diclofenac (100 microM) was incubated with hepatocytes, D-SG was detected in both rat and human incubation extracts by a sensitive LC-MS/MS technique. The initial formation rate of D-SG in rat and human hepatocyte incubations was rapid and reached maximum concentrations of 1 and 0.8 nM, respectively, after 4 min of incubation. By contrast, during incubations with rat hepatocytes, the formation of D-1-O-G increased over 30 min of incubation, reaching a maximum concentration of 14.6 microM. Co-incubation of diclofenac (50 microM) with (-)-borneol (400 microM), an inhibitor of glucuronidation, led to a 94% decrease in D-1-O-G formation, although no significant decrease in D-SG production was observed. Together, these results indicate that diclofenac becomes metabolically activated in vitro in rat and human hepatocytes to reactive acylating derivatives that transacylate glutathione forming D-SG, but which is not solely dependent on transacylation by the D-1-O-G metabolite. From these results, it is proposed that reactive acylating metabolites of diclofenac, besides D-1-O-G, may be significant in the protein acylation that occurs in vivo and therefore also be important with regard to the mechanism(s) of diclofenac-mediated idiosyncratic hepatotoxicity.