Diclofenac (2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic
acid), a nonsteroidal antiinflammatory
drug, undergoes bioactivation by
cytochrome P450 oxidation to chemically reactive metabolites that are capable of reacting with endogenous nucleophiles such as
glutathione (GSH) and
proteins and that may play a role in the idiosyncratic hepatotoxicity associated with the
drug. Here, we investigated the ability of
diclofenac to be metabolized to 2-(2,6-dichloro-phenylamino)benzyl-S-thioether
glutathione (
DPAB-SG) in incubations with rat liver microsomes (RLMs) and human liver microsomes (HLMs) fortified with
NADPH and GSH. Thus, after incubation of
diclofenac (50 microM) with liver microsomes (1 mg
protein/ml), the presence of
DPAB-SG was detected in both RLM and HLM incubation extracts by liquid chromatography-tandem mass spectrometry techniques. The formation of
DPAB-SG was
NADPH-, concentration-, and time-dependent. Coincubation of
diclofenac (10 microM) with
ketoconazole (1 microM), an inhibitor of
cytochrome P450 (P450) 3A4, with HLMs led to a 75% decrease in
DPAB-SG formation. However, in contrast, coincubation with the P450 2C9 inhibitor
sulfaphenazole (10 microM) or the P450 2D6 inhibitor
quinidine (40 microM) led to a 1.9- and 1.6-fold increase in
DPAB-SG production, respectively. From these data, we propose that P450 3A4 mediates the oxidative decarboxylation of
diclofenac, resulting in the formation of a transient benzylic
carbon-centered
free radical intermediate that partitions between elimination (o-
imine methide production) and recombination (alcohol formation) pathways. The
benzyl alcohol intermediate, which was not analyzed for in the present studies, if formed could undergo
dehydration to provide a reactive o-
imine methide species. The o-
imine methide intermediate then is proposed to react covalently with GSH, forming
DPAB-SG.