Diclofenac is a widely prescribed NSAID, which by itself and its reactive 
metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic 
hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced 
liver injury (DILI). In the present work, an investigation of the inhibitory 
effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) 
and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide 
(DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP 
synthesis in rat liver mitochondria was carried out. A mechanism based 
inhibition of ATP synthesis is exerted by diclofenac and its metabolites. 
Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) 
showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of 
the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The 
calculated kinetic constants of mechanism based inhibition of ATP synthesis by 
Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half 
maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of 
0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH 
exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our 
data from this study strongly indicate that Dic as well as its metabolites could 
be involved in the hepato-toxic action through inhibition of ATP synthesis.