γ-Glutamyltranspeptidase-Mediated Degradation of Diclofenac-S-acyl-glutathione in Vitro and in Vivo in Rat

• Published in: Chemical research in toxicology 2008-10, Vol.21 (10), p.1933-1938
• Main Authors: Grillo, Mark P, Hua, Fengmei, March, Kristi L, Benet, Leslie Z, Knutson, Charles G, Ware, Joseph A
• Format: Article
• Language: English
• Subjects: Animals; Bile - metabolism; Diclofenac - analogs & derivatives; Diclofenac - chemistry; Diclofenac - metabolism; Dipeptides - metabolism; gamma-Glutamyltransferase - metabolism; Glutathione - analogs & derivatives; Glutathione - chemistry; Glutathione - metabolism; Molecular Structure; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx800073p

Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic γ-glutamyltranspeptidase (γ-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic γ-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for γ-GT in vivo in rat and in vitro with commercially available γ-GT enzyme. We also examined the ability of D-SG to inhibit γ-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the γ-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D-N-CG), where a total of ∼8 μg was excreted 6 h postadministration. When D-SG (100 μM) was incubated with γ-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where ∼94 μM D-N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of γ-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the γ-GT enzyme. Results from theses studies indicate that D-SG is a substrate for γ-GT; however, the conjugate may not contribute significantly to the decrease in γ-GT activity reported to occur in vivo in rat.