γ-Glutamyltranspeptidase-Mediated Degradation of Diclofenac-S-acyl-glutathione in Vitro and in Vivo in Rat
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- 作者：Mark P. Grillo ; Fengmei Hua ; Kristi L. March ; Leslie Z. Benet ; Charles G. Knutson ; Joseph A. Ware
- 刊名：Chemical Research in Toxicology
- 出版年：2008
- 出版时间：October 20, 2008
- 年：2008
- 卷：21
- 期：10
- 页码：1933-1938
- 全文大小：158K
- 年卷期：v.21,no.10(October 20, 2008)
- ISSN：1520-5010
文摘
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.