Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat

Clofibric acid (CA) is metabolized to chemically reactive acylating product s that can transacylate glutathione to form clofibryl-S-acyl-glutathione (C A-SG) in vitro and in vivo. We investigated the first step in the degradati on of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcys teine (CA-SNAC), which is catalyzed by gamma -glutamyltranspeptidase (gamma -GT). After gamma -GT mediated cleavage of glutamate from CA-SG, the produ ct clofibryl-S-acylcysteinylglycine (CA-S-CG) should undergo an intramolecu lar rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to f orm clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro stud ies incubating CA-SG with gamma -GT to determine the products formed, and i n vivo studies examining the products excreted in urine after dosing rats w ith CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma -GT (0.1 uni t/mL) in buffer (pH 7.4, 25 degreesC) and analyzed for products formed by r eversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results sho wed that CA-SG is degraded completely after 6 h of incubation leading to th e formation of two products, CA-N-CG and its disulfide, with no detection o f CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to th e reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of C A-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, C A-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detec tion of clofibryl-N-acyl-cysteine (CAN-C), but no evidence for the formatio n of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma -GT mediated degradation of clofibryl-S-acyl-glutathione leads p rimarily to the formation and excretion of clofibryl-N-acyl-eysteine produc ts rather than the S-acyl-NAC conjugate.