Identification in Rat Bile of Glutathione Conjugates of Fluoromethyl 2,2-Difluoro-1-(trifluoromethyl)vinyl Ether, a Nephrotoxic Degradate of the Anesthetic Agent Sevoflurane

Abstract

Recent studies have indicated that the nephrotoxicity of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether (“Compound A”), a breakdown product of the inhaled anesthetic sevoflurane, may be mediated by a reactive intermediate(s) generated via the cysteine conjugate β-lyase pathway. In order to gain a better understanding of glutathione (GSH)-dependent metabolism of Compound A, the present study was carried out with the primary goal of detecting and characterizing Compound A−GSH conjugates. By means of ionspray LC-MS/MS and NMR spectroscopy, a total of four GSH conjugates (“A1−A4”) were identified from the bile of rats dosed intraperitoneally with Compound A. A1 and A2 were identified as two diastereomers of S-[1,1-difluoro-2-(fluoromethoxy)-2-(trifluoromethyl)ethyl]glutathione, while A3 and A4 were identified as (E)- and (Z)-S-[1-fluoro-2-(fluoromethoxy)-2-(trifluoromethyl)vinyl]glutathione, respectively. Quantitative analyses indicated that approximately 29% of the administered dose of Compound A was excreted into the bile in the form of the above GSH conjugates over a period of 6 h. Studies conducted in vitro demonstrated that the reaction of Compound A with GSH was catalyzed by both rat liver cytosolic and microsomal glutathione S-transferases (GST), with the two enzyme systems exhibiting different product selectivities. Formation of these GSH conjugates also occurred nonenzymatically at an appreciable rate. These results indicate that spontaneous and enzyme-mediated conjugation with GSH represents a major pathway of metabolism of Compound A in rats. Conjugation of Compound A with GSH in vivo appeared to be catalyzed preferentially by microsomal rather than cytosolic GST, based on comparison of biliary, microsomal, and cytosolic metabolic profiles. By analogy with other haloalkenes, further metabolism of the corresponding cysteine conjugates of Compound A by renal cysteine conjugate β-lyase may lead to the formation of reactive acylating agents, which would be expected to bind covalently to cellular macromolecules and cause organ-selective nephrotoxicity.