Short Communication Human Halothane Reduction In Vitro by Cytochrome P450 2A6 and 3A4: Identification of Low and High KM Isoforms

The volatile anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) is extensively metabolized in humans, with approximately 50% of an absorbed dose undergoing hepatic biotransformation (Carpenter et al., 1986). Halothane is a unique substrate, undergoing both oxidative and reductive P450-catalyzed dehalogenation during clinical anesthesia, with each metabolic pathway subtending a different form of hepatic toxicity (Cousins et al., 1989; Gut et al., 1995; Jenner et al., 1990; Ray and Drummond, 1991). Oxidative hepatic metabolism mediates a rare, often fatal, immune-based fulminant hepatic necrosis (“halothane hepatitis”) (Kenna et al., 1988; Ray and Drummond, 1991), whereas reductive metabolism mediates a common, mild, and subclinical hepatotoxicity (de Groot and Noll, 1983; Sato et al., 1990). The mechanism of halothane oxidation and hepatic necrosis has been well described (Bourdi, 1996; Gut et al., 1993, 1995; Ray and Drummond, 1991). Under sufficient oxygen tension, halothane undergoes P450-catalyzed oxidation to a reactive acyl chloride intermediate, which may trifluoroacetylate tissue proteins. In susceptible individuals, these act as neoantigens to stimulate formation of antitrifluoroacetylated antibodies that, upon re-exposure to halothane or other trifluoroacetylating volatile anesthetics (enflurane, isoflurane, or desflurane), mediate an immune response culminating in fulminant hepatic necrosis. The rate and extent of oxidative halothane metabolism are considered crucial factors in determining susceptibility to halothane hepatitis (Christ et al., 1988a, 1988b; Kenna et al., 1990; Pohl et al., 1989). The mechanism of halothane reduction and mild hepatotoxicity has been similarly well described (Ray and Drummond, 1991). Under anaerobic conditions, halothane undergoes P450-catalyzed reduction to an unstable radical intermediate (Ahr et al., 1982), which may 1) abstract a hydrogen atom to form the volatile metabolite 2-chloro1,1,1-trifluoroethane (CTE), 2) undergo a second P450-catalyzed reduction and loss of fluoride to give the volatile metabolite 2-chloro1,1-difluoroethylene (CDE), 3) bind covalently to microsomal phospholipids (Muller and Srier, 1982) or proteins such as P450 causing suicide inactivation (Baker et al., 1991; Manno et al., 1992), or 4) initiate microsomal lipid peroxidation (Akita et al., 1989; Awad et al., 1996; de Groot and Noll, 1983; Sato et al., 1990). These sequelae of halothane reduction are the putative causes of mild halothane hepatotoxicity, which is manifested by mildly elevated postoperative liver enzymes (Akita et al., 1989; de Groot and Noll, 1983; Sato et al., 1990). Of greater clinical significance is the impaired mixed function oxidase activity (Cousins et al., 1987), which ensues from P450halothane metabolite complex formation (Baker et al., 1991; Manno et al., 1992) and/or lipid peroxidation (Awad et al., 1996; de Groot and Noll, 1983). Halothane reduction accounts for approximately 1–6% of total metabolism (Wark et al., 1990), and mild hepatotoxicity occurs in up to 25% of patients undergoing halothane anesthesia (Ray and Drummond, 1991). Recent investigations identifying the P450 isoforms catalyzing halothane oxidation and reduction have revealed a curious isoform specificity. Human hepatic oxidative halothane metabolism is catalyzed predominantly by P450 2E1 in vitro and in vivo, and to a lesser extent by P450 2A6 (Kharasch et al., 1996; Madan and Parkinson et al., 1996; Spracklin et al., 1997). In contrast, human hepatic reductive halothane metabolism is catalyzed principally by P450s 2A6 and 3A4 (Spracklin et al., 1996). More specifically, P450s 2E1 and 2A6 were identified as the low and high Km isoforms, respectively, catalyzing halothane oxidation (Spracklin et al., 1997). In contrast, the identity of the low and high KM isoforms catalyzing halothane reduction remains unknown. The purpose of this investigation was to provide this identification.