Halothane-dependent lipid peroxidation in human liver microsomes is catalyzed by cytochrome P4502A6 (CYP2A6)

Background: Halothane is extensively (approximately 50%) metabolized in hum ans and undergoes both oxidative and reductive cytochrome P450-catalyzed he patic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrif luoroethane (CTE). The free radical is also thought to initiate lipid perox idation. Halothane-dependent lipid peroxidation has been shown in animals i n vitro and in vivo but has not been evaluated in humans. This investigatio n tested the hypothesis that halothane causes lipid peroxidation in human l iver microsomes, identified P450 isoforms responsible for halothane-depende nt lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction. Methods: Halothane metabolism was determined using human liver microsomes o r cxDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyd e (MDA) formation using high-pressure liquid chromatography-ultraviolet ana lysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry. Results: Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA product ion exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production wa s catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone . CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2 A6 antibody. Conclusions: Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction preven ts halothane-dependent lipid peroxidation in vitro.