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Pain Medicine  |   August 2001
Halothane-dependent Lipid Peroxidation in Human Liver Microsomes Is Catalyzed by Cytochrome P4502A6 (CYP2A6)
Author Affiliations & Notes
  • Yuko Minoda, M.D., Ph.D.
    *
  • Evan D. Kharasch, M.D., Ph.D.
  • * Visiting Professor, University of Washington, and Staff Anesthesiologist, Kagoshima University School of Medicine. † Professor of Anesthesiology and Medicinal Chemistry (Adjunct), University of Washington.
  • Received from the Department of Anesthesiology and Critical Care Medicine, Kagoshima University School of Medicine, Kagoshima, Japan; the Departments of Anesthesiology and Medicinal Chemistry, University of Washington, Seattle, Washington; and the Anesthesiology Service, Puget Sound Veterans Affairs Healthcare System, Seattle, Washington.
Article Information
Pain Medicine
Pain Medicine   |   August 2001
Halothane-dependent Lipid Peroxidation in Human Liver Microsomes Is Catalyzed by Cytochrome P4502A6 (CYP2A6)
Anesthesiology 8 2001, Vol.95, 509-514. doi:
Anesthesiology 8 2001, Vol.95, 509-514. doi:
HALOTHANE undergoes both oxidative and reductive metabolism in humans in vivo  . 1 Even with patients breathing 100% oxygen, there are portions of the liver with sufficiently low oxygen tension to permit reductive metabolism, owing in part to halothane-dependent reductions in hepatic blood flow. Halothane is reduced by cytochrome P450 (CYP) to the 2-chloro-1,1,1-trifluoroethyl radical, which has been demonstrated in rodent livers and bile in vitro  and in vivo  . 2–6 This radical may abstract hydrogen to form the stable metabolite chlorotrifluoroethane (CTE) or undergo further P450-catalyzed reduction to yield chlorodifluoroethene (CDE) and inorganic fluoride. Recently, human liver microsomal halothane reduction to CTE and CDE in vitro  was shown to be catalyzed principally by CYP2A6 and CYP3A4. 7 These were identified as the low- and high-Kmisoforms, respectively. 8 
Halothane radicals may also react with tissue macromolecules, such as cytochrome P450, to form a catalytically inactive enzyme-metabolite complex 9,10 and with lipids to form a number of products. 11,12 It is generally accepted that halothane radicals initiate lipid peroxidation, at least in animals. 12,13 Considerable evidence for halothane-dependent lipid peroxidation in rodents has been presented. Halothane-dependent lipid peroxidation was demonstrated in vitro  using microsomes and hepatocytes from uninduced and phenobarbital-induced rats, rabbits, and guinea pigs, 11,12,14–17 in livers excised from rats and guinea pigs treated with halothane in vivo  even under normoxic conditions, 18,19 and noninvasively in phenobarbital-induced hypoxic rats in vivo.  20 Awad et al.  19 recently demonstrated halothane-dependent lipid peroxidation in rats in vivo  , using quantification of F2-isoprostanes in plasma and excised livers, which was increased under hypoxic conditions. Enhancement of lipid peroxidation was specific to halothane and not observed with enflurane, isoflurane, or desflurane in microsomes or in vivo.  18,19 Lipid peroxidation in guinea pigs is greater than in rats and has been attributed to greater rates of reductive halothane metabolism. 21 Inhibition of halothane reduction in guinea pig liver microsomes decreased lipid peroxidation. 16 Halothane reduction and consequent lipid peroxidation are considered by some to mediate mild halothane hepatotoxicity. 1,18,20 
In contrast to rodents, little is known about halothane effects on lipid peroxidation in humans. Halothane-dependent lipid peroxidation has never been evaluated in human liver microsomes. The purpose of this investigation was to test the hypothesis that anaerobic reduction of halothane causes lipid peroxidation in human liver microsomes and to identify P450 isoforms responsible for halothane-dependent lipid peroxidation. In general, when anesthetic toxicity is related to anesthetic metabolism, modulation of anesthetic metabolism may have the more important effect of modifying metabolism-based toxicity. For example, inhibition of CYP2E1-dependent halothane oxidation decreased trifluoroacetylated neoantigen formation. 22 Although it has been shown that halothane reduction in vitro  is prevented by inhibiting CYP2A6 and CYP3A4 activity, 7 it is not known whether the consequence of halothane reduction, namely lipid peroxidation, is prevented by inhibiting P450 activity. Therefore, the second purpose of this investigation was to determine whether inhibition of halothane reduction prevents lipid peroxidation in human liver microsomes.
Materials and Methods
Halothane was obtained from Ayerst Laboratories, Inc. (New York, NY). CDE and CTE were purchased from PCR, Inc. (Gainesville, FL). Microsomes containing individual cDNA-expressed P450 isoforms, P450 reductase, or both were purchased from Gentest, Inc. (Woburn, MA). Monoclonal anti-CYP2A6 antibody was prepared and characterized as monospecific as previously described 23 and was the generous gift of Yang Sai, Ph.D. (University of Washington, Seattle, WA). Other reagents were purchased from Sigma Chemical Co. (St Louis, MO). A thiobarbituric acid (TBA) solution was prepared adding 6 ml NaOH, 1 m, to 1% aqueous TBA. After heating with stirring until dissolved, 0.1 ml concentrated HCl was added. The solution was decolorized with Norit activated charcoal, filtered, and adjusted to pH 2.5 with concentrated HCl. All buffers and reagents were prepared with high-purity water. Microsomes were prepared from human livers as described previously. 24 Experiments were done with several livers; data presented were from HL141. Animal experiments were approved by the University of Washington Animal Care and Use Committee in accordance with American Association for Accreditation of Laboratory Animal Care guidelines. Microsomes were prepared from livers of uninduced and phenobarbital-, pregnenolone-, and isoniazid-induced rats as described previously. 25 
Halothane metabolism was determined in reaction mixtures (0.5 ml) containing human liver microsomes (2 mg/ml) or cDNA-expressed P450 (1.6 mg/ml), halothane, and 2 mm NADPH in 50 mm Tris-HCl–150 mm KCl buffer (pH 7.4) as described previously. 7 Incubation vials were sealed with septa and flushed with prepurified nitrogen (99.995%) for 3 min. Halothane was then added, either 2 μl or diluted in acetonitrile (final aqueous acetonitrile concentration 0.2%) for experiments at subsaturating substrate concentrations. After 3 min preincubation, the reactions were initiated by injecting NADPH through the septum. Incubations were routinely performed at 37°C for 60 min, except for the time course experiment. Reactions were quenched by addition of 10% trichloroacetic acid (75 μl) into the sealed vials, followed by 2% butylated hydroxy toluene in methanol (10 μl). Gas samples were removed using a gas-tight syringe, and CDE and CTE were analyzed by headspace gas chromatography–mass spectrometry using a Hewlett-Packard (Wilmington, DE) 5890 gas chromatograph interfaced to an HP5971 mass-selective detector and an HP7694 headspace sampler, with a DB-VRX capillary column (30 m × 0.32 mm × 1.8 μm film thickness; J&W Scientific, Folsom, CA). 7 Halothane concentrations were also analyzed by this assay for the concentration experiment. After removal of the gas sample, the microsomal mixture was centrifuged at 14,000 rpm for 10 min. Malondialdehyde (MDA) was quantified as the TBA adducts by high-pressure liquid chromatography. 26,27 Microsomal supernatant (0.3 ml) was combined with 0.3 ml TBA solution and heated in a boiling water bath for 15 min. The subsequent TBA-derivatized products were cooled and extracted by vortexing with 0.75 ml n  -butanol. After centrifuging, the organic layer was evaporated to dryness, reconstituted in mobile phase, and analyzed by high-pressure liquid chromatography using a Hewlett-Packard 1050 system and a Microsorb MV C18 column (4.6 mm x 250 mm x 5 μm; Rainin, Walnut Creek, CA). The mobile phase was acetonitrile: 5 mm phosphate buffer (pH 7.0) (15:85) at a flow rate of 1.0 ml/min, and the MDA-TBA adduct was detected at 515 nm. An MDA standard curve (0, 0.2, 0.4 1, and 2 μm) was prepared daily by adding 1,1,3,3-tetramethoxypropane to the buffer and microsomes and analyzing as described for the unknown. MDA concentrations in zero time blanks or incubations without halothane were subtracted from those incubations containing halothane, as appropriate.
Experiments with isoform-selective P450 inhibitors used 5 μm methoxsalen (CYP2A6) and 100 μm troleandomycin (CYP3A4). Inhibitors were added in acetonitrile (final concentration 0.2%) to incubations that were then preincubated with NADPH at 37°C for 10 (methoxsalen) or 15 (troleandomycin) min under aerobic conditions to permit isoform-selective mechanism-based inhibition. The vials were then placed on ice, sealed, and purged with nitrogen, and then 2 μl halothane was added to initiate the reductive halothane reaction. Reactions were performed at 37°C for 60 min and then quenched as described previously. Experiments using anti-CYP2A6 antibody were preincubated at 37°C for 5 min before adding NADPH.
Regression and statistical analyses were performed using SigmaPlot and SigmaStat (SPSS, Chicago, IL). Experiments were typically performed in triplicate, and results are expressed as mean ± SD.
Results
Initial experiments characterized the time dependence of MDA production (fig. 1). Human liver microsomes (HL141) were incubated with halothane and NADPH under anaerobic conditions. MDA production increased linearly for 60 min. CTE and CDE formation paralleled that of MDA (not shown), as described previously. 7 MDA formation was also supported by NADH, although at lower rates (58% compared with NADPH), and was negligible in the absence of either cofactor (not shown). MDA formation was also markedly reduced by 76% when incubations were conducted using room air. Halothane-dependent lipid peroxidation in microsomes from other human livers was less than in HL141; therefore, this liver was used for subsequent kinetic and inhibition experiments.
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
×
Halothane-dependent MDA production by human liver microsomes was compared with that in rat liver microsomes (fig. 2). Microsomes from rats pretreated with isoniazid (CYP2E1 induction), phenobarbital (CYP2B, 2C, and 3A induction), pregnenolone (CYP3A induction), or nothing were evaluated. MDA formation with all rat liver microsomes, including uninduced rats, was higher than with human liver microsomes. Among induced rats, microsomes from phenobarbital- and pregnenolone-pretreated rats produced the greatest amounts of MDA.
Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
×
The halothane concentration dependence of MDA production was studied using microsomes from one human liver (fig. 3). MDA production was concentration- dependent and saturable, and exhibited biphasic kinetics with respect to substrate concentration. MDA production was fit to a two-enzyme system by nonlinear regression analysis. The parameters (± standard error of the parameter estimate) obtained were Vmax1= 0.86 ± 0.21 pmol · min−1· mg−1, Km1= 0.0062 ± 0.0047%, Vmax2= 2.41 ± 0.28 pmol · min−1· mg−1, and Km2= 0.81 ± 0.39%. CTE and CDE formation were also measured and similarly exhibited biphasic kinetics.
Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
×
To identify the P450 isoforms responsible for the halothane-dependent lipid peroxidation in human liver microsomes, the effect of isoform-selective P450 inhibitors was determined (fig. 4). Halothane-reductive metabolism in human liver microsome was previously shown to be catalyzed by CYP2A6 and CYP3A4 7; therefore, the effect of the CYP2A6 and CYP3A4 inhibitors, methoxsalen and troleandomycin, were evaluated. MDA production was inhibited 52% by methoxsalen but was not decreased by troleandomycin.
Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P  < 0.05) by analysis of variance.
Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P 
	< 0.05) by analysis of variance.
Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P  < 0.05) by analysis of variance.
×
To further identify the isoforms responsible for halothane-dependent lipid peroxidation, rates of MDA production catalyzed by cDNA-expressed P450 isoforms were determined (fig. 5). Experiments were performed using microsomes from cells expressing CYPs 2A6 or 3A4 and coexpressing P450 reductase. Halothane metabolism and MDA formation were catalyzed predominantly by CYP2A6. Microsomes containing CYP3A4 and reductase or reductase alone formed a negligible amount of MDA. Halothane metabolism and MDA formation by expressed CYP2A6 were also studied after preincubation with methoxsalen or anti-CYP2A6 monoclonal antibody (fig. 6). Methoxsalen inhibited CDE and CTE production by 87 and 91%, respectively, and inhibited MDA formation by 81%. Anti-CYP2A6 antibody inhibited CDE and CTE productions by 93% and completely inhibited halothane-dependent MDA production.
Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
×
Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P  < 0.05) by analysis of variance.
Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P 
	< 0.05) by analysis of variance.
Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P  < 0.05) by analysis of variance.
×
Discussion
Malondialdehyde results from the oxidative degradation of fatty acids. 28 As an index of lipid peroxidation, the most widely used method for the determination of MDA in biologic materials is based on its reaction with TBA to form a pink complex with an absorption maximum at 532–535 nm. However, numerous aldehydic compounds other than MDA can react with TBA to form a complex that absorbs in the 532- to 535-nm region. 29 Therefore, spectrophotometric assays for MDA express results only as nonspecific TBA-reactive substances. To separate the TBA–MDA adduct from other possible TBA-reactive substances, adduct samples were subsequently analyzed by high-pressure liquid chromatography, providing greater specificity and allowing quantitation of lipid peroxidation specifically as MDA. 26,27 
The results of this investigation clearly demonstrate halothane-dependent lipid peroxidation, assessed by formation of MDA, in human liver microsomes under decreased oxygen tension. Similar to results in previous studies with rat liver microsomes, lipid peroxidation was negligible under normoxic conditions (approximately 1.2 mm oxygen). 11,15–17 Thus, both halothane reduction 7,30 and lipid peroxidation require reduced oxygen tension (peroxidation was slightly enhanced at 10 mmHg and 12 μm oxygen but decreased at higher tensions). 15,17 In addition, similar kinetic profiles between MDA, CDE, and CTE production were observed, as well as parallel effects of cytochrome P450 inhibitors on halothane metabolism and lipid peroxidation catalyzed by human liver microsomes and expressed human CYP2A6. Together, these observations support the hypothesis that lipid peroxidation in human liver microsomes results from halothane reduction under decreased oxygen tensions. This seems to be the first report of halothane-dependent lipid peroxidation in human liver microsomes.
The results also suggest that CYP2A6 is a major catalyst of lipid peroxidation in human liver microsomes. Human liver microsomal MDA formation was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. In addition, expressed CYP2A6 was a better catalyst of MDA formation than was CYP3A4. CYPs 2A6 and 3A4 were the focus of these experiments because previous investigations showed that these two isoforms were the predominant catalysts of human liver microsomal halothane reduction. 7,8 Halothane-dependent lipid peroxidation catalyzed by CYP2A6 is consistent with previous identification of CYP2A6 as the low-Kmisoform responsible for halothane reduction.
Results obtained with rat liver microsomes are consistent with previous evaluations of halothane metabolism and lipid peroxidation in rats but reveal an important species difference in comparison with human liver microsomes. Rat liver microsomal MDA production was increased twofold to fourfold by phenobarbital (CYP2B, 2C, and 3A) or pregnenolone (CYP3A) induction but was unaffected by isoniazid (CYP2E1) induction. Previous investigations also showed that lipid peroxidation in rats in vivo  , measured by TBA-reactive substances or F2-isoprostane production, was enhanced twofold to fourfold by phenobarbital pretreatment but not increased by isoniazid, 19,20 and both phenobarbital and pregnenolone induction resulted in twofold to threefold increases in halothane metabolism by rat liver microsomes, whereas isoniazid had no effect. 9,30,31 These results are consistent with known halothane reduction by rat CYPs 2B1>3A2>2C11 but not 2E1. 32,33 Halothane-dependent MDA formation by human liver microsomes was substantially less than that by induced and even uninduced rat liver microsomes. This species difference may be attributable to the relative absence of CYP2B in human liver, to the fact that the orthologous forms of CYP2A6 in rats exhibits considerable differences in substrate specificity compared with the human form, or to both. Alternatively, or in addition, there may be species differences in the susceptibility of microsomal lipids to peroxidation by the halothane radical, as well as species differences in hepatic lipid composition. 34 Also, MDA formation per se  during lipid peroxidation is related in part to the number and position of double bonds and lipid chain length. 35 Similar species differences with other models of lipid peroxidation have been observed. 36 Further experiments are needed to elucidate the specific mechanism for the apparent species difference. Regardless of the mechanism, human liver microsomes seem to be a better model than the classically used induced rat liver microsomes for halothane-dependent in vitro  lipid peroxidation in humans. The species difference in halothane-dependent lipid peroxidation observed in vitro  is also consistent with the species difference recently observed in vivo  . Halothane caused a fourfold increase in plasma isoprostane concentrations (used as the index of peroxidation) in rats 19 but only a 50% increase in humans. 37 
Michaelis-Menten kinetic analysis of human liver microsomal halothane reduction 7,8 and lipid peroxidation shows biphasic kinetics, indicating participation of at least two enzymes. This was confirmed by identifying human halothane reduction metabolism in vitro  by CYPs 2A6 and 3A4. 7,8 The current investigation suggested a greater role for CYP2A6 than 3A4 in lipid peroxidation, consistent with the former enzyme being the low-Kmisoform; however, CYP3A4-dependent MDA formation was less than expected. Further experiments are necessary to identify definitively the second enzyme participating in halothane-dependent human liver microsomal lipid peroxidation. Nevertheless, CYP2A6 plays a greater role in halothane metabolism and lipid peroxidation. Similarly, additional investigations are required to determine whether halothane-induced lipid peroxidation in humans can be decreased in vivo  . 37 
In summary, this investigation showed that halothane causes lipid peroxidation in human liver microsomes in vitro  , identified a major role for CYP2A6 in human microsomal halothane-dependent lipid peroxidation, and showed that inhibition of CYP2A6-catalyzed halothane reduction decreased lipid peroxidation.
The authors thank Douglas Hankins (University of Washington, Seattle, WA) for excellent technical assistance. They thank Yang Sai, Ph.D. (University of Washington), for the generous gift of anti-CYP2A6 monoclonal antibody and Dr. Kenneth Thummel, Ph.D. (University of Washington), for the rat liver microsomes and numerous helpful discussions.
References
Ray DC, Drummond GB: Halothane hepatitis. Br J Anaesth 1991; 67: 84–99Ray, DC Drummond, GB
Poyer JL, McCay PB, Weddle CC, Downs PE:In vivo  spin-trapping of radicals formed during halothane metabolism. Biochem Pharmacol 1981; 30: 1517–9Poyer, JL McCay, PB Weddle, CC Downs, PE
Plummer JL, Beckwith ALJ, Bastin FN, Adams JF, Cousins MJ, Hall P: Free radical formation in vivo and hepatotoxicity due to anesthesia with halothane. A nesthesiology 1982; 57: 160–6Plummer, JL Beckwith, ALJ Bastin, FN Adams, JF Cousins, MJ Hall, P
Fujii K, Morio M, Kikuchi H, Ishihara S, Okida M, Ficor F: In vivo spin-trap study on anaerobic dehalogenation of halothane. Life Sci 1984; 35: 463–8Fujii, K Morio, M Kikuchi, H Ishihara, S Okida, M Ficor, F
Hughes HM, George IM, Evans JC, Rowlands CC, Powell GM, Curtis CG: The role of the liver in the production of free radicals during halothane anaesthesia in the rat: Quantification of N-tert-butyl-a-(4-nitrophenyl)nitrone (PBN)-trapped adducts in bile from halothane as compared with carbon tetrachloride. Biochem J 1991; 277: 795–800Hughes, HM George, IM Evans, JC Rowlands, CC Powell, GM Curtis, CG
Knecht KT, Degray JA, Mason RP: Free radical metabolism of halothane in vivo  : Radical adducts detected in bile. Mol Pharmacol 1992; 41: 943–9Knecht, KT Degray, JA Mason, RP
Spracklin D, Thummel KE, Kharasch ED: Human reductive halothane metabolism in vitro  is catalyzed by cytochrome P450 2A6 and 3A4. Drug Metab Dispos 1996; 24: 976–83Spracklin, D Thummel, KE Kharasch, ED
Spracklin D, Kharasch ED: Human halothane reduction in vitro  by cytochrome P450 2A6 and 3A4: Identification of low and high Kmisoforms. Drug Metab Dispos 1998; 26: 605–8Spracklin, D Kharasch, ED
Baker MT, Vasquez MT, Chiang C-K: Evidence for the stability and cytochrome P450 specificity of the phenobarbital-induced reductive halothane-cytochrome P450 complex formed in rat hepatic microsomes. Biochem Pharmacol 1991; 41: 1691–9Baker, MT Vasquez, MT Chiang, C-K
Manno M, Ferrara R, Cazzaro S, Rigotti P, Ancona E: Suicidal inactivation of human cytochrome P-450 by carbon tetrachloride and halothane in vitro  . Pharmacol Toxicol 1992; 70: 13–8Manno, M Ferrara, R Cazzaro, S Rigotti, P Ancona, E
Wood CL, Gandolfi AJ, Van Dyke RA: Lipid binding of a halothane metabolite: Relationship to lipid peroxidation in vitro  . Drug Metab Dispos 1976; 4: 305–13Wood, CL Gandolfi, AJ Van Dyke, RA
Trudell JR, Bosterling B, Trevor AJ: Reductive metabolism of halothane by human and rabbit cytochrome P-450: Binding of 1-chloro-2,2,2-trifluoroethyl radical to phospholipids. Mol Pharmacol 1982; 21: 710–7Trudell, JR Bosterling, B Trevor, AJ
de Groot H, Noll T: Halothane hepatotoxicity: Relation between metabolic activation, hypoxia, covalent binding, lipid peroxidation and liver cell damage. Hepatology 1983; 3: 601–6de Groot, H Noll, T
Tomasi A, Billing S, Garner A, Slater TF, Albano E: The metabolism of halothane by hepatocytes: A comparison between free radical spin trapping and lipid peroxidation in relation to cell damage. Chem Biol Interact 1983; 46: 353–68Tomasi, A Billing, S Garner, A Slater, TF Albano, E
de Groot H, Noll T: Halothane-induced lipid peroxidation and glucose-6-phosphatase inactivation in microsomes under hypoxic conditions. A nesthesiology 1985; 62: 44–8de Groot, H Noll, T
Sato N, Fujii K, Yuge O, Morio M: The association of halothane-induced lipid peroxidation with the anaerobic metabolism of halothane: An in vitro study in guinea pig liver microsomes. Hiroshima J Med Sci 1990; 39: 1–6Sato, N Fujii, K Yuge, O Morio, M
Gut J, Huwyler J: Leukotriene B4formation upon halothane-induced lipid peroxidation in liver membrane fractions under low O2concentrations in vitro  . Eur J Biochem 1994; 219: 287–95Gut, J Huwyler, J
Akita S, Morio M, Kawahara M, Takeshita T, Fujii K, Yamamoto M: Halothane-induced liver injury as a consequence of enhanced microsomal lipid peroxidation in guinea pigs. Res Commun Chem Path Pharmacol 1988; 61: 227–43Akita, S Morio, M Kawahara, M Takeshita, T Fujii, K Yamamoto, M
Awad JA, Horn J-L, Roberts J, II Franks JJ: Demonstration of halothane-induced hepatic lipid peroxidation in rats by quantification of F2-isoprostanes. A nesthesiology 1996; 84: 910–6Awad, JA Horn, J-L Roberts, J Franks, JJ
Younes M, Heger B, Wilhelm K-P, Siegers C-P: Enhanced in vivo  -lipid peroxidation associated with halothane hepatotoxicity in rats. Pharmacol Toxicol 1988; 63: 52–6Younes, M Heger, B Wilhelm, K-P Siegers, C-P
Akita S, Kawahara M, Takeshita T, Morio M, Fujii K: Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats. J Appl Toxicol 1989; 9: 9–14Akita, S Kawahara, M Takeshita, T Morio, M Fujii, K
Kharasch ED, Hankins D, Mautz D, Thummel KE: Identification of the enzyme responsible for oxidative halothane metabolism: Implications for prevention of halothane hepatitis. Lancet 1996; 347: 1367–71Kharasch, ED Hankins, D Mautz, D Thummel, KE
Sai Y, Yang TJ, Krausz KW, Gonzalez FJ, Gelboin HV: An inhibitory monoclonal antibody to human cytochrome P450 2A6 defines its role in the metabolism of coumarin, 7-ethoxycoumarin and 4-nitroanisole in human liver. Pharmacogenetics 1999; 9: 229–37Sai, Y Yang, TJ Krausz, KW Gonzalez, FJ Gelboin, HV
Thummel KE, Kharasch ED, Podoll T, Kunze K: Human liver microsomal enflurane defluorination catalyzed by cytochrome P-450 2E1. Drug Metab Dispos 1993; 21: 350–7Thummel, KE Kharasch, ED Podoll, T Kunze, K
Kharasch ED, Thummel K: Human alfentanil metabolism by cytochrome P450 3A3/4: An explanation for the interindividual variability in alfentanil clearance? Anesth Analg 1993; 76: 1033–9Kharasch, ED Thummel, K
Yu LW, Latriano L, Duncan S, Hartwick RA, Witz G: High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malonaldehyde and tran,trans  -muconaldehyde. Anal Biochem 1986; 156: 326Yu, LW Latriano, L Duncan, S Hartwick, RA Witz, G
Hartley DP, Kroll DJ, Petersen DR: Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: Detection of 4-hydroxynonenal- and malondialdehyde-protein adducts. Chem Res Toxicol 1997; 10: 895–905Hartley, DP Kroll, DJ Petersen, DR
Esterbauer H, Schaur RJ, Zollner H: Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991; 11: 81–128Esterbauer, H Schaur, RJ Zollner, H
Draper HH, Hadley M: Malonaldehyde determination as an index of lipid peroxidation. Methods Enzymol 1990; 186: 421–31Draper, HH Hadley, M
Lind RC, Gandolfi AJ, Sipes IG, Brown BR, Waters SJ: Oxygen concentrations required for reductive defluorination of halothane by rat hepatic microsomes. Anesth Analg 1986; 65: 835–9Lind, RC Gandolfi, AJ Sipes, IG Brown, BR Waters, SJ
Jenner MA, Plummer JL, Cousins MJ: Influence of isoniazid, phenobarbital, phenytoin, pregnenolone 16-α-carbonitrile, and β-naphthoflavone on halothane metabolism and hepatotoxicity. Drug Metab Dispos 1990; 18: 819–22Jenner, MA Plummer, JL Cousins, MJ
Van Dyke RA, Baker MT, Jansson I, Schenkman J: Reductive metabolism of halothane by purified cytochrome P-450. Biochem Pharmacol 1988; 37: 2357–61Van Dyke, RA Baker, MT Jansson, I Schenkman, J
Chow T, Imaoka S, Hiroi T, Funae Y: Reductive metabolism of halothane by cytochrome P450 isoforms in rats and humans. Res Commun Mol Pathol Pharmacol 1996; 93: 363–74Chow, T Imaoka, S Hiroi, T Funae, Y
Reiter R, Burk RF: Effect of oxygen tension on the generation of alkanes and malondialdehyde by peroxidizing rat liver microsomes. Biochem Pharmacol 1987; 36: 925–9Reiter, R Burk, RF
Liu J, Yeo HC, Doniger SJ, Ames BN: Assay of aldehydes from lipid peroxidation: Gas chromatography-mass spectrometry compared to thiobarbituric acid. Anal Biochem 1997; 245: 161–6Liu, J Yeo, HC Doniger, SJ Ames, BN
Caraceni P, Gasbarrini A, Nussler A, Di Silvio M, Bartoli F, Borle AB, Van Thiel DH: Human hepatocytes are more resistant than rat hepatocytes to anoxia-reoxygenation injury. Hepatology 1994; 20: 1247–54Caraceni, P Gasbarrini, A Nussler, A Di Silvio, M Bartoli, F Borle, AB Van Thiel, DH
Kharasch ED, Hankins D, Fenstamaker K, Cox K: Human halothane metabolism, lipid peroxidation, and cytochromes P4502A6 and P4503A4. Eur J Clin Pharmacol 2000; 55: 853–9Kharasch, ED Hankins, D Fenstamaker, K Cox, K
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
Fig. 1. Time dependence of malondialdehyde (MDA) production. Human liver microsomes were incubated with NADPH and halothane at saturating concentrations under anaerobic conditions. Each data point is the mean of triplicate incubations.
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Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
Fig. 2. Halothane-dependent malondialdehyde (MDA) formation by human (HLM) and rat (RLM) liver microsomes. Rats were induced with isoniazid (INH), phenobarbital (PB), pregnenolone (PCN), or nothing. The halothane concentration was 1%. Each data point is the mean of triplicate incubations.
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Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
Fig. 3. Dependence of human liver microsomal malondialdehyde (MDA) production on halothane concentration. Reactions were performed as described in Materials and Methods, with varying halothane concentration (0–2 μl, producing a concentration of 0–3.5% (v/v). Data points represent observed values. Lines represent values predicted from parameters determined by nonlinear regression analysis using a two-enzyme model. CTE = chlorotrifluoroethane; CDE = chlorodifluoroethene.
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Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P  < 0.05) by analysis of variance.
Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P 
	< 0.05) by analysis of variance.
Fig. 4. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation in human liver microsomes (n = 8). Methoxsalen (8MOX) and troleandomycin (TAO) were present at 5 and 100 μm, respectively, and halothane concentrations were saturating. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. *Significantly different from control (P  < 0.05) by analysis of variance.
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Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
Fig. 5. Halothane-dependent malondialdehyde (MDA) formation catalyzed by microsomes containing cDNA-expressed enzymes. Control microsomes contained vector only. Halothane concentrations were saturating. Background values obtained without halothane were subtracted from those with halothane. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA.
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Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P  < 0.05) by analysis of variance.
Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P 
	< 0.05) by analysis of variance.
Fig. 6. Effects of P450 inhibitors on halothane (HAL) metabolism and halothane-dependent malondialdehyde (MDA) formation catalyzed by coexpressed human CYP2A6 (0.3 nmol) and P450 reductase. Microsomes were preincubated with NADPH and methoxsalen (8MOX) or anti-CYP2A6 antibody, then flushed with nitrogen and incubated with halothane for 60 min. Background values without halothane were subtracted. Open and hatched bars represent chlorotrifluoroethane (CTE) and chlorodifluoroethene (CDE). Solid bars depict MDA. Results are the mean of triplicate incubations. *Significantly different from control (P  < 0.05) by analysis of variance.
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