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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 281:400 –411, 1997
Vol. 281, No. 1
Printed in U.S.A.
Cytochrome P450 2E1 is the Principal Catalyst of Human
Oxidative Halothane Metabolism in Vitro1
DOUGLAS K. SPRACKLIN, DOUGLAS C. HANKINS, JEANNINE M. FISHER, KENNETH E. THUMMEL and
EVAN D. KHARASCH
Departments of Anesthesiology (D.K.S., D.C.H., E.D.K.), Pharmaceutics (J.M.F., K.E.T.) and Medicinal Chemistry (E.D.K.), University of
Washington, Seattle, Washington
Accepted for publication December 6, 1996
Halothane is one of the more widely used volatile anesthetics in the world. Halothane undergoes extensive biotransformation with approximately 50% of an administered dose
metabolized by reductive and oxidative pathways, both evident during routine anesthesia (Carpenter et al., 1986). Numerous epidemiological, clinical and laboratory investigations have established that halothane metabolism mediates
both a mild and severe form of hepatic toxicity (for recent
Received for publication July 23, 1996.
1
This work was supported by grants from the National Institutes of Health
(R01 GM48712, GM 48349) and by a Pharmaceutical Research and Manufacturers of America Foundation Faculty Development Award to E.D.K.
exceeded that by P450 2A6. Among a panel of human liver
microsomes, there were significant linear correlations between
halothane oxidation and P450 2A6 activity and protein content
at saturating halothane concentrations (2.4 vol%), and a significant correlation between metabolite formation and P450 2E1
activity (but not P450 2A6 activity) at subsaturating concentrations (0.12 vol%). These experiments suggested P450 2A6 and
2E1 as the predominant catalysts at saturating and subsaturating halothane concentrations, respectively. Further kinetic analysis using cDNA-expressed P450 and liver microsomes clearly
demonstrated that P450 2E1 is the high affinity/low capacity
isoform (Km 5 0.030-0.053 vol%) and P450 2A6 is the low
affinity/high capacity isoform (Km 5 0.77-1.2 vol%). Evidence
was also obtained for substrate inhibition of P450 2E1. The in
vitro clearance estimates (Vmax/Km) for microsomal P450 2E1
(4.3-5.7 ml/min/g) were substantially greater than those for
microsomal P450 2A6 (0.12-0.21). These clearances, as well as
rates of apparent halothane oxidation predicted from kinetic
parameters in conjunction with plasma halothane concentrations measured during clinical anesthesia in humans, demonstrated that both P450 2E1 and P450 2A6 participate in human
halothane metabolism, and that P450 2E1 is the predominant
catalytic isoform.
reviews see Cousins et al., 1989; Ray and Drummond, 1991;
Gut et al., 1995). Mild hepatic reactions occur in up to 20% of
halothane anesthetics (Ray and Drummond, 1991) and are
evidenced clinically by elevated postoperative liver enzymes
(AST, ALT, GST) (de Groot and Noll, 1983; Akita et al., 1989;
Sato et al., 1990). Current theories suggest that this mild,
subclinical hepatotoxicity is attributable to anaerobic reductive halothane metabolism that results in free radical generation and lipid peroxidation (de Groot and Noll, 1983; Sato et
al., 1990; Awad et al., 1996). The severe form of hepatic
toxicity is a rare but often fatal fulminant hepatic necrosis,
commonly known as “halothane hepatitis” (Ray and Drum-
ABBREVIATIONS: cDNA, complementary deoxyribonucleic acid; GC/MS, gas chromatography-mass spectrometry; HLM, human liver microsomes; HPLC, high performance liquid chromatography; IOD, integrated optical density; NADPH, b-nicotinamide adenine dinucleotide, reduced
form; P450, cytochrome P450; TFA, trifluoroacetic acid; vol%, headspace concentration (v/v); AST, aspartate aminotransferase; ALT, alanine
aminotransferase; GST, glutathione S-transferase.
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ABSTRACT
The volatile anesthetic halothane undergoes substantial biotransformation generating metabolites that mediate hepatotoxicity. Aerobically, halothane undergoes cytochrome P450-catalyzed oxidation to trifluoroacetic acid (TFA), bromide and a
reactive intermediate that can acetylate liver proteins. These
protein neo-antigens stimulate an immune reaction that mediates severe hepatic necrosis (“halothane hepatitis”). This investigation identified the human P450 isoform(s) that catalyze oxidative halothane metabolism. Halothane oxidation by human
liver microsomes was assessed by TFA and bromide formation.
Eadie-Hofstee plots of TFA and bromide formation were both
nonlinear, suggesting the participation of multiple P450s. Microsomal TFA and bromide formation were inhibited 45 to 66%
and 21 to 26%, respectively, by the P450 2A6 inhibitors 8-methoxypsoralen and coumarin, 84 to 90% by the P450 2E1
inhibitor 4-methylpyrazole and 55% by diethyldithiocarbamate,
an inhibitor of both P450 2A6 and 2E1. Selective inhibitors of
P450s 1A, 2B6, 2C9/10, 2D6 and 3A4 did not affect halothane
oxidation. At saturating halothane concentrations (2.4 vol%)
only cDNA-expressed P450 2A6 and 2B6 catalyzed significant
rates of TFA and bromide formation, and P450 2E1 catalyzed
comparatively minimal oxidation. Conversely, at subsaturating
halothane concentrations (0.30 vol%), metabolism by P450 2E1
1997
Halothane Oxidation in Vitro by CYP 2E1
The mechanism and consequences of oxidative halothane
metabolism have been well studied; nonetheless, clinicians
are still unable to identify which patients will develop antiTFA proteins, or which patients will develop halothane hepatitis. However, it is clear that P450-catalyzed oxidative
halothane metabolism is the critical initiating event, and
inhibition of P450-catalyzed halothane oxidation is a promising potential clinical strategy to prevent halothane hepatitis (Kharasch et al., 1996). Nevertheless, the exact identity of
the P450 isoform(s) that catalyze oxidative metabolism of
halothane in humans is unknown. Previous investigations
have suggested a role for P450 2E1 with possible involvement
of P450 2A6 (Brown et al., 1995; Kharasch et al., 1996).
Disulfiram, an effective P450 2E1 inhibitor, significantly diminished halothane oxidation in humans (Kharasch et al.,
1996), however disulfiram may inhibit P450 2A6 as well as
P450 2E1. Therefore, the purpose of this investigation was to
clarify the human liver P450 isoform(s) responsible for oxidative halothane metabolism.
Materials and Methods
Halothane was purchased from Halocarbon Laboratories (N. Augusta, SC). Furafylline and sulfaphenazole were generous gifts from
Drs. Kent Kunze and William F. Trager, respectively (University of
Washington, Seattle, WA). Sodium trifluoroacetate, sodium bromide
and chlorodifluoroacetic acid were purchased from Fluka Chemical
Co. (Ronkonkoma, NY) and were of the highest purity available.
Microsomes containing individual cDNA-expressed cytochrome P450
isoforms were purchased from Gentest (Woburn, MA). Unless specified, all other reagents were purchased from Sigma Chemical Co.
(St. Louis, MO) and were of the highest purity available. All buffers
and reagents were prepared with high-purity ($18.2 MVzcm) water
(Milli-Q, Millipore, Bedford, MA).
Microsomes were prepared from human livers as described previously (Kharasch and Thummel, 1993). Microsomal protein concentrations were determined by the method of Lowry et al. (1951). Total
microsomal cytochrome P450 content was determined from the reduced minus oxidized carbon monoxide difference spectrum (Estabrook et al., 1972).
Halothane metabolism was determined in scintillation vials (24.4
ml) containing human liver microsomes (2 mg/ml), halothane and
NADPH (2 mM) in a final volume of 1.0 ml potassium phosphate
buffer (50 mM, pH 7.4). The reaction was initiated by the addition of
halothane that was added either undiluted (2 ml, producing a headspace concentration of 2.4 vol% at 37°C) for experiments at saturating substrate concentrations, or diluted in methanol (final aqueous
methanol concentration 0.2%) for experiments at subsaturating sub-
Fig. 1. Mechanism of TFA and bromide formation from P450-catalyzed halothane oxidation.
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mond, 1991). Clinically, halothane hepatitis occurs in approximately 1:6,000 to 35,000 halothane anesthetics (Ray
and Drummond, 1991) but is fatal in 75% of these cases
(Cousins et al., 1989). It is manifested by fever, jaundice and
grossly elevated serum transaminase concentrations. Pathologically, it is characterized by massive centrilobular necrosis. The cytotoxicity associated with halothane hepatitis is
consistent with an immunological reaction to trifluoroacetylated (TFA) liver protein neo-antigens. These TFA-antigens
derive from acylation of native liver proteins that, in susceptible individuals, serve as neo-antigens that stimulate the
formation of anti-TFA-protein antibodies. On reexposure to
halothane or certain other volatile anesthetics, these antibodies initiate an immunological cascade that ultimately results in “halothane hepatitis.” TFA-protein formation results
from oxidative halothane metabolism. Investigations suggest
that the amount of antigen formation, and thus, the rate and
extent of halothane metabolism may be a critical regulatory
factor in the onset of halothane hepatitis (Christ et al., 1988a,
1988b; Pohl et al., 1989; Kenna et al., 1990). For example, in
immunoblot analysis, the sera from six patients with halothane hepatitis cross-reacted with neo-antigens of halothaneand enflurane-treated rats but not with isoflurane-treated
animals (Christ et al., 1988a). The relative amount of crossreaction (halothane.enflurane.isoflurane) correlates with
the extent of metabolism of the three anesthetics. Therefore,
the seminal event in immune-based halothane hepatitis is
P450-catalyzed oxidative halothane metabolism (Kenna et
al., 1987).
The anaerobic reductive pathway of halothane metabolism
has been well-described (Ray and Drummond, 1991; Spracklin et al., 1996). The oxidative pathway is shown in figure 1.
Under sufficient oxygen tensions, halothane undergoes P450catalyzed oxidation to trifluoroacetyl chloride, with concomitant loss of bromine. This unstable intermediate undergoes
further reactions, including: 1) hydrolysis to yield the nontoxic metabolite TFA; 2) binding to phospholipids (Muller
and Srier, 1982) and 3) acetylation of tissue proteins to form
the TFA-protein adducts (Ray and Drummond, 1991). To
date, a number of the TFA-modified proteins have been identified. These include protein disulfide isomerase, microsomal
carboxylesterase, calreticulin, stress protein ERp72 and
ERp99/endoplasmin/GRP 94 in microsomes (Gut et al., 1995),
and glutathione-S-transferase in cytosol (Brown and Gandolfi, 1994). However, the precise antigens that cause halothane hepatitis are unknown.
401
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Spracklin et al.
mM), troleandomycin (P450 3A4, 100 mM), ketoconazole (P450 3A4,
90 nM), n-octylamine (P450, 3 mM). All inhibitors were added in
potassium phosphate buffer except 7,8-benzoflavone, 8-methoxypsoralen, (S)-mephenytoin, troleandomycin, ketoconazole and n-octylamine which were diluted in methanol (final methanol concentration 0.2%). Substrate and inhibitor concentrations were chosen to
theoretically suppress more than 80% of isoform activity based on
published Ki values. In experiments using the competitive inhibitors
7,8-benzoflavone, coumarin, 8-methoxypsoralen, sulfaphenazole, (S)mephenytoin, quinidine, 4-methylpyrazole and ketoconazole, the inhibitor was added followed by a solution of halothane [2 ml of 10%
(v/v) halothane/methanol, equivalent to 0.2 ml halothane or 0.24
vol%, final methanol concentration 0.2%], and the reaction was initiated by the addition of the NADPH. Reactions were carried out at
37°C for 60 min and then quenched with trichloroacetic acid as
described above. Incubations containing the mechanism-based inhibitors furafylline, orphenadrine, diethyldithiocarbamate, troleandomycin and n-octylamine were first preincubated at 37°C for 15 min
with NADPH under aerobic conditions, after which time halothane
was added (2 ml; 2.4 vol%). Reactions were carried out at 37°C for 60
min and then quenched as described previously. Kinetic and inhibitor experiments were performed in a single liver that was competent
in all the major drug metabolizing P450 isoforms.
Microsomal catalytic activities of P450s 1A2, 2A6, 2C9, 2D6, 2E1
and 3A4 were measured by (R)-warfarin 6-hydroxylation, coumarin
7-hydroxylation, (S)-warfarin 7-hydroxylation, metoprolol a-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 19-hydroxylation, respectively (Miles et al., 1990; Kharasch and Thummel,
1993; Kunze et al., 1996; Thummel et al., 1996). Microsomal P450
isoform content was determined by Western blot analysis as described previously (Kharasch and Thummel, 1993; Thummel et al.,
1993). In addition to P450 2E1, anti-P450 2E1 antibody also detected
a separately migrating lower molecular weight protein recently identified as P450 2A6 (K. E. Thummel, unpublished results). This antibody was used to quantitate P450 2A6 content using cDNA-expressed P450 2A6 as the standard.
All results are expressed as the mean 6 S.D.of three experiments.
Typical coefficients of variation in a set of triplicate measurements
were # 10%. Statistical analyses were carried out with SigmaStat
(version 1.02) and nonlinear regression analyses were carried out
with SigmaPlot (version 5.01) (Jandel Scientific, San Rafael, CA).
A clinical investigation was conducted to determine blood halothane concentrations during anesthesia. The investigation was approved by the institutional Human Subjects Committee and all patients provided written informed consent. Twenty normal weight
males, without hepatic or renal disease, ethanol abuse or current use
of medications known to alter hepatic drug metabolism who were
undergoing anesthesia for elective surgery that did not significantly
alter hepatic blood flow were studied. All patients were anesthetized
with 1.0% end-tidal halothane (determined by an infrared detector;
Capnomax, Datex Medical, Tewksbury, MA) for 3 hr. The inspired
halothane concentration was adjusted to maintain 1% end-tidal halothane concentration. Venous blood samples for determination of
blood halothane concentration were obtained before anesthesia and
at hourly intervals thereafter. Whole blood halothane concentrations
were determined by gas chromatography. Details have been published previously (Kharasch et al., 1996).
Results
Details of oxidative halothane metabolism by human liver
microsomes have not been reported previously. Therefore,
initial experiments characterized the NADPH-, time- and
protein-dependence of the reaction. Ion HPLC chromatograms showing trifluoroacetate (RT 5 6.1 min), and bromide
(RT 5 7.8 min) produced in a 60-min incubation of halothane
and human liver microsomes in the presence (fig. 2A) or
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strate concentrations. Preliminary experiments showed that this
methanol concentration did not affect the rate of halothane oxidation. Incubations were routinely carried out for 60 min at 37°C
unless indicated otherwise. Reactions were quenched by quantitatively transferring the reaction mixture to a 2-ml polypropylene vial
containing trichloroacetic acid (10 ml of a 6 N aqueous solution; 60
mmol) and mixing thoroughly for 10 sec. The internal standard
chlorodifluoroacetic acid (25 ml of a 1 mM aqueous solution; 25 nmol)
was then added and the solution vortexed for another 10 sec. The
vials were placed on ice for 15 min and then centrifuged at 16,000 3
g for 30 min. The resulting supernatant was filtered through a 0.2
mm DIMEX syringe filter (Millipore) directly into an autosampler
vial. Experiments using cDNA-expressed protein were carried out
similarly using typical protein concentrations of 1 mg/ml and incubation times of 60 or 90 min.
Metabolites and internal standards were analyzed by ion HPLC
with conductivity detection. Analyses were performed using a DX300 HPLC-IC system (Dionex Corp. Sunnyvale, CA), consisting of an
AGP gradient pump, LCM-3 chromatography module, CDM-3 conductivity detector, ASM-3 autosampler, IonPac AS11 analytical column and AG11 guard column and an ASRS-1 anion self-regenerating suppressor operating in the autosuppression recycle mode.
Dionex AI-450 software was used to control the hardware, detector
signal acquisition and chromatographic peak integration. Injections
of 50 ml were made via an autosampler utilizing 0.5-ml polypropylene vials equipped with 20-mm filter caps (PolyVial, Dionex
Corp.). The sodium hydroxide concentration was initially 0.75 mM
for 5 min, linearly decreased to 0.5 mM at 0.1 mM/min, linearly
increased to 3.0 mM over 10.5 min, increased to 80 mM at 25
mM/min and held at 80 mM for 5 min. The concentration was then
linearly decreased to 0.75 mM at 14.5 mM/min, and the column
allowed to reequilibrate at this concentration for 7 min. The eluant
flow rate was 2.0 ml/min and the detector sampling rate was 0.20 sec.
Under these conditions, the retention times for trifluoroacetate,
bromide and chlorodifluoroacetate were 6.1, 7.8 and 11.2 min, respectively. Trichloroacetic acid did not interfere with analyte quantitation. Standards were prepared by adding aqueous TFA (0.25-40
nmol), sodium bromide (0.25-40 nmol) and the internal standard to
microsomal mixtures prepared similarly to the incubation mixtures
except for the omission of halothane. Standard curves for TFA and
bromide were constructed from peak area ratios of metabolite to
internal standard. Standard curves were linear over the concentration range 0 to 40 mM (r2 5 0.99). The lower limit of quantitation (0.1
mM) was defined as a signal to noise ratio of 3:1. Quantitation of
metabolic TFA and bromide was accomplished by comparing sample
peak area ratios to those of the standard curve.
For kinetic experiments measuring TFA and bromide formation as
a function of substrate concentration, headspace halothane concentrations were measured as previously described (Spracklin et al.,
1996). Briefly, incubation mixtures were prepared identically to
those used to assess TFA and bromide formation except that NADPH
was omitted and the vial was sealed with a rubber septum instead of
a screw cap. After 10 min at 37°C, an aliquot of the headspace gas
from the reaction vial was transferred to another sealed vial. Headspace GC/MS analysis was used to quantitate halothane. Microsomal
halothane concentrations were measured by a previously reported
method used to measure whole blood halothane concentrations
(Kharasch et al., 1996). After 10 min at 37°C, an aliquot of the
reaction mixture was added to heptane. After centrifugation, an
aliquot of the heptane was analyzed by gas chromatography to quantitate halothane.
Experiments with isoform-selective P450 inhibitors were conducted at the following final concentrations: 7,8-benzoflavone (P450
1A, 16 mM), furafylline (P450 1A2, 20 mM), 8-methoxypsoralen (P450
2A6, 28 mM), coumarin (P450 2A6, 36 mM), orphenadrine (P450 2B6,
5 mM), sulfaphenazole (P450 2C9/10, 3.6 mM), (S)-mephenytoin
(P450 2C19, 100 mM), quinidine (P450 2D6, 45 nM), 4-methylpyrazole (P450 2E1, 540 mM), diethyldithiocarbamate (P450 2E1, 100
Vol. 281
1997
Halothane Oxidation in Vitro by CYP 2E1
403
absence (fig. 2B) of NADPH illustrate that no TFA or bromide formation was observed in the absence of NADPH. TFA
and bromide formation increased linearly with time for 60
min (fig. 2C). Formation of both metabolites was also linear
with protein concentration up to 5 mg/ml (fig. 2D). Adequate
sensitivity was obtained at 2.0 mg/ml which was used for
subsequent incubations.
Anesthetic doses delivered during surgery are measured by
the concentration (in volumes percent) in inspired gas. Tissue anesthetic concentrations are most closely reflected by
those in expired alveolar gas (end-tidal concentration), but
actual hepatic concentrations corresponding to those in respiratory gas are unknown. Therefore halothane was added
to microsomes in sealed vials and the concentration of halothane in both the headspace gas and the microsomal suspensions was measured (fig. 3). There was a linear relationship
between halothane concentrations (vol%) in the headspace
gas and the amount of halothane added up to 3 ml. Similarly,
Fig. 3. Relationship between halothane addition and measured halothane concentrations. Halothane concentrations were measured in the
headspace (triangles) and in the microsomal suspension (circles) after
addition of halothane to sealed vials containing human liver microsomes (2 mg/ml) and equilibrated at 37°C for 10 min. The inset shows
the relationship between halothane concentrations measured in the
headspace and the microsomal suspension (r 5 0.99; P , .001).
there was a linear relationship between halothane concentrations (mM) in the microsomal suspension and the amount
of halothane added, for halothane additions up to 1 ml. Aqueous solubility was limited at higher halothane concentrations. The relationship between headspace and solution concentrations is shown in the inset to figure 3. For example, a
headspace concentration of 0.6 vol% corresponded to a suspension concentration of 400 mM, resulting from 0.5 ml of
halothane. Calculation of a microsome:gas partition coefficient (l) from the measured microsomal halothane concentrations yielded a value of 2.2. This is in good agreement with
the blood:gas partition coefficient (l 5 2.3) reported for halothane (Eger, 1985). Thus the blood:gas partition coefficient
appears to be a reasonable approximation for the microsome:
gas partition coefficient.
The substrate concentration dependence of halothane oxidation in human liver microsomes was examined over the
range of 0 to 3 ml added, producing a headspace concentration
of 0 to 3.6 vol% (fig. 4). Eadie-Hofstee plots for both TFA and
bromide formation were nonlinear, suggesting the participation of multiple enzymes in halothane oxidation. Experimental data were fit to a two-enzyme Michaelis-Menten model by
nonlinear regression analysis. The parameters obtained are
summarized in table 1. For TFA formation, the apparent
parameters obtained were Vmax(1) 5130 pmol/min/mg protein, Km(1) 5 0.045 vol% (30 mM); Vmax(2) 5 94 pmol/min/mg
protein, Km(2) 5 1.2 vol% (800 mM). For bromide, the apparent parameters were Vmax(1) 5 170 pmol/min/mg protein,
Km(1) 5 0.045 vol% (30 mM); Vmax(2) 5 130 pmol/min/mg
protein, Km(2) 5 0.94 vol% (630 mM).
To identify the P450 isoforms responsible for oxidative
halothane metabolism, the effect of isoform-selective P450
inhibitors on rates of TFA and bromide formation were determined (fig. 5). The P450 2A6-selective inhibitor 8-methoxypsoralen (Maenpaa et al., 1994), used as a competitive
inhibitor, decreased the rate of TFA and bromide formation
by 45 and 66%, respectively. The P450 2A6 substrate couma-
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Fig. 2. NADPH-, time- and protein-dependence of TFA and bromide formation
from P450-catalyzed halothane oxidation. Ion HPLC chromatograms showing
TFA and bromide formation in a 60 min
incubation of halothane with human liver
microsomes as described in “Methods.”
Incubations were carried out in a single
liver that was competent in all the major
drug metabolizing P450 isoforms. Incubations were performed in the (A) presence or (B) absence of NADPH. Also
shown are rates of TFA and bromide
formation as a function of (C) time (2
mg/ml protein) and (D) protein concentration (incubation time was 60 min).
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Spracklin et al.
Vol. 281
Fig. 4. Substrate dependence of TFA
and bromide formation from halothane
oxidation. Reactions were carried out as
described in “Methods” with varying
halothane concentrations (0-3 ml, producing a gas phase concentration of
0-3.6 vol%). Incubations were carried
out in a single liver that was competent
in all the major drug metabolizing P450
isoforms. Symbols denote observed metabolite formation. Lines represent rates
predicted using Michaelis-Menten kinetic parameters derived from nonlinear
regression analysis of the experimental
data (TFA, r 5 0.99; Br, r 5 0.99, P ,
.05). The inset shows Eadie-Hofstee
plots for TFA and bromide formation as a
function of halothane concentration
(TFA, r 5 0.94; Br, r 5 0.99, P , .05).
TFA
Enzyme
HLMb
cDNAc
HLM
cDNA
High affinity
2E1
Low affinity
2A6
Vmaxa
Km
(vol%)
Km
(mM)
130
0.30
94
0.59
0.045
0.053
1.2
1.2
30
35
800
800
Br
Ki
(vol%)
Vmax/Km
(ml/min/g)
4.3
0.36
0.12
Vmax
Km
(vol%)
Km
(mM)
170
0.52
130
0.87
0.045
0.030
0.94
0.77
30
20
630
510
Ki
(vol%)
Vmax/Km
(ml/min/g)
5.7
1.3
0.21
a
Rates of halothane oxidation by human liver microsomes expressed as pmol/min/mg protein; Rates of halothane oxidation by cDNA-expressed P450 expressed
as pmol/min/pmol P450.
b
Human liver microsomes.
c
cDNA-expressed P450.
rin decreased the rate of TFA and bromide formation by 26
and 21%, respectively. Rates of TFA and bromide formation
were both inhibited 84 to 90% by the P450 2E1-selective
inhibitor 4-methylpyrazole. Additionally, TFA and bromide
formation were each inhibited 55% by diethyldithiocarbamate, an inhibitor of P450 2E1 and 2A6. In contrast, the P450
1A-, 2B6-, 2C9/10-, 2C19-, 2D6-, 3A4-selective inhibitors 7,8benzoflavone, furafylline, orphenadrine, sulfaphenazole, (S)mephenytoin, quinidine, troleandomycin and ketoconazole
had no significant effect on rates of TFA or bromide formation. Additionally, the nonselective P450 inhibitor n-octylamine (Jefcoate et al., 1969) almost completely inhibited
TFA and bromide formation. These results suggested the
involvement of both P450 2E1 and 2A6 in halothane oxidation, and did not provide evidence for the participation of
other P450 isoforms.
To identify further the isoforms responsible for oxidative
halothane metabolism, the rates of TFA and bromide formation by cDNA-expressed P450 isoforms were examined (fig.
6). At saturating halothane concentrations (2 ml 5 2.4 vol%),
P450 2A6 (0.45 pmol/min/pmol P450) and P450 2B6 (0.47
pmol/min/pmol P450) catalyzed significant and comparable
rates of TFA formation. P450 1A2 (0.050 pmol/min/pmol
P450), P450 2C9 (,0.001 pmol/min/pmol P450), P450 2D6
(0.036 pmol/min/pmol P450), P450 2E1 (0.084 pmol/min/pmol
P450) and P450 3A4 (,0.001 pmol/min/pmol P450) catalyzed
much lower amounts of TFA formation. A similar pattern for
bromide formation was observed. At saturating halothane
concentrations, P450 2A6 (0.34 pmol/min/pmol P450) and
P450 2B6 (0.37 pmol/min/pmol P450) catalyzed significant
and comparable rates of bromide formation. P450 1A2 (0.028
pmol/min/pmol P450), P450 2C9 (,0.001 pmol/min/pmol
P450), P450 2D6 (0.0024 pmol/min/pmol P450), P450 2E1
(0.032 pmol/min/pmol P450) and P450 3A4 (0.010 pmol/min/
pmol P450) catalyzed much less bromide formation. However, different results were obtained at lower, subsaturating
halothane concentrations (0.25 ml 5 0.30 vol%). At these
lower substrate concentrations (fig. 6, inset), the rates of TFA
formation catalyzed by P450 2E1 (0.19 pmol/min/pmol P450)
markedly exceeded those catalyzed by 2A6 (0.054 pmol/min/
pmol P450). Similarly, the rates of bromide formation catalyzed by P450 2E1 (0.28 pmol/min/pmol P450) exceeded those
catalyzed by 2A6 (0.14 pmol/min/pmol P450). These results
suggested a predominant role in halothane oxidation for
P450 2E1 at subsaturating halothane concentrations, and
P450 2A6 at saturating halothane concentrations.
Halothane metabolism by microsomes from a panel of several human livers was examined to assess the relationship
between metabolite formation and P450 content. This relationship was examined at both high (2 ml 5 2.4 vol%) and low
(0.10 ml 5 0.12 vol%) halothane concentrations. At saturating
halothane concentrations (2.4 vol%), among a panel of 20
livers, there was a highly significant linear correlation between coumarin hydroxylase (P450 2A6) activity and both
TFA (r 5 0.90; P , .001) (fig. 7A) and bromide formation (r 5
0.85; P , .001) (fig. 7D). Similarly, there was a significant
linear correlation between P450 2A6 protein content and
both TFA (r 5 0.71; P , .001) and bromide (r 5 0.63; P ,
.003) formation (fig. 7, B and E). Conversely, at these halothane concentrations, the correlation between chlorzoxazone
hydroxylase (P450 2E1) activity and either TFA or bromide
formation was less significant (P , .11 and .05, respectively)
and exhibited considerably greater scatter (fig. 7, C and F).
As a further refinement of the analysis, multiple linear re-
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TABLE 1
Kinetic Parameters for TFA and bromide formation from P450-catalyzed halothane oxidation by HLM and cDNA-expressed P450
1997
Halothane Oxidation in Vitro by CYP 2E1
405
Fig. 5. Effects of isoform-selective inhibitors and competitive substrates on TFA and bromide formation. Rates of TFA (solid) and bromide (cross-hatch) formation are expressed as a percentage of control
mixtures lacking the inhibitor. Reactions were carried out as described
in “Methods.” Incubations were carried out in a single liver that was
competent in all the major drug metabolizing P450 isoforms. Final
concentrations of each inhibitor were: 7,8-benzoflavone, 16 mM;
furafylline, 20 mM; 8-methoxypsoralen, 28 mM; coumarin, 36 mM; orphenadrine, 5 mM; sulfaphenazole, 3.6 mM; (S)-mephenytoin, 100 mM;
quinidine, 45 nM; 4-methylpyrazole, 540 mM; diethyldithiocarbamate
(DDC), 100 mM; troleandomycin, 100 mM; ketoconazole, 90 nm; noctylamine, 3 mM.
gression analysis for P450 2A6 and 2E1 activity yielded
slightly improved correlation coefficients compared with linear regression analysis for P450 2A6 alone. The correlation
coefficients (r) obtained using multiple linear regression were
0.93 (P , .001) for TFA formation and 0.91 (P , .001) for
bromide formation. There was no significant correlation between either TFA or bromide formation and P450 1A2, 2C9
or 2D6 activities. There was a significant correlation between
TFA and bromide formation and P450 3A4 activity. However,
this was due to coexpression of P450s 2A6 and 3A4 activities
(r 5 0.59; P , .006). The relationship between metabolite
formation and P450 content was also examined at subsaturating halothane concentrations (fig. 8). For reactions at this
lower halothane concentration (0.12 vol%), the relationship
between metabolite formation and P450 2A6 and 2E1 activities was opposite to that observed at saturating halothane
concentrations. Among a panel of 15 livers, there was a
positive correlation between chlorzoxazone hydroxylase
(P450 2E1) activity and both TFA (r 5 0.48; P , .08) and
bromide (r 5 0.63; P , .02) formation (fig. 8, B and D).
Conversely, there was no significant correlation between coumarin hydroxylase (P450 2A6) activity and either TFA or
bromide formation (fig. 8, A and C). There was not a signif-
icant correlation between TFA or bromide formation and
P450 1A2, 2C9, 2D6 or 3A4 activities. These results further
supported the predominant catalytic participation of P450
2E1 at low, subsaturating halothane concentrations, and
P450 2A6 at higher halothane concentrations.
To define further the roles of P450 2A6 and 2E1 in oxidative halothane metabolism, the substrate dependence of halothane oxidation was examined using cDNA-expressed P450s
2A6 and 2E1 (figs. 9 and 10). Eadie-Hofstee plots for TFA and
bromide formation by cDNA-expressed P450 2A6 were linear,
and the data were fit to a one enzyme Michaelis-Menten
model using nonlinear regression analysis (fig. 9). Kinetic
parameters are summarized in table 1. For TFA formation,
Vmax was 0.59 pmol/min/pmol P450 and Km was 1.2 vol%
(800 mM). For bromide formation, Vmax was 0.87 pmol/min/
pmol P450 and Km was 0.77 vol% (510 mM). By comparison,
the oxidation of halothane by P450 2E1 was more complex
(fig. 10). At very low halothane concentrations (0.012-0.25
vol%), TFA and bromide formation exhibited single-enzyme
Michaelis-Menten kinetics and linear Eadie-Hofstee plots.
However, at higher halothane concentrations TFA and bromide formation decreased, consistent with substrate inhibition. Therefore, experimental data for metabolite formation
by cDNA-expressed P450 2E1 were fit by nonlinear regression analysis to a one enzyme Michaelis-Menten model that
incorporated a term for substrate inhibition (Andersen et al.,
1987). The parameters obtained are summarized in table 1.
For TFA, the apparent parameters were: Vmax 5 0.30 pmol/
min/pmol P450; Km 5 0.053 vol% (35 mM); Ki 5 0.36 vol%
(240 mM). For Br, the apparent parameters were: Vmax 5
0.52 pmol/min/pmol P450; Km 5 0.030 vol% (20 mM); Ki 5 1.3
vol% (870 mM). These results demonstrated that among the
P450 isoforms that catalyze oxidative halothane metabolism,
P450 2E1 is the high affinity/low capacity P450 isoform and
P450 2A6 is the low affinity/high capacity isoform.
The relationship between rates of TFA and bromide formation by microsomes from each liver within a panel of human
livers was examined (fig. 11). The results are consistent with
the known mechanism of halothane oxidation in which TFA
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Fig. 6. Rates of TFA and bromide formation from halothane oxidation
catalyzed by cDNA-expressed P450. Shown are rates of TFA (solid) and
bromide (cross-hatch) formation from incubations of halothane at saturating concentrations (2 ml 5 2.4 vol%) and cDNA-expressed P450, as
described in “Methods.” The inset shows rates of TFA and bromide
formation catalyzed by cDNA-expressed P450s 2A6 and 2E1 at subsaturating halothane concentrations (0.25 ml 5 0.30 vol%).
406
Spracklin et al.
Vol. 281
and bromide are formed in equimolar amounts. At both saturating (2 ml 5 2.4 vol%) and subsaturating (0.10 ml 5 0.12
vol%) halothane concentrations, there was a highly significant linear correlation between TFA and bromide formation
rates among the liver microsomes examined.
Discussion
The results of this investigation demonstrate that human
liver microsomal oxidative halothane metabolism is catalyzed by multiple P450 isoforms. Near complete inhibition of
TFA and bromide formation by the nonselective P450 inhibitor n-octylamine suggests that oxidative halothane metabolism is catalyzed exclusively by P450. Eadie-Hofstee plots of
human liver microsomal TFA and bromide formation were
both nonlinear, consistent with a reaction catalyzed by two or
more P450 isoforms. TFA and bromide formation were each
decreased by inhibitors of two different isoforms. Experiments using cDNA-expressed P450 proteins showed that
multiple P450 isoforms catalyzed oxidative halothane metabolism. Finally, in a panel of human livers, there was a correlation of TFA and bromide formation with one isoform at
saturating halothane concentrations and a correlation with a
different isoform at subsaturating halothane concentrations.
Several lines of investigation were used to determine that
P450s 2A6 and 2E1 are the isoforms which catalyze oxidative
halothane metabolism in human liver microsomes. These
included: 1) Effects of isoform-selective inhibitors and competitive substrates on rates of TFA and bromide formation, 2)
rates of TFA and bromide formation by cDNA-expressed
P450 isoforms and 3) correlation of TFA and bromide formation with isoform activity in a panel of human liver microsomes. Moreover, a similar experimental strategy using different halothane concentrations suggested that P450 2E1 is
the predominant catalyst at subsaturating halothane concentrations and 2A6 is the predominant catalyst at saturating
halothane concentrations. Inhibitors of P450 2A6 (8-methoxypsoralen and coumarin) and P450 2E1 (4-methylpyrazole and DDC) significantly decreased the rates of both TFA
and bromide formation. At saturating halothane concentrations, high rates of TFA and bromide formation were catalyzed by cDNA-expressed P450 2A6. However, at subsaturating halothane concentrations, the rate of metabolite
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Fig. 7. Correlation of TFA and bromide formation with P450 2A6 and 2E1 activities and protein contents at saturating halothane concentrations.
P450 2A6 and 2E1 activities were measured by coumarin 7-hydroxylation and chlorzoxazone 6-hydroxylation, respectively. The correlation
coefficients and statistical significance are indicated (n 5 20). For reactions at saturating halothane concentrations (2 ml 5 2.4 vol%), the upper
panels show correlation of TFA formation with: A, P450 2A6 activity; B, P450 2A6 protein content; C, P450 2E1 activity. At the same halothane
concentrations, the lower panels show correlation of bromide formation with: D, P450 2A6 activity; E, P450 2A6 protein content; F, P450 2E1
activity.
1997
Halothane Oxidation in Vitro by CYP 2E1
407
Fig. 9. TFA and bromide formation from halothane
oxidation catalyzed by cDNA-expressed P450 2A6.
Reactions were carried out as described in “Methods”
with varying halothane concentrations (0-4 ml, 0-4.9
vol%). Symbols denote observed metabolite formation. Lines represent rates predicted using MichaelisMenten kinetic parameters derived from nonlinear regression analysis of the experimental data (TFA, r 5
0.99; Br, r 5 0.98, P , .05). The inset shows EadieHofstee plots for TFA and bromide formation as a
function of halothane concentration (TFA, r 5 0.99; Br,
r 5 0.91, P , .05).
formation by P450 2E1 exceeded that of 2A6. At saturating
halothane concentrations, cDNA-expressed P450 2B6 catalyzed rates of metabolite formation comparable to that of
2A6. However, the P450 2B6-selective inhibitor orphenadrine had no effect on halothane oxidation, and P450 2B6 is
minimally expressed in human livers (Yamano et al., 1989;
Mimura et al., 1993; Shimada et al., 1994). The cumulative
evidence suggests that P450 2B6 does not play a significant
role in oxidative halothane metabolism in human liver microsomes. Finally, in a panel of human liver microsomes, for
reactions at saturating halothane concentrations, there was
a significant linear correlation between both TFA and bromide formation and P450 2A6 activity and content. The correlation was improved by a multiple linear regression anal-
ysis using both P450 2A6 and 2E1 activities. At
subsaturating halothane concentrations, there was a significant linear correlation between both TFA and bromide formation and P450 2E1 activity. A previous investigation suggested that P450 2E1 is not the principal catalyst of oxidative
metabolism of halothane by human liver microsomes (Brown
et al., 1995). The present data do not support this conclusion
and clearly demonstrate that both P450s 2A6 and 2E1 are
the isoforms that catalyze oxidative halothane metabolism in
human liver microsomes. These results suggest further that
at low halothane concentrations, P450 2E1 is the predominant catalyst and at high concentrations, P450 2A6 is the
predominant catalyst of halothane oxidation.
Kinetic analysis of halothane oxidation by human liver
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Fig. 8. Correlation of TFA and bromide formation
with P450 2A6 and 2E1 activities and protein contents at subsaturating halothane concentrations.
The correlation coefficients and statistical significance are indicated (n 5 15). For reactions at subsaturating halothane concentrations (0.10 ml 5 .12
vol%), the upper panels show correlation of TFA
formation with: A, P450 2A6 activity; B, P450 2E1
activity. At the same halothane concentrations, the
lower panels show correlation of bromide formation with: C, P450 2A6 activity; D, P450 2E1 activity.
408
Spracklin et al.
Vol. 281
Fig. 10. TFA and bromide formation from halothane
oxidation catalyzed by cDNA-expressed P450 2E1.
Reactions were carried out as described in “Methods”
with varying halothane concentrations (0-2 ml 5 0-2.4
vol%). Symbols denote observed metabolite formation. Lines represent predicted rates using MichaelisMenten kinetic parameters derived from nonlinear regression analysis of the experimental data, including a
term for substrate inhibition (TFA, r 5 0.98; Br, r 5
0.98, P , .05).
microsomes and cDNA-expressed P450 clearly showed that
P450 2E1 is the high affinity, low capacity enzyme and P450
2A6 is the low affinity, high capacity enzyme (table 1) that
catalyzes halothane oxidation. There was excellent agreement between the high affinity Km values for TFA formation
obtained for both microsomes and cDNA-expressed P450 2E1
(0.045 and 0.053 vol%, respectively). Similarly, the low affinity Km values derived from both enzyme sources are in superb agreement (1.2 vol% for both). There was also excellent
agreement between the high affinity Km values for bromide
formation obtained for both microsomes and cDNA-expressed
P450 2E1 (0.045 and 0.030 vol%, respectively). Similarly, the
low affinity Km values derived from both sources are in excellent agreement (0.94 and 0.77 vol%, respectively). The Km
values obtained for TFA and bromide formation were comparable in both microsomes and expressed P450 in every case,
consistent with the known mechanism of halothane oxidation
whereby TFA and bromide are formed in equimolar amounts.
Most impressively, the Km value for the high affinity isoform
catalyzing microsomal halothane oxidation (0.045 vol%) is in
remarkable agreement with that reported (Km 5 0.029%) for
human halothane metabolism in vivo, in which halothane
was administered over the alveolar concentration range
0.0007 to 0.13% (Cahalan et al., 1982). These investigations
explicitly demonstrated that P450 2E1 is the high affinity,
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Fig. 11. Comparison of TFA and bromide formation from P450-catalyzed halothane oxidation by a panel of human liver microsomes. Reactions were performed at saturating (2 ml 5 2.4 vol%) halothane
concentrations (n 5 20, r 5 0.99; P , .001) and (inset) at subsaturating
halothane (0.10 ml 5 0.12 vol%) concentrations (n 5 15, r 5 0.98; P ,
.001) as described in “Methods.”
low capacity catalyst and P450 2A6 is the low affinity, high
capacity catalyst of halothane oxidation.
Compelling evidence was obtained to suggest that human
liver microsomal and cDNA-expressed P450 2E1 are subject
to substrate inhibition at high concentrations of halothane.
Furthermore, this substrate inhibition occurs at halothane
concentrations that occur during anesthesia. Simple Michaelis-Menten kinetics did not adequately model the kinetic data
for cDNA-expressed P450 2E1. Rather, the experimental
data for cDNA-expressed P450 2E1-catalyzed halothane oxidation to both TFA and bromide were best modeled by incorporating a term for substrate inhibition. Evidence for substrate inhibition of P450 2E1 in human liver microsomes was
also obtained. At halothane concentrations exceeding 4 vol%,
the rates of microsomal TFA and bromide formation decreased (data not shown). Furthermore, the two enzyme
Michaelis-Menten model of the microsomal data predicted
that unlike the data for cDNA-expressed enzymes, the high
affinity isoform was the high capacity isoform. Although the
apparent parameters correctly modeled the data, the absolute values for Vmax were inaccurate because the simple
model did not include a term for substrate inhibition. Attempts to model microsomal halothane oxidation including a
component for substrate inhibition were unsuccessful because there was not a unique solution to the equation. Substrate inhibition has also been observed previously for the
P450-catalyzed metabolism of 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123), a halothane congener (Vinegar et al., 1994).
HCFC-123 metabolism could only be accurately described
when the physiological based pharmacokinetic model included a term for substrate inhibition (Andersen et al., 1987).
These results provide a biochemical rationale for observations in patients where halothane metabolism decreased as
administered halothane concentrations increased (Cascorbi
et al., 1970; Cahalan et al., 1981; Carpenter et al., 1986).
Patients receiving 0.11% halothane metabolized a greater
proportion (55%) of halothane than did those receiving 0.44%
halothane (41%) (Cahalan et al., 1981). Also, in a valiant
study by Cascorbi and co-workers (1970), the authors were
injected with radioactive halothane, both with and without
concomitant halothane anesthesia. Halothane metabolism
was greater when the subjects were not anesthetized. Two
investigations in animals (Eckes and Buch, 1985; Lind and
Gandolfi, 1993) have also demonstrated that at high halothane concentrations, halothane metabolism was inhibited;
however, as halothane concentrations decreased after cessation of anesthesia, metabolism increased. The present results
1997
Halothane Oxidation in Vitro by CYP 2E1
409
provide a biochemical explanation for all these observations.
In vivo halothane metabolism decreases as halothane concentrations increase because the P450 isoform that catalyzes
the majority of halothane oxidation, P450 2E1, is subject to
substrate inhibition.
The ultimate objective of human microsomal investiga-
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Fig. 12. Predicted halothane oxidation
catalyzed by P450s 2E1 and 2A6. A,
Blood halothane concentrations for patients undergoing a 3-hr halothane anesthetic were measured up to 9 hr postanesthesia and extrapolated to 4 days
(inset). B, Rates of predicted TFA formation from halothane oxidation catalyzed
by microsomal P450s 2E1 and 2A6. The
rates were calculated using blood halothane concentrations in (A) and kinetic
parameters obtained from nonlinear regression analysis of microsomal TFA
formation. C, Rates of predicted TFA formation from halothane oxidation catalyzed by cDNA-expressed P450s 2E1
and 2A6. For various relative contents of
P450s 2E1 and 2A6, the rates of halothane oxidation were calculated using
blood halothane concentrations in (A)
and kinetic parameters obtained from
nonlinear regression analysis of TFA formation catalyzed by cDNA-expressed
P450s 2E1 and 2A6.
410
Spracklin et al.
P450 2E1 in halothane hepatotoxicity may be relevant to
humans. Animal models have also demonstrated a role for
phenobarbital-induced P450 isoforms in halothane oxidation
in vitro (Gruenke et al., 1988) and in vivo (Jenner et al.,
1990), suggesting the participation of non-2E1 isoforms.
P450 2B enzymes are the major phenobarbital-induced isoforms in rats. Experiments with cDNA-expressed P450 2B6,
as well as previous experiments (Gruenke et al., 1988; Jenner
et al., 1990), demonstrated the activity of P450 2B toward
halothane oxidation. Indeed, other P450 2E1 substrates can
also be metabolized by P450 2B at high substrate concentrations (Nakajima et al., 1990; Nakajima et al., 1992). However,
P450 2B6 is not routinely expressed in human liver, and
thus, does not appear to be a significant catalyst of human
halothane oxidation. Therefore, animal models describing
the role of phenobarbital-induced P450s in halothane oxidation must be interpreted with caution.
The mild and severe forms of hepatotoxicity mediated by
halothane metabolism are believed to arise from different
routes of biotransformation, with different clinical sequale.
The present investigation, coupled with recent findings
(Spracklin et al., 1996), provide biochemical evidence that the
mild and severe halothane hepatotoxicities indeed arise from
different routes of biotransformation. Furthermore, these
disparate pathways of metabolism are catalyzed by different
P450 isoforms. P450 2A6 and P450 3A4 catalyze reductive
halothane metabolism while P450 2E1 and 2A6 catalyze
oxidative halothane metabolism. Halothane is a unique substrate in that it readily undergoes both oxidative and reductive metabolism. The underlying basis for this apparent oxygen-dependence of isoform specificity is currently unknown.
The identification of the enzymes that catalyze halothane
oxidation is an important step in understanding halothane
hepatitis. These investigations have identified P450 2E1 as
the major catalyst of oxidative halothane metabolism using
an in vitro model. This model has been used to rationalize in
vivo results in humans, where P450 2E1 was identified as the
predominant catalyst of oxidative halothane metabolism.
Thus, the microsomal in vitro model is an accurate predictor
of in vivo human halothane oxidation.
Acknowledgments
The authors thank Dr. Douglas S. Mautz for the measurement of
blood halothane concentrations.
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Halothane Oxidation in Vitro by CYP 2E1