Carcinogenesis vol.18 no.9 pp.1695–1700, 1997
ACCELERATED PAPER
Identification of benzene oxide as a product of benzene
metabolism by mouse, rat, and human liver microsomes
Mark R.Lovern1,2, Max J. Turner2, Mark Meyer3,
Gregory L.Kedderis2, William E.Bechtold3 and
Paul M.Schlosser2,4
1Biomathematics
Program, North Carolina State University, Raleigh, NC,
NC and
2Chemical Industry Institute of Toxicology, Research Triangle Park,
3Lovelace Respiratory Research Institute, Albuquerque, NM, USA
4To
whom correspondence should be addressed
Benzene is a ubiquitous environmental pollutant that is
known to cause hematotoxicity and leukemia in humans.
The initial oxidative metabolite of benzene has long been
suspected to be benzene oxide (3,5-cyclohexadiene-1,2oxide). During in vitro experiments designed to characterize
the oxidative metabolism of [14C]benzene, a metabolite was
detected by HPLC-radioactivity analysis that did not elute
with other known oxidative metabolites. The purpose of
our investigation was to prove the hypothesis that this
metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male
B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM),
and NADPH (1 mM) in 0.1 M sodium phosphate buffer
(pH 7.4) and then extracted with methylene chloride. Gas
chromatography–mass spectrometry analysis of incubation
extracts for mice, rats, and humans detected a metabolite
whose elution time and mass spectrum matched that of
synthetic benzene oxide. The elution time of the benzene
oxide peak was ~4.1 min, while phenol eluted at ~8 min.
Benzene oxide also coeluted with the HPLC peak of the
previously unidentified metabolite. Based on the 14C activity
of this peak, the concentration of benzene oxide was
determined to be ~18 µM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes
with 1 mM benzene. The metabolite was not observed in
incubations using heat-inactivated microsomes. This is the
first demonstration that benzene oxide is a product of
hepatic benzene metabolism in vitro. The level of benzene
oxide detected suggests that benzene oxide is sufficiently
stable to reach significant levels in the blood of mice, rats,
and humans and may be translocated to the bone marrow.
Therefore benzene oxide should not be excluded as a
possible metabolite involved in benzene-induced leukemogenesis.
Introduction
Benzene was used extensively in the past for production of
paints, resins, rubber, inks, and dyes. It is currently 1% of
gasoline by volume and a feedstock for synthetic organic
chemical production (1). Benzene is a constituent of gasoline
*Abbreviations: AML, acute myelogenous leukemia; CYP2E1, cytochrome
P450 2E1; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide
adenine dinucleotide phosphate; BHT, butylated hydroxytoluene; GC–MS,
gas chromatography–mass spectrometry; LSS, liquid scintillation spectrometry; ACN, acetonitrile; EA, ethyl acetate.
© Oxford University Press
fumes, automobile exhaust, and both mainstream and side
stream tobacco smoke (2). Exposure to high levels of benzene
for extended periods of time is associated with aplastic anemia
and acute myelogenous leukemia (AML*) in humans (3).
Workplace exposures to benzene in the United States are now
limited through safety regulations (4), but recent reports of
increases in hematotoxicity and leukemia among benzeneexposed workers in China provide further supporting evidence
that it causes these health effects (5,6). As in previous studies,
AML is associated with constant, high-level exposures or high
cumulative exposures in the Chinese cohort.
Benzene is hematotoxic (e.g. results in aplastic anemia) and
myelotoxic (e.g. causes decreases in bone marrow cellularity)
in mice and rats (7,8). Because hematotoxicity is often observed
prior to the onset of AML in humans, these endpoints are
presumed to be predictive of AML risk for humans. Sammett
et al. (9) showed that partial hepatectomy protected rats against
benzene-induced hematotoxicity, leading to the hypothesis that
hepatic benzene metabolism is a primary step in the induction
of hematotoxicity. This hypothesis is further supported by
the recently observed correlation between increased risk of
benzene-induced hematotoxicity in humans and cytochrome
P450 2E1 (CYP2E1) activity as measured by chlorzoxazone
metabolism (10). These results suggest that benzene-induced
myelotoxicity, and hence the risk of benzene-induced leukemia
in humans, depends on the dose of benzene metabolites
reaching the bone marrow, which, in turn, depends on the
rates of various steps in hepatic benzene metabolism. Therefore
quantification of benzene metabolic rates and subsequent
predictions of benzene metabolite dosimetry in the bone
marrow through the use of biologically based mathematical
models could considerably improve benzene risk assessment
by allowing risk to be correlated with those target-tissue
metabolite levels.
Benzene metabolism was first studied by Williams and
coworkers over 40 years ago (11,12) and Jerina and coworkers
proposed that the first step in benzene metabolism was oxidation to benzene oxide nearly 30 years ago (13). Yet only
recently has benzene oxide been detected in vivo (14). Jerina
et al. (13) showed that benzene oxide can be enzymatically
conjugated with glutathione to form S-(1,2-dihydro-2-hydroxyphenyl)-glutathione (pre-phenylmercapturic acid), indicating
that it is an intermediate in the metabolism of benzene to Sphenyl-N-acetylcysteine (phenylmercapturic acid), a significant
pathway for benzene elimination. Davies and Whitham (15)
showed that benzene oxide can react with mild oxidizing
agents to form muconaldehyde, suggesting that it is also an
intermediate in the conversion of benzene to muconic acid.
This conversion is a significant pathway for benzene elimination. Thus the ability to detect and ultimately quantify levels
of the primary benzene metabolite, benzene oxide, would
contribute significantly to the development of a biologically
based pharmacokinetic model for benzene.
An oxidative metabolite of benzene that did not elute with
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M.R.Lovern et al.
Fig. 1. (a) Gas chromatogram for the authentic standard for benzene oxide. (b) Mass spectrum of the gas chromatogram peak eluting at ~4.1 min.
other known oxidative metabolites was previously noted after
incubations of [14C]benzene with hepatic microsomes of male
B6C3F1 mice, male Fischer-344 rats, and 10 human tissue
donors (16). The metabolite appeared rapidly after the initiation
of benzene metabolism, reached a peak concentration after
~16 min, and declined thereafter. This time course indicated
that the metabolite was produced from benzene via a single
reaction step and subsequently reacted to form some other
product (16). The purpose of our investigation was to test the
hypothesis that this oxidative metabolite was benzene oxide
and that it is produced by microsomes from all three species.
Materials and methods
Chemicals
[14C]Benzene (102.1 mCi/mmol, ù98% purity) was purchased from Chemsyn
Science Laboratories (Lenexa, KS). Benzene (ù99.9% purity), L-ascorbic acid
(sodium salt), reduced β-nicotinamide adenine dinucleotide (NADH) and β-
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nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from
Sigma Chemical Co. (St Louis, MO). Sodium phosphate was obtained from
Fisher Scientific (Fair Lawn, NJ). Methylene chloride (HPLC grade) was
obtained from Baxter Healthcare Corporation (Muskegon, MI). Acetonitrile
was purchased from J.T.Baker Inc. (Phillipsburg, NJ, ‘Baker Analyzed’®
HPLC Reagent) and KH2PO4 was purchased from Aldrich Chemical Co.,
Inc. (Milwaukee, WI). All other chemicals were of the highest quality
commercially available.
Benzene oxide was synthesized by a three-step procedure similar to that
used by Platt and Oesch (17). 4,5-Dibromocyclohexene was synthesized
by dibromination of 1,4-cyclohexadiene in chloroform at 0°C (18). 4,5Dibromocyclohexane-1,2-oxide was prepared by oxidation of 4,5-dibromocyclohexene using meta-chloroperoxybenzoic acid (19). Benzene oxide was
prepared by dehydrohalogenation of 4,5-dibromocyclohexane-1,2-oxide using
sodium methoxide in boiling ether (20) or 1,8-diazobicyclo [5.4.0] undec-7ene in ether at room temperature (19). Benzene oxide was purified by vacuum
distillation at 0.5–1.0 Torr followed by preparatory gas chromatography (21).
Stock solutions were prepared immediately prior to each incubation by
diluting benzene in 0.1 M sodium phosphate buffer (pH 7.4) to a concentration
of 2 mM (based on liquid volume and the density of benzene) in a glass
bottle that was filled to capacity (no head space) and sealed with a Teflon™
Benzene oxide production from benzene metabolism in liver
Fig. 2. (a) Gas chromatogram of a methylene chloride extract from an incubation of benzene with active microsomes. (b) Mass spectrum for the gas
chromatogram peak eluting at ~4.1 min.
Silicon septum (Pierce, Rockford, IL). For incubations with [14C]benzene, the
stock was spiked with [14C]benzene to a final specific activity of 0.45 µCi/
µmol. Dissolution of benzene was facilitated by sonication.
Animals
Male Fischer-344 rats (CDF [F-344]/CrlBR) 10–12 weeks old (200–250 g)
and male B6C3F1 mice (B6C3F1/CrlBR) 7–9 weeks old (22–30 g) (Charles
River Laboratories, Raleigh, NC) were used as sources of liver tissue. Animals
were housed in mass air displacement, temperature and humidity controlled
rooms (22 6 1 °C and 60 6 15%, respectively), fed pelleted NIH-07 rodent
chow (Ziegler Bros., Gardner, PA) and purified water ad libitum, and kept on
a 12-h light–dark cycle. Animals were allowed to acclimate for at least
2 weeks prior to use. Sentinel animals were screened weekly for viral infection
(Standard Rat Screen, Microbiological Associates, Bethesda, MD) and were
negative throughout the study.
Microsome preparation
Mouse and rat microsomes were prepared from pooled liver samples according
to the method of Csanády et al. (22). Frozen human liver samples were
obtained from Dr F.P.Guengerich (Vanderbilt University, Nashville, TN)
through Tennessee Donor Services (Nashville, TN). Human liver microsomes
from samples HL98, HL100, and HL104 were prepared as described by
Kedderis et al. (23). Protein content was determined with a protein assay kit
(Sigma Analytical, St Louis, MO) using a modified micro-Lowry method
(24). Microsomes were stored at –80°C until time of use. For control
experiments, microsomes were inactivated by heating at 100°C for 20 min.
Incubation procedure
The incubation procedure followed closely that of Schlosser et al. (25).
Microsomes (1 mg/ml final protein concentration) and sodium ascorbate
(0.1 M final concentration in 0.1 M sodium phosphate, pH 7.4) were placed
in screw-top vials with Teflon™-rubber septa (Pierce). Incubation mixtures
were made to a total volume of 1 ml, 5 ml, or 10 ml. Vials were sealed and
benzene working stock was added using a gas-tight syringe to yield a final
benzene concentration of 1 mM. Vials were placed in a 37°C water bath and
allowed to equilibrate for 5 min. Reactions were then initiated by addition of
NADH and NADPH (both at 1 mM final concentration in 0.1 M sodium
phosphate, pH 7.4). Incubations were terminated after 18 min by extraction
with either ice-cold ethyl acetate containing internal standards and butylated
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M.R.Lovern et al.
equipped with an HP5-MS capillary column (30 m, 0.25 mm i.d., 0.25 mm
film; Hewlett Packard, Wilmington, DE). Samples (1 ml) were injected oncolumn using a Hewlett-Packard liquid autosampler. The on-column inlet was
operated at 40°C using helium at an inlet pressure of 8.0 psi. The column
was held at 35°C for 0.2 min following injection, raised at 50°/min to 50°C
and held for 1.5 min, then raised at 10°/min to 150°C and held for 0.1 min,
and then raised at 30°/min to 200°C and held for 1.0 min. The mass
spectrometer was operated in the electron ionization mode (EI, 70 eV) and
scanned over the range m/z 35–200 at a rate of 1.8 scans/s.
HPLC and LSS analysis
Radiolabeled metabolites in ethyl acetate extracts were separated by the
method of Bechtold et al. (26), with the following modification in the solvent
program: a gradient from 10% acetonitrile (ACN):90% buffer (B) at 0 min to
12% ACN:88% B at 3 min, followed by a gradient to 62% ACN:38% B from
3 to 8.5 min, then a gradient to 72% ACN:28% B from 8.5 to 11.5 min, a
gradient to 100% ACN from 11.5 to 14 min, and finally held at 100% ACN
until 24 min. Quantitation of metabolite concentrations was performed by
LSS as described previously (25) after correcting the specific activity of the
[14C]benzene for the dilution with unlabeled benzene during preparation of
the stock solution.
Results
Fig. 3. UV and radiochromatograms for incubations with (a) heatinactivated and (b) active mouse microsomes. Incubation was at 1 mM
benzene, with specific activity 0.45 µCi/µmole. Incubation mixture was
extracted into 1 ml ethyl acetate. Results are for a 100-µl injection. UVtrace is that of internal standards (a) without or (b) with benzene oxide.
hydroxytoluene (BHT) or pure, ice-cold methylene chloride. The incubation
time length was chosen to be close to that at which Seaton observed the
maximum concentration of the unidentified metabolite (16). The internal
standard mixture contained benzene (8.9 mg/ml), phenol (0.63 mg/ml),
catechol (1.6 mg/ml), hydroquinone (2.9 mg/ml), trihydroxybenzene (1.3 mg/
ml), and BHT (20 mg/ml). Samples were vortexed for 1 min and then
centrifuged at 3000 rpm for 5 min to separate the aqueous and organic
fractions and to pellet the protein.
Control incubations using heat-inactivated microsomes were performed to
ensure that benzene oxide did not originate from another source. One
incubation each was performed in 5 ml (final volume) with active microsomes
from mice, rats, and three human donors; these were extracted with 0.5 ml of
methylene chloride and analysed directly. Two 10-ml (final volume) incubations
with mouse microsomes were extracted into 5 ml methylene chloride, and the
extract was concentrated under nitrogen to a volume of 200 ml prior to
analysis. All samples extracted in methylene chloride were analysed by gas
chromatography–mass spectrometry (GC–MS). Two additional incubations
were performed with [14C]benzene and mouse microsomes in 1 ml final
volume, extracted into 1 ml ethyl acetate with internal standards and butylated
hydroxy toluene, and analysed directly by HPLC (collected in 12-s fractions)
and liquid scintillation spectrometry (LSS). The UV absorbance (263 nm)
chromatogram and elution time for the HPLC samples were compared to
those of internal standard solutions in ethyl acetate with or without addition
of the benzene oxide standard (in methylene chloride).
GC-MS analysis of incubation extracts
Incubation extracts in methylene chloride were analyzed using a Hewlett
Packard 5989B mass spectrometer coupled to an HP 5890 gas chromatograph
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GC–MS analysis of synthetic benzene oxide
The authentic standard for benzene oxide had an approximate
elution time of 4.1 min (Figure 1a). The EI mass spectrum for
the standard produced ions at m/z 94 (M), 78 (M–O), 68
(M–C2H2), 66 (M–CO), 65 (M–CHO), 40 (66–C2H2), and 39
(65–C2H2) (Figure 1b). This spectrum is consistent with
previously published EI mass spectra for benzene oxide (19).
GC–MS analysis of incubation extracts
The mass chromatogram of a methylene chloride extract from
a mouse microsomal incubation with benzene showed a peak
with an elution time corresponding to that of benzene oxide
(~4.1 min) (Figure 2a). The mass spectrum of this peak (Figure
2b) is consistent with that of authentic benzene oxide (Figure
1b). The peak corresponding to benzene oxide was observed
in all incubations using active microsomes, including the three
individual human samples. No substance with a similar elution
time or mass spectrum was observed in the extract from the
incubation with heat-inactivated microsomes. The elution time
of phenol was ~8 min (data not shown).
HPLC and LSS analysis
A chromatogram from an incubation of [14C]benzene with heatinactivated microsomes extracted with ethyl acetate containing
unlabeled internal standards (Figure 3a) showed a single peak
with an retention time of ~18 min. The 14C activity from an
incubation with active microsomes extracted with ethyl acetate
containing unlabeled internal standards and synthetic benzene
oxide is shown in Figure 3b. The 14C activity eluted in several
peaks, one of which had the same retention time as benzene
oxide (~15 min). For mouse liver microsomes, the concentration of benzene oxide produced by a 1 mM incubation of
benzene was estimated from radioactivity to be 18 mM, or
7% of all metabolites formed.
Discussion
Benzene oxide was found in all extracts from incubations with
active mouse, rat, and human liver microsomes, but not in
extracts from control incubations, indicating that it is a product
of oxidative metabolism. While benzene oxide has long been
assumed to be the initial product of oxidative metabolism of
benzene, this is the first confirmation that it is produced by
hepatic microsomal metabolism. Also, benzene oxide coeluted
with a hepatic microsomal metabolite of benzene previously
Benzene oxide production from benzene metabolism in liver
Table I. Benzene oxide produced by 16 minute incubations of 4 mM
benzene with mouse, rat, and human liver microsomesa
Sample
Benzene oxide
concentration (µM)
% of Total
Metabolites produced
rat
mouse
Human
Human
Human
Human
Human
Human
Human
Human
Human
1
2
3
4
5
6
7
8
9
0.142
0.191
0.203
0.083
0.158
0.113
0.178
0.211
0.117
0.135
0.159
11
8
5
10
6
8
7
5
6
8
7
aSeaton
et al. (16).
noted but unidentified in all three species (16). In those
experiments, 16 min incubations of 4 µM benzene with rat
and mouse microsomes produced metabolite concentrations of
0.14 and 0.19 µM, respectively (Table I). This corresponded
to 11% of the total metabolites formed in rat samples, and 8%
of those in mouse samples. Identical experiments conducted
with liver microsomes from 9 human individuals resulted in
concentrations ranging 0.08–0.21 µM, and the metabolite
accounted for 5–10% of the metabolites produced. At 1 mM
benzene after an 18-min incubation, mouse microsomes produced 18 µM benzene oxide, ~7% of the total metabolites.
Since human exposures would likely limit benzene blood
levels to no higher than micromolar levels, the findings at
4 µM are likely more relevant to humans.
The [14C]metabolite peak in the HPLC was first observed
serendipitously when the method of Bechtold and coworkers
(26) was modified as described here to obtain better resolution
between catechol and phenol. With that change, what had been
an intermittent shoulder on the back of the phenol peak
resolved into a distinct peak (given the sensitivity of detection
with [14C]benzene as the substrate). Benzene oxide could not
be reliably detected by GC–MS in ethyl acetate (EA) extracts
(used for the HPLC method). In particular, attempts to concentrate the metabolite in these extracts were unsuccessful since
the metabolite was lost with EA evaporation. Detection was
successful in methylene chloride, a solvent that is sometimes
avoided because it has greater density than the aqueous phase.
This suggests that application of this method to blood samples
may lead to successful detection of benzene oxide in vivo,
provided that the extraction is performed immediately after
sampling, and that the sample is analysed shortly thereafter.
Many attempts were made to obtain positive identification
for the metabolite identified by the HPLC fraction that coelutes
with benzene oxide. The peak elutes at ~14 min, when the
gradient is programmed to just reach 100% ACN, but there is
still some water in the fraction. But because of the volatility
or the instability of benzene oxide, we were unsuccessful in
concentrating it and obtaining positive identification in extracts
of the fraction.
Detection of 0.25 µM benzene oxide in the blood of rats
after oral administration of 400 mg benzene/kg body wt has
recently been reported by Lindstrom et al. (14), showing that
benzene oxide is formed in vivo and is sufficiently stable to
exit the rat liver. We have shown here that benzene oxide is a
product of hepatic microsomal metabolism in mice and humans
as well as rats. Based on previous studies (16), one would
expect concentrations similar to those observed in rats. The
half-life of benzene oxide in aqueous buffer at physiological
pH has been shown to be 8.25 min (27), and 7.9 min in the
blood of rats (14). If the half-life of benzene oxide in human
blood is also close to 8 min, the recirculation time for blood
in humans of ~1 min (28) would allow 83% of the benzene
oxide released to the blood from the liver to reach the bone
marrow in humans.
Focusing on metabolites that were demonstrably present,
various studies have indicated that benzene toxicity may result
from the combined effects of several metabolites: muconaldehyde and hydroquinone act additively when the hydroquinone concentration is 100 mg/kg, and synergistically when
the concentration is reduced to 50 mg/kg (29,30). Phenol and
hydroquinone also act synergistically (31,32). But no other
oxidative benzene metabolite or combination of metabolites
has been shown to be the agent of benzene leukemogenicity.
Because benzene oxide was thought to be too reactive to reach
significant blood levels, its potential involvement has not been
proposed.
Many other epoxide compounds have been shown to form
DNA adducts and act as genotoxins (33). DNA adducts have
been detected in the bone marrow and white blood cells of
benzene-treated mice (34). If benzene oxide does reach the
bone marrow in significant quantities and exhibits reactivity
similar to other epoxides, then further investigation of benzene
oxide as a potential myelotoxin and leukemogen may be
warranted. Toward this end, it is the authors’ intention to
extend the work published here by re-structuring the previously
published model of benzene in vitro oxidative metabolism (16)
so that the data for the unknown metabolite is identified with
benzene oxide. The model described by Seaton et al. (16)
presumed that the metabolite now believed to be benzene
oxide was not intermediate in the production of phenol. While
Seaton’s model performed well at predicting experimental data
for benzene oxide, the assumed model structure was not
correct, and therefore the values of some of the fitted parameters
have no biological interpretation. Therefore, it will be necessary
to modify the model so that the kinetics of benzene oxide and
benzene’s other oxidative metabolites can be determined.
Acknowledgements
Mark Lovern, a graduate student at North Carolina State University (NCSU),
has been supported via an unrestricted grant from CIIT to NCSU. The work
of William Bechtold and Mark Meyer was sponsored in part by the US
Department of Energy, Office of Health and Environmental Research, under
contract DE-AC04-76EV01013. We thank Drs Bernard Golding and Christine
Dalby for their direction in the synthesis of benzene oxide and for supplying
a sample of dibromocyclohexane oxide. Dr F.P.Guengerich, Vanderbilt University, graciously supplied the human liver tissues used in this study. We thank
Dr James Mathews, Research Triangle Institute, Dr Stephen Rappaport,
University of North Carolina at Chapel Hill, and Drs James Bond and Michele
Medinsky, CIIT, for helpful discussions and suggestions. Valuable assistance
in the laboratory was provided by Mr Horace Parkinson. Editorial assistance
was kindly provided by Dr Barbara Kuyper, CIIT.
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Received on May 2, 1997; revised on June 16, 1997; accepted on June 24, 1997