Toxicology 226 (2006) 99–106
Investigation of bioactivation and toxicity of styrene
in CYP2E1 transgenic cells
Jou-Ku Chung a , Wei Yuan a , Guangxian Liu a , Jiang Zheng a,b,∗
a
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA 02115, United States
b Center for Developmental Pharmacology and Toxicology, Children’s Hospital and Regional Medical Center,
University of Washington, Seattle, WA 98105, United States
Received 22 February 2006; received in revised form 17 May 2006; accepted 3 June 2006
Available online 9 June 2006
Abstract
Styrene has been found to be toxic to the respiratory system, and the toxicity of styrene is metabolism-dependent. CYP2E1 is
suggested to be one of the cytochrome P450 enzymes responsible for the bioactivation of styrene. Our work focused on the roles
of CYP2E1 and epoxide, a metabolite of styrene epoxidation, in the cytotoxicity of styrene. Styrene was found to be more toxic
to h2E1 cells than to the wild type, while there was no difference found when styrene oxide was administered. Both soluble and
microsomal epoxide hydrolase inhibitors dramatically enhanced styrene toxicity. Glutathione and glutathione ethyl ester showed
protection against styrene cytotoxicity. Cytotoxicity of a selection of styrene analogues, such as ethylbenzene, vinylcyclohexane, and
ethylcyclohexane, was assessed to determine if unsaturation is required for styrene toxicity. Ethylbenzene and vinylcyclohexane were
found to be as toxic as styrene to h2E1 cells, whereas little toxicity of ethylcyclohexane to h2E1 cells was observed. This indicates the
importance of vinyl group of styrene in its cytotoxicity, but saturation of the vinyl group does not necessarily eliminate styrene toxicity.
An N-acetylcysteine conjugate derived from styrene oxide was identified by LC/MS/MS in the sample obtained from the incubation
of h2E1 cell lysate with styrene in the presence of N-acetylcysteine. Formation of the N-acetylcysteine conjugate was found to be
NADPH-dependent. These studies provided strong evidence in support of toxic role of styrene epoxide metabolite in styrene toxicity.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Styrene; Styrene-7,8-oxide; CYP2E1
1. Introduction
Styrene is widely used in the production of rubbers, plastic, insulation, fiberglass, pipes, automobile
Abbreviations: GSH, Glutathione; GST, Glutathione S-transferase;
LC/MS/MS, liquid chromatography/tandem mass spectrometry;
mEHI, microsomal epoxide hydrolase inhibitor; MRM, multiple reaction monitoring; NADPH, nicotinamide adenine dinucleotide phosphate; sEHI, soluble epoxide hydrolase inhibitor; ST, styrene
∗ Corresponding author. Tel.: +1 617 373 5258;
fax: +1 617 373 8886.
E-mail address: [email protected] (J. Zheng).
parts, food containers, and carpet backing (Kolstad et al.,
1995). Approximately 21 million tonnes of styrene were
consumed worldwide in 2000, a 50% increase since 1993
(Kolstad et al., 1995). There are an estimated 30,000
workers who are exposed to styrene on a regular basis
and approximately 300,000 on a part-time basis in the
United States (NIOSH, 1983). In 1996, an estimated
90,000 workers were potentially exposed to styrene in
USA (OSHA, 1996).
Styrene exposure has been associated with mucous
membrane and upper respiratory tract irritation in
humans. The toxic symptoms exist as chronic bronchitis,
obstructive pulmonary changes, decreased lung ventila-
0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2006.06.001
100
J.-K. Chung et al. / Toxicology 226 (2006) 99–106
tion, asthma, tiredness, nasal secretion and nose irritation
(Carpenter et al., 1944; Wilson, 1944; Stewart et al.,
1968; Gotell et al., 1972; Alarie, 1973; Chmielewski
and Hac, 1976; IARC, 1987–1997; Hayes et al., 1991;
IARC, 2002b; IARC, 2002a; Kaufmann et al., 2005).
There has been extensive research in experimental animals on the respiratory toxicity of styrene. Intraperitoneal administration of styrene in mice caused elevated
levels of ␥-glutamyltranspeptidase (GGT) and lactate
dehydrogenase (LDH) in bronchoalveolar lavage fluid
(BALF) (Gadberry et al., 1996; Carlson, 1997a). Multifocal necrosis and cell loss in bronchiolar epithelium
were observed in CD-1 mice after exposure to 40 or
160 ppm inhaled styrene (Green et al., 2001). Systemic
studies in CD-1 and B6C3F1 mice consistently showed
cell crowding, decreased staining, and increased cell proliferation in the Clara cells of the mouse bronchiolar
epithelium (Cruzan et al., 1997; Green et al., 2001). Also,
Green and coworkers reported increased proliferation of
the Clara cells in mice after administration with styrene
by oral gavage (Green et al., 2001).
Styrene has also been found to be toxic to the nasal
olfactory epithelium. A single exposure of CD-1 mice
to 80 ppm styrene resulted in early atrophy and degeneration of olfactory epithelial cells with dilation of Bowman’s glands. Continued exposure caused replacement
of olfactory cells by ciliated columnar cells, Bowman’s
gland hyperplasia, and debris (Green et al., 2001). After
exposure to styrene at 100–200 ppm for 13 weeks, all
CD-1 mice had atrophy of olfactory epithelium and dilation of Bowman’s glands (Cruzan et al., 1997).
Metabolism of styrene has been extensively studied
in mice, rats and humans (Sumner and Fennell, 1994).
Styrene is primarily metabolized by the CYP monooxygenase system to styrene 7,8-oxide (2, Scheme 1).
Styrene 7,8-oxide can be further metabolized to styrene
glycol (3) by epoxide hydrolase. Conjugation with glutathione to glutathione conjugates 4a and 4b has been
Scheme 1.
documented, and a total of four urinary diasteriomeric
styrene mercapturic acids were apparently identified
in animals after treatment with styrene (Manini et al.,
2000). In vivo metabolism studies have demonstrated
that the majority of styrene oxide is hydrated by epoxide hydrolase. Another pathway of detoxification of
styrene oxide is conjugation with glutathione (GSH).
Both epoxide hydrolase and glutathione S-transferase
are important in elimination of electrophilic reactivity
of styrene oxide. Ring epoxidation was also reported in
styrene metabolism (Vogie et al., 2004; Cruzan et al.,
2005). 4-Vinylphenol was found as a minor metabolite, which showed 5- to 10-fold more toxic than styrene
7,8-oxide.
Lung microsomes were found to catalyze the transformation of styrene to styrene oxide (Carlson, 1997a,b;
Carlson et al., 2000; Green et al., 2001), and CYP2F2 and
CYP2E1 were identified to be responsible for the pulmonary metabolism of styrene to styrene oxide (Carlson,
1997b; Green et al., 2001). Chemically, styrene oxide
exists as enantiomers. Both R- and S-enantiomers of
styrene oxide have been identified in in vitro and in vivo
studies. Interestingly, the R-stereoisomer was found to
be more toxic to the mouse respiratory system than the
S-stereoisomer (Gadberry et al., 1996).
Pulmonary toxicity studies showed that mice were
more susceptible to styrene than rats. Clara-cell-enriched
fractions from mice were found to produce styrene oxide
at levels five-fold higher than fractions from rats after
exposure to styrene (Hynes et al., 1999). Furthermore,
the mouse Clara cells produced about three times more
of the R-isomer than the S-isomer. In contrast, the production of the S-isomer was predominant in rats (Hynes
et al., 1999).
Clara cells were reported to be responsible for pulmonary metabolism of styrene. Type II cells, however,
showed little metabolizing activity of styrene when compared to Clara cells in both rats and mice (Hynes et al.,
1999). This is consistent with the report that Clara cells
contain CYP2E1 and CYP2F2 while type II cells do not
(Buckpitt et al., 1995; Forkert, 1995). In addition, the
levels of both CYP2E1 and CYP2F2 were found to be
higher in the mouse Clara cells than rats. These match the
report that styrene exposure produces toxicity in mouse
Clara cells but not in type II cells or rat lung cells.
Acute pneumotoxicity of styrene is evidently supported by animal studies. However, limited information is known regarding the biochemical mechanism
of acute cytotoxicity induced by styrene. The objectives of the present study were to examine the role of
CYP2E1 in styrene toxicity, to identify styrene oxide as
a metabolite of styrene, to confirm CYP2E1 is respon-
J.-K. Chung et al. / Toxicology 226 (2006) 99–106
101
sible for the formation of oxide metabolite of styrene,
and to verify the cytotoxic effect of styrene oxide
metabolite.
2. Methods and materials
2.1. Materials
Styrene (99%), styrene oxide (98%), ethylbenzene (99.8%),
vinylcyclohexane (99%), ethylcyclohexane (99%), dimethyl
sulfoxide (DMSO), ␤-nicotinamide adenine dinucleotide
phosphate (␤-NADPH), glutathione ethyl ester, glutathione
(GSH), N-acetylcysteine were purchased from Sigma Chemical Co. (St. Louis, MO). HPLC-grade acetonitrile, formic
acid and Secureseal were obtained from Fisher Scientific Co.
(Pittsburgh, PA). CellTiter 96 Aqueous one solution cell proliferation assay kit was purchased from Promega Co. (Madison, WI). Transgenic cell line expressing CYP2E1 (h2E1)
and the wild-type cell line (cHo1, human B-lymphoblastoid)
were obtained from BD-Gentest (Palo Alto, CA). Trypan Blue
stain was purchased from Bio Whittaker (Walkersville, MD).
Media RPMI 1640 and DMEM were purchased from MediaTech (Herndon, VA). Compound 8, [(13-oxotetradecyl)amino]N-tricyclo[5.3.1.0(4,9)] undecylcarboxamide, and compound
9 (9E)octadec-9-enamide, were kindly provided as a gift
from Dr. Bruce Hammock at University of California at
Davis.
2.2. Instruments
The HPLC system consisted of an Agilent 1100 LC Binary
pump system and an Agilent 1100 Autosampler (Palo Alto,
CA). The HPLC system was interfaced with a PE Sciex API
2000LC/MS/MS System including an electrospray ionization
source and a triple quadrupole mass analyzer (Applied Biosystems, Foster City, CA). Data were gathered by software Analyst
1.2. A microplate reader (VERSAMax , Molecular Devices, Sunnyvale, CA) was utilized for cell viability studies, enzyme
activity assays and protein assays.
2.3. Synthesis of styrene mercapturic acid (10, Scheme 2)
N-Acetylcysteine (232 mg, 1.4 mmol) was dissolved in
10 mL of phosphate buffer (pH 10). Styrene oxide (321 mg,
2.7 mmol) dissolved in 1.5 mL of DMSO was added to the Nacetylcysteine solution. The mixture was poured into 100 mL
of water after being stirred at room temperature under nitrogen
for 14 h. The resulting aqueous solution was washed with ethyl
acetate (3× 40 mL), and then acidified to pH 2.0 by 2.0N HCl.
The acidic solution was extracted by CHCl3 (3× 40 mL), and
extracts were pooled and dehydrated by anhydrous Na2 SO4 .
The organic solvent was removed by rotary evaporation, and
the mercapturic acid was purified by HPLC. 1 H NMR (CD3 OD)
δ 7.32 (m, 5H), 4.78 (m, 1H), 4.51 (m, 1H), 2.98 (m, 2H), 2.86
(m, 2H), 1.95 (s, 3H).
Scheme 2.
2.4. Cell culture
h2E1 and wild-type cells were cultured in RPMI 1640
supplemented with l-glutamine and 10% fetal bovine serum
(Hyclone Laboratory, Logan, UT) under 95% air/5% CO2 at
37 ◦ C.
2.5. Cytotoxicity assay
Before treatment, cells were counted with Trypan Blue
staining using a hemocytometer to ensure that a density of
2 × 105 cells/mL was reached. Chemicals of interest were
added to 96-well microplates containing h2E1 cells. The resulting plate was sealed with Secureseal to prevent possible evaporation of the chemicals tested. Cell viability was determined
by both Trypan Blue staining using a hemocytometer and by
CellTiter 96 Aqueous One solution assay. Cell viability assays
by CellTiter 96 Aqueous One were performed based on the
protocol provided by the manufacturer. Briefly, a CellTiter
96 reagent (20 ␮L) was added into each well of the 96-well
plate containing the cell samples in 100 ␮L of culture medium,
followed by incubation at 37 ◦ C in a humidified, 5% CO2
atmosphere for 4 h. The resulting absorbance at 490 nm was
measured by using 96-well microplate reader.
2.6. GSH/GSH ester protection study
Cells were counted to reach a density of 2 × 105 cells/mL.
Styrene (100 ␮M) was dissolved in DMSO (0.1%, v/v). Glutathione or glutathione ethyl ester was dissolved in phosphate
buffer to reach a final concentration of 0.5 mM. Cells were
incubated 24 h in 5% CO2 incubator at 37 ◦ C. Cell viability
was assessed by using both Trypan Blue staining and CellTiter
96 Aqueous One assay.
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2.7. Epoxide hydrolase inhibition
Similar procedures were performed as above. Compounds 8
or 9 in DMSO was added to cell samples followed by incorporation of styrene. The final concentrations of 8, 9, styrene, and
DMSO were 10 ␮M, 10 ␮M, 10 ␮M and 0.1% (v/v), respectively. Cell viability was determined after a 24 h incubation in
5% CO2 incubator at 37 ◦ C.
2.8. Reactive metabolite trapping and identification
h2E1 cells (6 × 106 cells) were harvested and re-suspended
in 1.0 mL phosphate buffer (10 mM, pH 7.4). Cells were lysed
by sonication in an ice-cold water bath, and the resulting
lysate was mixed with N-acetylcysteine (2.0 mM) and styrene
(500 ␮M) in phosphate buffer (10 mM, pH 7.4). The reaction
was initiated by the addition of NADPH (10 mM). The same
volume of vehicle was added to a separate reaction. After a 4 h
incubation period at 37 ◦ C, resulting samples were acidified to
pH 2 with 2N HCl, followed by extraction with ethyl acetate
(3× 0.5 mL). Extracts were pooled, and organic solvent was
evaporated using nitrogen. Residues were reconstituted using
50% acetonitrile in water and subjected to LC/MS/MS analysis.
An LC/MS/MS scan operated in multiple reaction
monitoring (MRM) mode for the ion transitions 284/162
(MH+ /MH+ -122) m/z was acquired. The LC conditions and
mass spectrometer parameters used were as follows. Five
to 20 ␮L samples were injected into a C18 column (3 ␮m,
2.1 mm × 100 mm, Alltech, Deerfield, IL). Samples were
eluted from the column with a gradient of solvent A (0.2%
aqueous formic acid) and solvent B (0.2% formic acid in
acetonitrile) at a flow rate of 200 ␮L/min. The flow was
directed to the mass spectrometer. The mass spectrometer
operating conditions were as follows: positive ionization
mode, ionization voltage at 5 kV, orifice potential at 30 V, ion
source temperature at 250 ◦ C. Collision activated dissociation
(CAD) of parent ions with m/z of 284 Da was performed using
N2 as collision gas in Q2 (collision cell). The collision energy
(CE) was 30 V. The mass transition monitored was 284/162
m/z, and the dwell time per transition was 200 ms.
3. Results and discussion
CYP2E1 has been reported to be one of major
cytochrome P450 enzymes responsible for the pulmonary bioactivation of styrene (Carlson, 1997b; Green
et al., 2001). To investigate if CYP2E1 is involved in
the cytotoxicity of styrene, we compared the toxicity of
styrene to h2E1 cells with that to wild-type cells. Dosedependent studies showed that styrene was toxic to h2E1
cells (IC50 = 121.8 ␮M), while no significant cell death
was observed after the wild-type cells were exposed to
styrene at the same concentration and even at a concentration as high as 1000 ␮M (Fig. 1a). This clearly
indicates that CYP2E1 plays a critical role in cytotoxicity induced by styrene in these cell lines. We also
Fig. 1. (a) Dose-dependent cell viability of h2E1 cells () and the wildtype (䊉) following exposure to styrene for 24 h; (b) dose-dependent
cell viability after h2E1 cells () and the wild-type (䊉) were exposed
to styrene-7,8-oxide for 24 h.
compared the susceptibility of h2E1 cells and the wild
type to styrene-7,8-oxide. Unlike the data obtained from
styrene studies, we found that h2E1 cells were as susceptible to styrene-7,8-oxide as the wild type (Fig. 1b).
This indicates the important role of styrene-7,8-oxide
metabolite in styrene-induced cytotoxicity. Our result is
consistent with the findings reported by Carlson; that
there are no differences in in vivo toxicity of styrene
oxide between the CYP2E1 knockout mice and the wild
type (Carlson, 2003, 2004c). Recent studies showed that
styrene respiratory tract toxicity in mice and rats, including mouse lung tumors, are mediated by CYP2F (Cruzan
et al., 2002). CYP2B was also reported to play a minor
role in styrene metabolism (Carlson et al., 1998). In vivo
studies demonstrated that CYP2E1 knockout mice can
metabolize styrene to a similar extent as the wild-type
mice (Carlson, 2003, 2004a,b). This indicates other CYP
enzymes, particularly CPY2F, also involve in bioactivation of styrene besides CYP2E1.
Styrene-7,8-oxide is the primary metabolite of
styrene and a possible metabolite responsible for styrene-
J.-K. Chung et al. / Toxicology 226 (2006) 99–106
induced toxicity. Styrene oxide has been reported
to induce genotoxicity and oxidative effects in vitro
(Trenga et al., 1991; Chakrabarti et al., 1993; Speit and
Henderson, 2005; Godderis et al., 2006). To investigate
the role of styrene oxide metabolite in cytotoxicity of
styrene, we conducted following studies. Both microsomal and soluble epoxide hydrolase are known to catalyze
hydration of epoxide compounds and to quench electrophilicity of epoxides. We reasoned that inhibition of
epoxide hydrolase may cause the accumulation of electrophilic epoxides. Employment of epoxide hydrolase
inhibitors allowed us to test the toxic role of epoxides in cytotoxicity induced by styrene. Compounds
8 and 9 have been reported to be selective inhibitors
of soluble epoxide hydrolase and microsomal epoxide
hydrolase, respectively (Morisseau et al., 2001, 2002).
The potentiating effect of epoxide hydrolase inhibitors
on the cytotoxicity of styrene was determined in h2E1
cells. Cells were exposed to styrene for 24 h in the presence or absence of the epoxide hydrolase inhibitors, and
then subjected to cell viability assays. There potentiating effect studies showed that neither styrene alone at
10 ␮M nor compounds 8 and 9 at 10 ␮M were found to
be toxic to the cells. As expected, the presence of either
compound 8 or compound 9 was found to dramatically
increase the cytotoxicity of styrene (Fig. 2). This indicates that epoxide hydrolase is important in the detoxification of styrene oxide, although spontaneous hydration
of styrene oxide was observed in aqueous solution at
physiological pH (data not shown). This also supports the
hypothesis that the styrene-7,8-oxide metabolite plays a
critic role in styrene toxicity.
Besides hydration, glutathione conjugation is another
important metabolic pathway that detoxifies electrophilic epoxides by oxirane ring opening. Glutathione
Fig. 2. Viability of h2E1 cells after exposure to styrene (10 ␮M), soluble epoxide hydrolase inhibitor (sEHI, 10 ␮M), microsomal epoxide
hydrolase inhibitor (mEHI, 10 ␮M) or mixtures for 24 h.
103
Fig. 3. Cell viability after exposure to GSH (500 ␮M) alone, GSH ester
(500 ␮M) alone, styrene (100 ␮M) alone, styrene (100 ␮M) + GSH
(500 ␮M), and styrene (100 ␮M) + GSH ester (500 ␮M).
protection was conducted to investigate the toxic effects
of epoxide metabolite of styrene. We tested the toxicity
of styrene in h2E1 cells in the presence of glutathione or
glutathione ethyl ester. Electrophilic epoxides are often
metabolized by nucleophilic glutathione spontaneously
and/or catalyzed by glutathione S-transferase. Cells were
pretreated with glutathione or its ester employed as a
modulator, followed by incubation with styrene. Glutathione was found to protect the cells from styrene, and
glutathione ester had a better protective effect (Fig. 3).
As shown in Fig. 3, styrene alone at a concentration of
100 ␮M resulted in cell death compared to the vehicle
control. The presence of GSH was found to decrease
styrene toxicity (25% less cell death than the control),
and the presence of GSH esters almost reversed the toxicity by styrene (2% cell death). In other words, GSH
ethyl ester protected cells against styrene cytotoxicity
to a greater degree than GSH. This may be attributed
to higher lipophilicity of GSH ester than that of GSH.
Higher lipophilicity increases GSH ester uptake into
the cells. Chemically, unsaturated carbon–carbon bonds,
Fig. 4. Dose-dependent cell viability after h2E1 cells were exposed
to styrene (), ethyl benzene (), vinyl cyclohexane (), and ethyl
cyclohexane (䊉) for 24 h.
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J.-K. Chung et al. / Toxicology 226 (2006) 99–106
including double and triple, are precursors for the formation of epoxides. We developed structure–activity
(toxicity) relationships of styrene to test if unsaturation is a prerequisite for styrene toxicity. A selection of
styrene analogues including ethylbenzene, vinyl cyclohexane, and ethyl cyclohexane were used as models for
the structure–activity relationship study. h2E1 cells were
exposed to styrene, ethylbenzene, vinyl cyclohexane,
and ethyl cyclohexane individually, and cell viability was
monitored. Cells were found to have different susceptibilities to the chemicals tested (Fig. 4). Ethylbenzene and
vinyl cyclohexane were as toxic as styrene, but toxicity
of ethyl cyclohexane to h2E1 cells was found negligible.
Ethylcyclohexane, which lacks a ␲ system as a precursor of epoxide metabolite, shows little cytotoxicity. This
indicates that the vinyl group of styrene is important in
its toxicity. However, the observed cytotoxicity of ethylbenzene implies that saturation of the vinyl group of
styrene does not necessarily eliminate styrene toxicity,
although the side chain has been considered as a precursor of epoxidation responsible for styrene toxicity. 4ethylphenol was reported as a metabolite of ethylbenzene
in microsome suggesting the formation of ethylbenzene3,4-oxide, a ring-oxide intermediate (Midorikawa et al.,
2004). Ethylbenzene has been reported to cause carcinoma in a number of organs and tissues including kidney,
testis, lung and liver (Chan et al., 1998; Stott et al., 2003).
It is suggested that carcinogenesis of ethylbenzene is
associated with its reactive metabolites through oxidation of its phenyl ring mediated by cytochrome P450
enzymes (Sequeira et al., 1994).
These observed potentiating effects of epoxide hydrolase inhibitors on styrene toxicity along with the
structure–activity relationship studies provides evidence
for the toxic effect of one or more epoxide metabolites
of styrene. To confirm the toxic role of styrene oxide
metabolite in styrene toxicity, we chemically identified the reactive metabolite. Many epoxides are unstable
Fig. 5. LC/MRM ion chromatogram profiles of: (a) incubation of h2E1 cell lysate with styrene (500 ␮M) trapped with N-acetylcysteine; (b)
incubation of h2E1 cell lysate with styrene (500 ␮M) and NADPH (10 mM) trapped with N-acetylcysteine; (c) authentic standard of styrene oxide
N-acetylcysteine conjugate. The mass transition 284/164 m/z was monitored under positive mode.
J.-K. Chung et al. / Toxicology 226 (2006) 99–106
and/or react with nucleophilic molecules in situ. Epoxide
instability makes it difficult to isolate and identify epoxide metabolites of styrene. To identify epoxide metabolites, the metabolites can be trapped by N-acetylcysteine,
and the structures of the epoxide metabolites may be
inferred by the corresponding N-acetylcysteine conjugates. We chemically synthesized styrene oxide Nacetylcysteine conjugate by reaction of styrene oxide
with N-acetylcysteine. The structure of the conjugate was
confirmed by NMR and mass spectrometry. To confirm
the formation of styrene oxide metabolite in h2E1 cells,
styrene was incubated with h2E1 cell homogenates in
the presence of N-acetylcysteine as a trapping agent for
styrene oxide metabolite. The resulting mixtures were
analyzed by LC/MS/MS under MRM scanning mode.
The ion pairs monitored were 284/162 m/z in positive mode. A peak (retention time = 16.8 min, Fig. 5a)
responsible for styrene oxide-derived N-acetylcysteine
conjugate was observed in the incubation of h2E1
cell homogenates with styrene in the presence of Nacetylcysteine. Its identity in both chromatographic and
mass spectrometric behavior was found to be the same
as that of the synthetic standard (Fig. 5c). In addition,
we determined whether the formation of styrene oxide
metabolite is NADPH-dependent and found that the
presence of NADPH significantly increased the formation of styrene oxide-derived N-acetylcysteine conjugate
(about three-fold) detected by LC/MS/MS (Fig. 5b). This
further supports the hypothesized role of cytochrome
P450 in bioactivation of styrene.
In summary, cytotoxicity of styrene requires
metabolic activation mediated by cytochrome P450
enzymes, and CYP2E1 is one of oxidative enzymes
bioactivating styrene. Inhibition of both soluble and
microsomal epoxide hydrolases potentiates cytotoxicity of styrene, and glutathione conjugation diminishes
styrene toxicity. Therefore, the epoxide metabolite is
responsible for the styrene-induced cytotoxicity.
Acknowledgements
This work was supported by the National Institutes
of Health (Grant HL 080226). We also thank Dr. Bruce
Hammock of UC Davis for his generous gift of epoxide
hydrolase inhibitors.
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