New urinary metabolites formed from ring-oxidized metabolic intermediates of styrene

Article abstract of DOI:10.1021/tx9004192

The urine from mice exposed to styrene vapors (600 and 1200 mg/m ³, 6 h) was analyzed for ring-oxidized metabolites of styrene. To facilitate the identification of metabolites in urine, the following potential metabolites were prepared: 2-, 3-, and 4-vinylphenol (2-, 3-, and 4-VP), 4-vinylpyrocatechol, and 2-, 3-, and 4-vinylphenylmercapturic acid (2-, 3-, and 4-VPMA). For the analysis of vinylphenols β-glucuronidase-treated urine was extracted and derivatized with acetanhydride/triethylamine before injection into GC/MS. Three isomers, 2-, 3-, and 4-VP, were found in the exposed urine using authentic standards. Additionally, three novel minor urinary metabolites, arylmercapturic acids 2-, 3-, and 4-VPMA, were identified by LC-ESI-MS ² by comparison with authentic standards. Excretion of the most abundant isomer, 4-VPMA, amounted to 535 ± 47 nmol/kg and 984 ± 78 nmol/kg, representing approximately 0.047 and 0.043% of the absorbed dose for the exposure levels of 600 and 1200 mg/m³, respectively. The ratio of 2-VPMA, 3-VPMA, and 4-VPMA was approximately 2:1:6. In model reactions of styrene 3,4-oxide (3,4-STO) with N-acetylcysteine in aqueous solutions and of its methyl ester in methanol, 4-vinylphenol was always the main product, while 3-vinylphenol has never been detected. No mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine in aqueous solution at pH 7.4 or 9.7, but a small amount of 4-VPMA methyl ester was detected by LC-ESI-MS after the reaction of 3,4-STO with N-acetylcysteine methyl ester. In contrast, no mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine in aqueous solution at pH 7.4 or 9.7. These findings indicate a capability of 3,4-STO to react with cellular thiol groups despite its rapid isomerization to vinylphenol in an aqueous environment. Moreover, the in vivo formation of 2- and 3-isomers of both VP and VPMA, neither of which was formed from 3,4-STO in vitro, strongly suggests that another arene oxide, styrene 2,3-oxide, might be a minor metabolic intermediate of styrene.

Full text of DOI:10.1021/tx9004192

Chem. Res. Toxicol. 2010, 23, 251–257
251
New Urinary Metabolites Formed from Ring-Oxidized Metabolic
Intermediates of Styrene
,†
‡
†
†
‡
ˇ
Igor Linhart,* Jaroslav Mra´z, Jan Scharff, Jan Krouzˇelka, Sa´rka Dusˇkova´,
Hana Nohova´,‡ and L’udmila Vodicˇkova´‡
Department of Organic Chemistry, Faculty of Chemical Technology, Institute of Chemical Technology, Prague,
ˇ
Technicka´ 1905, CZ-166 28 Prague, Czech Republic, and National Institute of Public Health, Sroba´roVa 48,
CZ-100 42 Prague, Czech Republic
ReceiVed NoVember 23, 2009
The urine from mice exposed to styrene vapors (600 and 1200 mg/m3, 6 h) was analyzed for ring-oxidized
metabolites of styrene. To facilitate the identification of metabolites in urine, the following potential metabolites
were prepared: 2-, 3-, and 4-vinylphenol (2-, 3-, and 4-VP), 4-vinylpyrocatechol, and 2-, 3-, and
4-vinylphenylmercapturic acid (2-, 3-, and 4-VPMA). For the analysis of vinylphenols ꢀ-glucuronidase-treated
urine was extracted and derivatized with acetanhydride/triethylamine before injection into GC/MS. Three
isomers, 2-, 3-, and 4-VP, were found in the exposed urine using authentic standards. Additionally, three
novel minor urinary metabolites, arylmercapturic acids 2-, 3-, and 4-VPMA, were identified by LC-ESI-MS2
by comparison with authentic standards. Excretion of the most abundant isomer, 4-VPMA, amounted to 535
( 47 nmol/kg and 984 ( 78 nmol/kg, representing approximately 0.047 and 0.043% of the absorbed dose for
the exposure levels of 600 and 1200 mg/m3, respectively. The ratio of 2-VPMA, 3-VPMA, and 4-VPMA was
approximately 2:1:6. In model reactions of styrene 3,4-oxide (3,4-STO) with N-acetylcysteine in aqueous
solutions and of its methyl ester in methanol, 4-vinylphenol was always the main product, while 3-vinylphenol
has never been detected. No mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine in
aqueous solution at pH 7.4 or 9.7, but a small amount of 4-VPMA methyl ester was detected by LC-ESI-MS
after the reaction of 3,4-STO with N-acetylcysteine methyl ester. In contrast, no mercapturic acid was found
in the reaction of 3,4-STO with N-acetylcysteine in aqueous solution at pH 7.4 or 9.7. These findings indicate
a capability of 3,4-STO to react with cellular thiol groups despite its rapid isomerization to vinylphenol in an
aqueous environment. Moreover, the in ViVo formation of 2- and 3-isomers of both VP and VPMA, neither
of which was formed from 3,4-STO in Vitro, strongly suggests that another arene oxide, styrene 2,3-oxide,
might be a minor metabolic intermediate of styrene.
Scheme 1. Main Metabolic Routes of Styrenea
Introduction
Biotransformation of styrene in both humans and rodents
(Scheme 1) proceeds mainly through an oxidation at the vinyl
group. Styrene 7,8-oxide (7,8-STO1) is the key reactive me-
tabolite, which is further biotransformed by two main metabolic
routes, i.e., epoxide hydrolase-catalyzed hydrolysis and glu-
tathione (GSH) conjugation. More than 90% of the styrene
biotransformation proceeds via 7,8-STO (1). 4-Vinylphenol (4-
VP) is the only metabolite with an intact vinyl group hitherto
identified. Its detection in rat urine was first reported by Bakke
and Scheline (2). Pantarotto et al. suggested that 4-VP is
indicative of arene oxidation in the metabolism of styrene (3).
It is most likely formed by a rearrangement of styrene 3,4-oxide
(3,4-STO), known as NIH shift (4, 5). 4-VP was found also in
the urine of workers exposed to styrene (6–8).
Watabe et al. studied the mutagenicity of 3,4-STO and its
isomers, 1,2-STO and 7,8-STO, toward Salmonella typhimurium.
* Corresponding author. Igor Linhart, Department of Organic Chemistry,
ICT Prague, Technicka´ 1905, CZ-166 28, Prague, Czech Republic. Tel:
a More than 90% of biotransformation proceeds via 7,8-STO, while 3,4-
STO is a minor metabolic intermediate.
+420 220 444 165. E-mail: linharti@vscht.cz.
† Institute of Chemical Technology, Prague.
‡ National Institute of Public Health.
1 Abbreviations: STO, styrene oxide; VP, vinylphenol; VPC, vinylpy-
rocatechol; VPMA, vinylphenylmercapturic acid; ACC, N-acetyl-L-cysteine;
MeACC, N-acetyl-L-cysteine methyl ester; MAP, mercapturic acid pathway;
MNA,mandelicacid;DCM,dichloromethane;DBU,1,8-diazabicyclo[5.4.0]undec-
7-ene; MCPBA, m-chloroperoxybenzoic acid.
Despite its instability in aqueous solutions (isomerization to
4-VP with a half time as short as 4 s at pH 7.4), 3,4-STO was
found to be more mutagenic and more cytotoxic than its more
stable isomers (9). It has been assumed that although 4-VP
10.1021/tx9004192  2010 American Chemical Society
Published on Web 12/18/2009

252 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Linhart et al.
CH2S); 3.34 (dd, J ) 13.8 and 4.6 Hz, 1H, CH2S); 4.12 (m, 1H,
SCH2CH); 5.26 (d, J ) 10.8 Hz, 1H, CH2dCH); 5.87 (d, J ) 17.6
Hz, 1H, CH2dCH); 6.68 (dd, J ) 17.7 and 10.9 Hz, 1H, CH2dCH);
7.20 (m, 3H, aromatic CH), 7.40 (s, 1H, aromatic CH).
ESI-MS: m/z 266 (M + H)+, 264 (M - H)-. MS2 at 266: m/z
248 (MH - H2O)+, 224 (MH - CH2CO)+; 130 (MH -
CH2CHC6H4SH)+. MS2 at 264: m/z 135 (M - CH2CH(COOH)-
NHCOCH3)-.
reflects a minor biotransformation pathway of styrene, i.e., the
metabolic activation to 3,4-STO, this pathway may underlie a
substantial part of the cytogenetic damage associated with
styrene exposure (8). The role of arene oxidation in the
biotransformation of styrene and its toxicological significance
should be clarified. Some other phenolic metabolites, e.g.,
4-hydroxymandelic acid (3) and 2-(4-hydroxyphenyl)ethanol
(10) have been identified; however, no VP isomers, which would
indicate a formation of arene oxides other than 3,4-STO, have
been reported.
The aim of this study was to assess the possible formation
of other metabolites derived from ring oxidation of styrene,
namely, vinylphenols or vinylphenylmercapturic acids. These
metabolites would indicate the ring oxidation of styrene, which
has been shown to be a relevant toxicity-related event (8, 9).
4-Vinylphenylmercapturic Acid (4-VPMA). 1H NMR (DMSO-
d6): δ ) 1.74 (s, 3H, CH3CO); 3.11 (dd, J ) 12.3 and 6.5 Hz, 1H,
CH2S); another signal of CH2S (AB system) overlapped by HOD;
4.05 (m, 1H, SCH2CH); 5.16 (d, J ) 11.1 Hz, 1H, CH2dCH); 5.76
(d, J ) 17.9 Hz, 1H, CH2dCH); 6.65 (dd, J ) 17.7 and 10.8 Hz,
1H, CH2dCH); 7.23 (d, J ) 8.2 Hz, 2H, aromatic CH); 7.34 (d, J
) 8.2 Hz, 2H, aromatic CH).
ESI-MS: m/z 266 (M + H)+, 264 (M - H)-. MS2 at 266: m/z
248 (MH - H2O)+, 224 (MH - CH2CO)+; 130 (MH - CH2-
CHC6H4SH)+. MS2 at 264: m/z 135 (M - CH2CH(COOH)-
NHCOCH3)-.
Experimental Procedures
General. Acetonitrile for LC/MS Chromasolv was from Riedel
de Hae¨n, formic acid, puriss. p.a. from Fluka, and 3-chloroperoxy-
benzoic acid also purchased from Fluka, and the actual content of
the peracid was determined by iodometric titration. 3- and 4-Vi-
nylphenylboronic acids were from Aldrich, and 2-vinylphenylbo-
ronic acid was from Molekula (Germany). Other chemicals were
of analytical grade and were used as received. Redistilled water
was used for LC/MS and for SPE. ꢀ-Glucuronidase type H-2 from
Helix pomatia (EC 3.2.1.31), glucuronidase activity g100,000 units/
mL with residual sulfatase activity e7,500 units/mL, was from
Sigma. Analytical standards of vinylphenols, 2-VP (11), 3-VP (12),
4-VP (13), and 4-VPC (11), were prepared as described in the
literature and identified comparing their 1H NMR spectra with those
reported therein.
GC/MS analyses were carried out using a gas chromatograph-ion
trap mass spectrometric system GCQ (Thermo Finnigan). LC/MS
analyses were carried out on a Thermo Scientific LXQ linear trap
mass spectrometer in tandem with a Janeiro LC system consisting
of two Rheos 2200 pumps and a CTC PAL autosampler. NMR
spectra were taken on a Varian Gemini 300 MHz spectrometer.
Vinylphenylmercapturic Acids. Three isomeric mercapturic
acids, 2-, 3-, and 4-VPMA, were prepared by alkaline hydrolysis
of their methyl esters whose synthesis is described elsewhere (14).
The methyl ester of the corresponding mercapturic acid (30.6 mg,
0.110 mmol) was dissolved in a mixture of 2 mL of methanol and
4 mL of water, 120 µL of 1 M aqueous solution of NaOH was
added, and the mixture was stirred at room temperature for 2 h.
The solution was then neutralized with 120 µL of 1 M hydrochloric
acid, diluted with 30% aqueous methanol to 58 mL, resulting in a
0.5 mg/mL solution of VPMA which was then used for calibration.
Analytical samples of 3- and 4-VPMA in the form of ammonium
salts were obtained as follows. Aliquots (50 mL) of VPMA solutions
were partially evaporated in a vacuum to remove methanol.
Resulting turbid solutions were then diluted with water to 50 mL,
acidified with 1 M HCl to pH 2.5, and extracted with DCM (3 ×
20 mL). Extracts were dried with anhydrous sodium sulfate and
filtered, diluted ammonium hydroxide (1:5, 100 µL) was added,
and the extracts were evaporated to dryness in a vacuum to afford
solid residues.
Preparation of Styrene 3,4-Oxide (3,4-STO). 3,4-STO was
synthesized in 4 steps from 2-(cyclohexa-1,4-dien-1-yl)ethanol. In
brief, its bromination according to Watabe et al. (15) afforded a
mixture of 2-(1,6-dibromo-cyclohex-3-en-1-yl)ethanol and 2-(4,5-
dibromo-cyclohex-1-en-1-yl)ethanol. Isolation by column chroma-
tography on silica gel afforded 30% of the former.
Bromination of 2-(1,6-Dibromo-cyclohex-3-en-1-yl)ethanol
with Bromotriphenyl-phosphonium Bromide. To a suspension
of freshly prepared Ph3PBr2 (6,03 g, 13.3 mmol) in 30 mL of DCM,
a solution of 3.38 g (11.9 mmol) of 2-(1,6-dibromo-cyclohex-3-
en-1-yl)ethanol in 20 mL of DCM was added dropwise under
external cooling with crushed ice. The cooling bath was then
removed, and the reaction mixture was stirred for 20 h at room
temperature. The resulting yellowish solution was washed subse-
quently with saturated aqueous solutions of sodium bicarbonate and
sodium chloride, and dried with potassium carbonate. DCM was
removed by evaporation in a vacuum, and the solid residue was
then extracted with three 40 mL portions of hexane. The resulting
hexane solution was evaporated to dryness yielding 3.76 g (91%)
of the crude product, which was purified by flash chromatography
(10:1 hexane/DCM) to yield 1.66 g (40%) of pure 4,5-dibromo-
4-(2-bromoethyl)-1-cyclohexene identified by comparing its 1H
NMR spectra with that reported in the literature (15).
Oxidation of 4,5-Dibromo-4-(2-bromoethyl)-1-cyclohexene
with m-Chloroperoxybenzoic Acid (MCPBA). A solution of 4,5-
dibromo-4-(2-bromoethyl)-1-cyclohexene (1.032 g, 2.97 mmol) and
MCPBA (0.914 g, 80% pure, 4.24 mmol) in 15 mL of chloroform
was stirred for 24 h at 40 °C. The reaction mixture was then washed
subsequently with saturated aqueous solutions of sodium bisulphite
(20 mL), sodium bicarbonate (20 mL), and sodium chloride (20
mL), and dried with magnesium sulfate. Evaporation of the solvent
in a vacuum yielded 0.979 g (91%) of a crude product, which was
purified by flash chromatography (5:1 hexane/ethyl acetate) to afford
0.705 g (65%) of white crystals, mp 83-85 °C, identified by
comparing the 1H NMR spectrum with that reported in the literature
as 4,5-dibromo-4-(2-bromoethyl)cyclohexane 1,2-oxide.
Elimination with 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU).
4,5-Dibromo-4-(2-bromoethyl)cyclohexane 1,2-oxide (135 mg,
0.372 mmol) was placed in a reaction flask, which was then
evacuated and rinsed with argon to remove air and moisture. Dry
ether (4 mL) was added through a septum followed by 330 µL
(2.21 mmol) of DBU. The reaction mixture was stirred for 20 h at
room temperature under argon. A white precipitate was gradually
formed while the solution became intensely yellow. The reaction
flask was then evacuated to remove the ether, and the residue was
extracted with three portions of pentane (3 × 9 mL). Combined
pentane solutions were washed quickly with a saturated aqueous
bicarbonate solution (10 mL) followed by brine (10 mL). After
1 h of drying over sodium sulfate, a clear yellow solution was
evaporated in a vacuum evaporator, which was thoroughly dried
and protected from atmospheric moisture by a drying tube filled
with molecular sieves. Styrene 3,4-oxide (30 mg, 67%) was obtained
2-Vinylphenylmercapturic Acid (2-VPMA). 1H NMR (DMSO-
d6): δ ) 1.76 (s, 3H, CH3CO); 3.08 (dd, J ) 12.9 and 7.6 Hz, 1H,
CH2S); 3.31 (dd, J ) 12.9 and 5.3 Hz, 1H, CH2S); 4.14 (m, 1H,
SCH2CH); 5.30 (d, J ) 11.0 Hz, 1H, CH2dCH); 5.70 (d, J ) 17.6
Hz, 1H, CH2dCH); 7.09 (dd, J ) 17.7 and 11.0 Hz, 1H, CH2dCH);
7.20 (m, 1H, aromatic CH), 7.40 (d, J ) 6.7 Jz, 1H, aromatic CH);
7.52 (dd, J ) 7.3 and 1.5 Hz, 1H, aromatic CH); 7.82 (d, J ) 7.6
Hz, 1H, aromatic CH).
ESI-MS: m/z 266 (M + H)+, 264 (M - H)-. MS2 at 266: m/z
248 (MH - H2O)+, 224 (MH - CH2CO)+, 130 (MH - CH2CHC6-
H4SH)+. MS2 at 264: m/z 135 (M - CH2CH(COOH)NHCOCH3)-.
3-Vinylphenylmercapturic Acid (3-VPMA). 1H NMR (DMSO-
d6): δ ) 1.75 (s, 3H, CH3CO); 3.13 (dd, J ) 13.8 and 9.1 Hz, 1H,

Ring Oxidized Metabolites of Styrene
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 253
1
as a yellow liquid and identified by H NMR and GC/MS as 3,4-
respectively. The detector was operated in EI mode (70 eV), and
full scan data were collected in the mass range m/z 50-400.
Analyses for vinylphenols were carried out at m/z 120 (M -
CH2CO)+. For VPC (M ) 220), the transition 178 f 136 was
monitored in the SRM mode.
STO (15). It contained only a trace of 4-VP.
Model Reaction of 3,4-STO with N-Acetylcysteine Methyl
Ester (MeACC). To a solution of MeACC (26 mg, 0.148 mmol)
in methanol (5 mL), sodium hydride (5.9 mg, 60% suspension in
mineral oil, 0.148 mmol) was added to generate the thiolate anion.
The reaction mixture was stirred under argon and cooled externally
with crushed ice. Freshly prepared 3,4-STO (15 mg, 0.125 mmol)
in pentane (4 mL) was added. After 2 h, the solvents were removed
in a vacuum, the residue was redissolved in diethyl ether (10 mL),
and the resulting turbid ether solution was washed with brine (10
mL) followed by water (10 mL) and dried with magnesium sulfate.
The extract was then filtered, evaporated to dryness in a vacuum
yielding 25 mg of oily product. 1H NMR analysis confirmed 4-VP
as the predominating product. The product was further analyzed
by LC/MS using a 250 × 2 mm Phenomenex Gemini C18 column,
particle size 5 µm, eluted with aqueous methanol, the concentration
of which was increased linearly from 50 to 80% in 20 min and
then held for another 10 min at a flow rate of 200 µL/min. ESI-
MS (positive ions) was taken in full scan m/z 100-400. A peak
corresponding to 4-VPMA methyl ester was detected showing ions
at m/z 280 (M + H)+, 302 (M + Na)+, and 318 (M + K)+.
Model Reaction of 3,4-STO with N-Acetylcysteine (ACC)
in Aqueous Solutions. To a 0.1 M solution of ACC (2 mL), the
pH of which was adjusted with ammonium hydroxide to 7.4 or
9.7, freshly prepared 3,4-STO (4 mg) in pentane (4 mL) was added
in four portions of 1 mL each. After each addition, the reaction
mixture was stirred vigorously for 20 min, and pentane was removed
by evaporation in a vacuum. The yellow color of the pentane
solution disappeared rapidly. To ensure the conversion of possible
primary hydroxycyclohexadienyl adducts to vinylphenylmercapturic
acids, the reaction mixtures were acidified with acetic acid to pH
2, incubated for 1 h at 37 °C, and then neutralized with aqueous
ammonia. In a control experiment without ACC, 3,4-STO was
added in the same way to a 20 mM ammonium formate buffer (pH
7.4 or 9.7). The resulting solutions were analyzed by LC with UV
detection and by LC/MS. A peak coeluting with 4-VP was detected
by UV detection as a sole major product uniformly in all samples,
whereas VPMA was not detected.
Animal Treatment. Adult male NMRI mice (mean weight 29 g)
were exposed for 6 h to styrene vapor in a dynamic exposure
chamber with controlled concentrations of 600 and 1200 mg/m3.
Animals were divided into five groups, six animals per group. Each
group was placed into a glass metabolic cage with free access to
food and water. To enhance diuresis, sucrose (8 mg/mL) was added
to the drinking water. Animals were exposed by inhalation, two
groups at each concentration level, while one group remained
unexposed. Urine was collected 24 h after the beginning of the
exposure. During sample collection, we filtered the urine through
a gauze filter to remove pieces of feces and crumbs of food pellets.
The walls of the metabolic cages were rinsed with distilled water,
and the resulting solution was added to the main portion of collected
urine. Samples were stored at -20 °C until analyzed. Animal
experiments were approved by the Central Committee for Animal
Protection of the Czech Republic.
Analysis for the Phenolic Metabolites. To the urine samples
(2 mL) adjusted with 10 M acetic acid to pH 5.0-5.5, a suspension
of ꢀ-glucuronidase from Helix pomatia was added (10 µL). The
samples were incubated at 37 °C for 1 h and then extracted with
ethyl acetate (4 mL). The extract was evaporated to dryness, and
the residue was acetylated with a mixture of acetonitrile/acetan-
hydride/triethylamine at 3:1:1 (100 µL) at room temperature for
20 min. The solvents were again evaporated to dryness, and the
residue was dissolved in ethyl acetate (50 µL) and injected onto a
DB-5 fused silica capillary column 30 m × 0.25 mm I.D., film
thickness of 0.25 µm (J&W). The injector was set in splitless mode
(1 min). Helium was used as a carrier gas at a linear velocity of 30
cm/s. Temperature of the column was held at 60 °C for 1 min,
then raised at 10 °C/min to 100 °C, and thereafter at 40 °C/min to
300 °C, which was held for 4 min. Temperatures of the injector,
transfer line, and ion source were 300 °C, 250 °C, and 180 °C,
Analysis of Vinylphenylmercapturic Acids by LC/MS. Urine
samples were filtered through 0.2 µm Nylon membrane filters, and
1 mL aliquots were diluted with 2 mL of 0.1 M HCl resulting in
pH 1-2 and left standing for 1 h. Samples were then transferred
onto Oasis HLB 60 mg SPE columns (Waters), which were
preconditioned with 2 mL of methanol followed by 2 mL of water.
Columns were washed with 2 mL of 20% aqueous methanol and
then eluted with 2 mL of 80% aqueous methanol. Eluates were
evaporated to dryness in a stream of nitrogen at 37 °C and
redissolved in 1 mL of 20% aqueous methanol. Calibration solutions
of 4-VPMA in blank urine were worked up in the same way. The
calibration curve was linear in the range of concentration 0.2-5
µg/mL, R > 0.999. Aliquots of 10 µL were injected on a 250 × 2
mm Phenomenex Luna C18(2) column, particle size 5 µm, which
was eluted isocratically with 30% acetonitrile in 0.1% aqueous
formic acid at a flow rate of 200 µL/min. Full scan ESI-MS spectra
were collected in the mass range of m/z 100-400. Collision
activated daughter spectra (MS2) were generated from the parent
ions m/z 264 and 266 for negative and positive ion modes,
respectively, using a collision energy of 20 V for both modes.
Helium was used as the collision gas. Both negative and positive
ions were used for the detection and identification of VPMA
isomers, whereas only negative ions were used for the quantification
of 4-VPMA.
Results and Discussion
Authentic Standards. To ensure unequivocal identification
of the possible metabolites arising from ring oxidation of styrene,
authentic samples of 2-, 3-, and 4-vinylphenol, 4-vinylpyrocat-
echol, as well as 2-, 3-, and 4-vinylphenylmercapturic acid were
prepared and characterized by NMR and mass spectra. 4-VP
was prepared by hydrolysis of commercially available 4-ac-
etoxystyrene (13). 2-VP and 4-VPC were prepared by micro-
wave induced decarboxylation of 2-hydroxycinnamic and caffeic
acid, respectively (11). The same procedure failed to give a
preparatively useful yield of 3-vinylphenol; therefore, this isomer
was prepared from 3-hydroxycinnamic acid by a classical
copper-catalyzed decarboxylation (12). Preparation of 3-VPMA
and 4-VPMA methyl esters by S-arylation of MeACC with
corresponding vinylphenylboronic acids is described elsewhere
(14). Their alkaline hydrolysis afforded authentic samples of
2-, 3-, and 4-VPMA.
Phenolic Metabolites. Mice were exposed to styrene vapor
at two concentration levels, 600 and 1200 mg/m3 for 6 h. To
release phenolic metabolites from their conjugates, urine was
treated with ꢀ-glucuronidase/arylsulfatase, neutralized, and
extracted with ethyl acetate. The extracts were then acetylated
to improve the chromatographic properties of the analytes.
Systematic GC/MS analysis of the neutral extracts using
authentic standards indicated the presence of 3-VP, 4-VP, and
probably also 2-VP isomer. The latter could not be unequivo-
cally identified because of its insufficient separation from other
styrene metabolites. In the exposed urine samples, a cluster of
incompletely separated peaks appeared at m/z 120 near the
retention time of the authentic 2-VP (Figure 1). Our attempts
to improve separation by changing the column temperature
gradient were unsuccessful. 4-VPC was present in both exposed
and control urine at similar concentration levels so that its
occurrence cannot be attributed to styrene exposure. In fact, it
has been reported that in both rodents and humans this
compound is formed by intestinal microflora from caffeic acid,
which is present in the diet (16).

254 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Linhart et al.
Figure 1. GC/MS chromatograms of vinylphenols (m/z 120) in the
neutral extracts of urine: (a) control urine; (b) urine from mice exposed
to styrene at the concentration of 1200 mg/m3 for 6 h; (c) urine from
exposed mice spiked with authentic 2-, 3-, and 4-VP; (d) standards of
2-, 3-, and 4-VP.
Mercapturic Acids. In our initial search for VPMAs, acidic
extracts of mice urine were methylated and analyzed by GC/
MS. No VPMA was detected in the full scan or SIM modes,
but in SRM mode using a single transition of m/z 220 f 161,
3- and 4-VPMA isomers were detected in the form of their
methyl esters. Ultimate identification of all three VPMA isomers
was achieved by LC/MS. Urine samples were purified by SPE,
and the extracts were analyzed by LC-ESI-MS2 in both positive
and negative ion modes. Peaks coeluting with the authentic
standards of 2-, 3-, and 4-VPMA were detected (Figure 2).
Fragmentation of the quasimolecular ion (M + H)+ at m/z 266
in all VPMA isomers gave characteristic main fragments at m/z
248 (MH - H2O)+ and 224 (MH - CH2CO)+. Further
fragmentation of these daughter ions revealed significant dif-
ferences between 2-VPMA and the other two isomers. In Figures
3 and 4, the fragmentation patterns (MS2 and MS3) of 2-VPMA
with those of 3- and 4-VPMA are compared. While 2-VPMA
tends to stabilize the fragment ions by intramolecular interaction
with the ortho vinyl group giving thiatropylium ions and/or by
the stabilization of three membered thiiranium ions through
interaction with the vinyl π-electron (Figure 3), these stabilizing
effects are not possible with the more distant vinyl group in
the 3- and 4-isomers, which did not differ significantly in their
fragmentation (Figure 4). However, in the negative ion mode,
Figure 2. LC/MS chromatograms, MS2 at m/z 264 for (M - H)- of
VPMA. Blank urine (upper trace), urine from mice exposed to styrene,
1200 mg/m3 for 6 h (middle trace), and authentic samples of 2-, 3-,
and 4-VPMA (lower trace).
the quasimolecular ion (M - H)- at m/z 264 gave uniformly a
single fragment at m/z 135 (CH2dCHC6H4S-). Negative ion
MS2 spectra, which gave a better signal-to-noise ratio in urine
samples, were used for quantitative determination of the major
isomer, 4-VPMA. Its concentrations found in diluted urine
samples were 0.75 ( 0.10 mg/L and 1.09 ( 0.07 mg/L
corresponding to the excretion of 535 ( 47 nmol/kg and 984
( 78 nmol/kg for the lower and higher exposures, respectively.
As the formation of VPMAs proceeds through dehydration
of the corresponding premercapturic acids (Scheme 2), a portion
of the latter may have been still present in the native urine. To
ensure the full conversion of the premercapturic acids to
VPMAs, urine samples were acidified to pH 1-2 and allowed
to stand in these acidic solutions for 1 h. Then, the total
4-VPMA should correspond to the sum of 4-VPMA and its
premercapturic acid, i.e., N-acetyl-S-(4-vinyl-6-hydroxycyclo-
hexa-2,4-dien-1-yl)cysteine, which may have also been present
in the urine.

Ring Oxidized Metabolites of Styrene
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 255
Figure 3. Mass fragmentation pattern and MS2 and MS3 spectra of 2-VPMA.
Figure 4. Mass fragmentation pattern and MS2 and MS3 spectra of 3-VPMA and 4-VPMA. As these isomers showed the same mass fragmentation,
MS2 and MS3 spectra of 3-VPMA only are shown.

256 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Linhart et al.
Scheme 2. Proposed Biotransformation Routes Leading to Three Isomeric Vinylphenylmercapturic Acids from Styrenea
a GS ) S-glutathionyl; M.A.P. ) mercapturic acid pathway.
Assuming that the retention of styrene in lungs was 0.55 and
the minute ventilation volume of mice was 1 L/min per kg body
weight (17), the absorbed dose of styrene during the exposure
can be estimated to 1.14 and 2.28 mmol/kg at 600 and 1200
mg/m3, respectively. Hence, metabolic conversion to 4-VPMA
amounted to approximately 0.05 and 0.04% of absorbed dose
for the lower and the higher exposure levels, respectively. The
ratio of 4-VPMA/3-VPMA/2-VPMA was approximately 6:1:2
as determined from the corresponding peak areas.
Model Reactions of Styrene 3,4-Oxide. The chemical
reactivity of 3,4-STO was tested in model reactions with ACC
in aqueous solutions at pH 7.4 and 9.7 and with MeACC in
methanol. 3,4-Styrene oxide was prepared by a substantially
modified procedure of Watabe et al. (15) immediately before
addition to the model reaction mixture. Freshly prepared samples
contained only traces of 4-VP but isomerized to 4-VP within
24 h in deuterochloroform (in the NMR cuvette). The yellow
color, which indicated the presence of oxepin (a tautomeric form
of 3,4-STO), disappeared immediately after dissolution in water,
methanol, or in the model reaction mixture giving 4-VP as the
only product, which could be isolated and identified by 1H NMR.
4-VP has always been the predominating product, whereas only
a trace of 4-VPMA methyl ester was detected by LC-ESI-MS
when the reaction was performed in methanol. Collisionally
activated MS2 spectrum of the quasimolecular ion MH+ at m/z
280 gave fragments at m/z 248 (MH - CH3OH)+, 238 (MH -
CH2CO)+, 221 (M - CH3CONH2)+, 206 (MH - CH3OH -
CH2CO)+, and 144 (MH - CH2CHC6H4SH)+. In contrast, in
the model reactions performed in aqueous solutions no VPMA
was detected. Unlike in mice urine, no isomers other than 4-VP
or 4-VPMA could be detected in the model reactions.
lead exclusively to 4-VP. Formation of the 3-VP isomer in ViVo
can be therefore explained by an enzymatic process with a
different mechanism than NIH shift, namely, an epoxide hydrase
catalyzed hydration-dehydration sequence, starting either from
3,4- or 2,3-STO. Ring oxidation to 2,3-STO could also explain
the formation of 2-VP as a product of NIH shift as well as both
2- and 3-VPMA isomers.
2,3-STO and 3,4-STO may react with cellular GSH yielding
two isomeric primary conjugates each as shown in Scheme 2.
Glutathione conjugation, which is probably catalyzed by glu-
tathione-S-transferases, gives rise ultimately to three mercapturic
acids, 2-, 3-, and 4-VPMA. A similar biotransformation pathway
consisting of dehydration of the primary conjugates and
subsequent cleavage of γ-glutamyl and glycyl moieties followed
by N-acetylation has been proposed for naphthalene (19) and
benzene (20). Spontaneous dehydration may occur at all stages
of the metabolic sequence leading from glutathione conjugates
to corresponding arylmercapturic acids, which are the metabolic
end-products excreted in urine, as well as during acidic sample
treatment. After complete conversion of premercapturic acids
to the ultimate arylmercapturic acids, the latter urinary metabo-
lites reflect the extent of arene oxidation. For example, phe-
nylmercapturic acid is a useful urinary biomarker of occupa-
tional and environmental exposure to benzene and its metabolic
activation to benzene oxide (20, 21).
Conclusions. New ring oxidized metabolites of styrene, 2-
and 3-VP and 2-, 3-, and 4-VPMA bring new evidence for the
formation of electrophilic arene oxides in the course of styrene
metabolism. In addition to styrene 3,4-oxide, which has been
assumed to be a precursor to 4-VP as the only previously
identified VP isomer, another arene oxide, most likely styrene
2,3-oxide, is needed to plausibly explain our findings of new
metabolites, namely, 2- and 3-VPMA, 2-VP, and possibly also
3-VP.
Mechanistic Considerations. Formation of VPMA isomers
in ViVo represents an additional strong evidence of metabolic
ring oxidation of styrene. Theoretically, three isomeric arene
oxides, 1,2-, 2,3-, and 3,4-STO can be formed by this metabolic
process. However, formation of 1,2-STO is rather unlikely
because 1-substituted arene oxides cannot be easily formed by
arene oxidases because of steric hindrance (18).
In accordance with theoretical predictions, which were also
confirmed experimentally by model reactions of toluene 3,4-
oxide (18) as well as of 3,4-STO, NIH shift of 3,4-STO should
Acknowledgment. Financial support of this work by grant
numbers 2B08051 and MSM 604 613 73 01 from the Ministry
of Education, Youth and Sports of the Czech Republic is
gratefully acknowledged. We thank Mrs. Nad′a Kellerova´ and
Dr. Emil Frant´ık for their assistance with animal exposure and
handling.

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Chem. Res. Toxicol., Vol. 23, No. 1, 2010 257
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