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4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene
TOXICOLOGY
AND
APPLIED
4-Vinylphenol
PIRKKO Institute
60,85-90(1981)
PHARMACOLOGY
Excretion Suggestive Workers Occupationally
PFAFFLI,
of Occupational
ANTTI
HESSO, HARRI VAINIO.
Health, Department *Municipal Health
Received
of Arene Oxide Formation Exposed to Styrene
November
AND MARTTI
of Industrial Hygiene and Toxicology, Center, Lo&a, Finland
28. 1980; accepted
March
in
HYV~NEN* Helsinki:
and
24, 1981
4-Vinylphenol Excretion Suggestive of Arene Oxide Formation in Workers Occupationally Exposed to Styrene. FWFFLI, P., HESSO, A., VAINIO, H., AND HYVBNEN, M. (1981). Toxicol. Appl. Pharmacol. 60, 85-90. The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,Coxide is easily converted to corresponding phenols. In this study the presence of Cvinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both Cvinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandehc acid and 4-vinylphenol was fairly good (r = 0.93): increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4vinylphenoVmandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.
The metabolism of styrene has been extensively studied both in experimental animals and in man (cf. Leibman, 1975; Bardodej, 1978; Vainio, 1978). The major metabolic route is styrene oxidation to styrene glycol, mandelic acid, phenylglyoxylic acid, benzoic acid, and hippuric acid. The toxicity of styrene in most cases has been attributed to the formation of reactive epoxide intermediate, styrene-7,8oxide (Vainio, 1978; Norppa et al., 1980). On the other hand. it has been suggested that also arene oxides will be formed in styrene metabolism (Pantarotto et al., 1978). Alkylated arene oxides are easily converted to phenols (Kasperek and Bruice, 1972; Kaubisch et nl., 1972). Bakke and Scheline (1969) and Pantarotto et N/. (1978) have 85
identified 4-vinylphenol as a metabolite of styrene in the rat. Particular interest in arene oxide toxicity has led us to study the presence of 4-vinylphenol in the urine of workers occupationally exposed to styrene and the relation of 4vinylphenol to mandelic acid excretion. METHODS Subject.v
The urine samples for this study were obtained from workers in two reinforced plastics factories producing boats, car parts, and building materials. The workers were engaged in rolling polyester plastics by hand or by spray application. The average air concentration of styrene in these factories during the routine lamination process was around 130 ppm (Pfaffli et (I/.. 1979). 0041-008X/81/100085-06$02.00/0 Copyright All rights
‘0 1981 by Academic Press, Inc. of reproduction m any form recerved
86
PFAFFLI
ET AL.
B lb I 0
4 JLL-4
8 5
I1 15
10
min
C
4
3
4
il.J.i
0
5
10
15 min
FIG. 1. Gas chromatographic separation of acetylated Cvinylphenol with glass capillary column coated with OV-225. Peak 4 indicates Cacetoxystyrene (retention time, 13.5 min).
FIG. 2. Gas chromatographic separation 4-vinylphenol from urine extracts (A and analysis of a reference compound (B) using glass capillary column. Peak 3 indicatesp-cresol tion time, 9.5 min) and peak 4 4-vinylphenol tion time, 13.9 min).
The subjects provided urine samples at the end of the 8hr work shift. The urine samples were preserved at +4”C and analyzed within a week, with the exception of cases where the preservation of 4-vinylphenol in the urine was studied.
tion of 800, the accelerating voltage being 820 V and the electron beam energy 70 eV. The ion source and interface temperatures were 200 and 220°C respec-
Qualitative
Analysis
of Urinary
of free C) and OV-210 (reten(reten-
I-Vinylphenol
The 4-vinylphenol in the urine samples was identified by gclms in comparison to the retention times and mass spectra with a reference substance. The identification was made both as phenol direct and as its acetylated derivative (see later). The reference substance, I-vinylphenol, was synthesized fromp-hydroxybenzaldehyde as described by Sovish (1959). The identity and purity (more than 95%) were confirmed by gcims and ‘H NMR analysis. The low-resolution electron impact mass spectra were obtained with a Varian MAT 112S-SS 166 mass spectrometer-data system. The mass spectrometer was integrated with a Varian 1400 gas chromatograph equipped with a glass capillary column with open coupling to the mass spectrometer. Analyses were carried out at a helium flow of 2 mYmin and with a temperature program from 80 to 200°C rising 6Wmin. The mass spectrometer was operated under the resolu-
FIG. 3. A part of the reconstructed gc/ms trace of the urine sample of a worker exposed to styrene analyzed by OV-210 glass capillary column. Mass spectrum taken from peak 4 (vinylphenol) is presented in Fig. 4.
ARENE OXIDE
FORMATION
AND CVINYLPHENOL
87
120
OH
FIG. 4. Mass spectrum of 4-vinylphenol reference substance (B).
(A) taken from peak 4 in Fig. 3 and mass spectrum of the
tively High-resolution measurements and data acquisition were carried out on a Jeol 01 SG-2 mass spectrometer using photoplate detection. Operating conditions were: electron energy, 75 eV; accelerating voltage, 7.8 kV; resolution, 6000; and ion source temperature, 260°C. The capillaries used in gc/ms were coated with OV-210. The length was 30 m and the inner diameter 0.32 mm. The ‘H NMR spectra were obtained on a Jeol PFI-100 spectrometer (100 MHz) using tetramethylsilane as an internal standard. The synthesis and the ‘H NMR analysis of Cvinylphenol were performed in the Department of Chemistry of the University of Helsinki.
Quantitative Mandelic
analysis Acid
of Urinary
4-Viny/phenol
and
Enzymatic hydrolysis of phenol conjugates (Bakke and Scheline. 1969) was effected by adjusting a lo-ml portion of urine to pH 5 adding 2 ml 0.2 M acetate buffer (pH 5.0), 0.5 ml neomycin sulfate solution (1.5 mg/ml), and 12,500 units of fi-glucuronidase,’ and incubating the mixture for 20 hr at 37°C. AAer hydrolysis the samples were acidified to pH 1 by adding a few drops of sulfuric acid (diluted 1: 1 with water) and extracted three times with 25-ml portions of dichloromethane. The combined extracts were shaken three times with SO-ml portions of 5% NaHCO, solution to remove acids. In case of emulsion formation the layers were separated by centrifugation. The dichloromethane extract was dried over Na,S04, evaporated in a rotating evaporator until dry and dissolved in 1 ml of dichloromethane. These dichloromethane solutions ’ Sigma Chemical Co., St. Louis, MO., No. G-0876; activity, 120,000 units/ml; containing sulfatase.
(0.25 ml) were acetylated in room temperature by adding 0.05 ml acetic anhydride and 0.02 ml pyridine. The acetylated derivatives were assayed by capillary gas chromatography using external standards prepared from synthesized Cvinylphenol in dichloromethane. A gas chromatograph* was equipped for analysis with a glass capillary (length, 29 m; inner diameter, 0.2 mm) which was coated with OV-225. Helium was used as the carrier gas (1 mkmin). The l-w1 injection was split 1 to 10. The oven temperature was programmed from 140 to 180°C rising lO”C/min and finally holding at 180°C for 5 min. The detection was made by flame ionization. Urinary mandelic acid concentrations were analyzed as silyl derivatives by gas chromatography according to Engstrom and Rantanen (1974). Both urinary 4-vinylphenol and mandelic acid concentrations were corrected for excretion volume by creatinine determination.
RESULTS The presence of 4-vinylphenol in the urine samples of workers exposed to styrene was proved by gas chromatographic and by gc/ms analysis. Figure 1 shows the gas chromatographic separation of acetylated 4-vinylphenol (Cacetoxystyrene) on OV-225 glass capillary and Fig. 2 the separation of free 4-vinylphenol on OV-210 glass capillary column. The detection was made by flame ionization. Figure 3 shows a part of the reconstructed gc/ms trace separated on OV* Perkin-Elmer
Sigma 2 gas chromatograph.
PFAFFLI
88 ‘F
ET AL.
A
12C
I
OCOCHO
r 50 m/e 162
0
FIG. 5. Mass spectrum of 4-acetoxystyrene:
210 glass capillary from the same urine extract before acetylation. The mass spectrum of peak 4 in Fig. 3, which indicates 4-vinylphenol, is presented in Fig. 4A. The mass spectrum for the acetylated derivative is presented in Fig. 5. 4-Acetoxystyrene gives a molecular ion with a 15% intensity at rnle 162 and a base peak at mle 120. 4Vinylphenol gives molecular ion as the base peak at m/e 120. The spectra of these two compounds are otherwise quite similar. The elemental composition of the most important ions of 4-vinylphenol are presented in Table 1. The proposed fragmentation pathways for 4-acetoxystyrene are shown in the Scheme 1. The expulsion of ketene from the radical cation of acetylated 4-vinylphenol gives a rearrangement ion at m/e 120 which probably has the same structure as the molecular ion of 4-vinylphenol. The rearrangement reaction TABLE HIGH-RESOLUTION
DATA
(A) sample, (B) reference compound.
may involve a six-centered transition state (Scheme 2, II) in which hydrogen is transferred to carbon on the ortho position or a four-centered transition state (Scheme 2, I) where hydrogen is transferred to oxygen. Gamble et af. (1971) have suggested that the four-centered transition state provides the preferred pathway in the fragmentation of some substituted phenylacetates. The elimination of acetylene from the molecular ion of 4-vinylphenol gives phenol at m/e 94 and the loss of formyl radical gives an ion at mle 91 and a further ion at mle 65. Both mandelic acid and 4-vinylphenol could be detected in all the urine samples studied. No 4-vinylphenol could be detected in urine of unexposed individuals. The lowest detectable concentration in the analysis of added samples was 0.001 mmol/liter. The coefficient of variation was 0.02 at the concentration of 0.01 mmol/liter (n = 9).
1 FOR~VINYLPHENOL
m/e
Measured mass
Diff.” Onmu)
120 119 94 91 89
120.0576 119.0484 94.0421 91.0549 89.0369
0.1 1.2 0.2 0.1 -2.1
Elemental composition W-W C&O G&O W-L W-L
a Mass measurement error in millimass units (mmu).
CH=CH* -I” -cflzo 1; 0OCOCH3 m/e
t2
CkClq s 0:IOH
+
C7”?
m/e 91
m/e 120 -CH=CH
1
' : l" Q OH m/e 94
1 C5";1' m/e 65
SCHEME 1. Proposed fragmentation pathways of 4acetoxystyrene.
ARENE
OXIDE
FORMATION
Figure 6 shows the correlation between two styrene metabolites, mandelic acid, and 4-vinylphenol. The correlation was fairly good (Y = 0.93). Increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation in the 4vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3% (Table 2). DISCUSSION
CH=CH2”’
o+H”2 (1)
x9
4-VINYLPHENOL
-12.0
I /,”
T p -10.0 : .c 2 - 8.0 g Ps
. . / . .
6.0 /
Mandelic
The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that a fraction of styrene is metabolized via arene oxidation, styrene-3,4-oxide functioning as an intermediate. The metabolic route via the oxidation of the vinyl group is, however, at least quantitatively the most important pathway, because the amount of 4-vinylphenol is only about 0.3% of that of mandelic acid. There is, in addition, a possibility that pathways to 4-vinylphenol which do not involve arene oxides are also present to a certain extent in liver (Jerina, 1974). Mandelic acid is formed by the oxidation of styrene glycol or by the dehydrogenation of phenylglyoxylic acid (Leibman, 1975). Styrene glycol is formed from the epoxide intermediate styrene-7,8-oxide by the action of the epoxide hydrolase (Oesch, 1973). The importance of arene oxide intermediates in the toxicity of styrene has been suggested (Pantarotto et al., 1978). In light of the present data, provided that the urinary concentration of 4-vinylphenol gives any hint of flow in the arene oxide pathway, the impact of arene oxidation
1; 0
AND
CH=CHJ*’ : 8
’ ,.j 432 ‘F 0 (II)
SCHEME 2. Possible transition states during the rearrangement reaction.
.
// y= 0.31x * 0.17 r - 0.93 N=29
.
2p acid
2B (mM/g
3p 35 creatinine)
40
FI,G. 6. The correlation between 4-vinylphenol and mandelic acid in the urine samples of occupationally exposed workers.
appears less significant. It may, however, also be possible that the cellular “targetsite” concentrations of styrene-3,4-oxide are greater than what can be inferred on the basis of urinary 4-vinylphenol. Styrene-3.4oxide, in addition to isomerizing to corresponding phenols, will also form glutathione conjugates as well as react readily with proteins and nucleic acids. Styrene-7,8-oxide has recently been shown to be accumulated in human lymphocytes when incubated in vitro in the presence of styrene (Norppa et al., 1980). Styrene7,8-oxide as well as styrene-3,4-oxide are both known to possess mutagenic activity (Vainio et al., 1976; Watabe et al., 1978) as well as to be able to bind covalently to macromolecules (Marniemi et al., 1977; Pantarotto et (ll., 1978; Watabe et nl., 1978). The steady-state concentrations of intermediate epoxides or arene oxides are related to the rates at which they are formed, their ability to isomerize to phenols, to react with nucleophiles such as glutathione, and to undergo enzymatic hydration. However, when the arene oxidation of styrene is compared to vinyl group oxidation, the latter appears at least quantitatively far more important. The major metabolic pathway certainly proceeds via the formation of styrene7,8-oxide. On the basis of the present data. the at-me
90
PFAFFLI
ET AL.
TABLE URINARY
CONCENTRATION
OF 4-VINYLPHENOL TO THAT
Work place
Number of urine samples
A Bl” B*”
9 7 13
2
IN STYRENE-EXPOSED OF MANDELIC ACID
Mandelic acid (nmohg creatinine) 19.5 2 10.4” 8.0 r 5.7 7.5 + 5.2
WORKERS
AND ITS COMPARISON
4-Vinylphenol (nmol/g creatinine)
4-Vinylphenol/ mandelic acid (%)
0.060 f 0.028” 0.027 2 0.023 0.024 f 0.017
0.31 0.34 0.32
D Mean + SD. * Urine samples from factory B were obtained twice.
oxide intermediate in styrene metabolism in humans appears evident and its role in the toxicity of styrene remains a possibility. ACKNOWLEDGMENTS We wish to thank Ms. Kaija Pekari, MSc., and Ms. Helena Kivisto, M.Sc., for the analysis of mandelic acid and creatinine in the urine samples. We want also to thank Ms. Raija Vaaranrinta and Mr. Reijo Kuronen for their skillful technical assistance.
R. S. (1969). Analysis of simple phenols of interest in metabolism. Anal. 0. M.,
Biochem. BARDODEJ,
AND SCHELINE,
27, 451-462.
Z. (1978). Styrene, its metabolism and the evaluation of the hazards in industry. Stand. J. Work Environ. Health 4, Suppl. 2, 95- 103. DONNER, M., SORSA, M., AND VAINIO, H. (1979). Recessive lethals induced by styrene and styrene oxide in Drosophila melanogaster. Mutat. Res. 67, 373-376. ENGSTR~M, K., AND RANTANEN, J. (1974). A new gas chromatographic method for determination of mandelic acid in urine. Int. Arch. Arbeitsmed. 33, 163- 167. GAMBLE,
A.,
GILBERT,
J.,
AND
TILLET,
J. (1971).
Substituent effects on the mass spectra of substituted phenyl acetates. Org. Mass Spectrom. 5,1093- 1099. JERINA, D. M. (1974). Biological formation and disposition of arene oxides. Lloydia 37, 212-218. KASPEREK, G. J., AND BRUICE, T. C. (1972). Differentiation between the concerted and stepwise mechanisms for aromatization (NIH-shift) of arene epoxides. J. Chem. Sot. Chem. Commun., 784-785. KALJBISCH,
N.,
DALY,
J. W.,
11, 3080-3088. LEIBMAN, K. C. (1975). styrene. Environ. Health MARNIEMI, J., SUOLINNA, AND VAINIO, H. (1977).
Metabolism Perspect.
and toxicity of 119.
11, 1ISKAARTINEN,
E.-M., N., Covalent binding of styrene oxide to rat liver macromolecules in vivo and in vitro. In Microsomes and Drug Oxidutions (V. Ullrich, I. Roots, A. Hildebrandt, R. W. Estabrook, and A. H. Conney, eds.), pp. 698-703. Pergamon, New York.
NORPPA,
H., SORSA, M.,
PF~~FFLI,
P., AND VAINIO,
H.
(1980). Styrene and styrene oxide induce SCEs and are metabolised in human lymphocyte cultures. Carcinogenesis 1, 357-361. OESCH, F. (1973). Mammalian
REFERENCES BAKKE,
tion of methyl-substituted arene oxides. Biochemistry
AND
JERINA,
D.
M.
(1972). Arene oxides as intermediates in the oxidative metabolism of aromatic compounds. Isomeriza-
epoxide hydratase: Inducible enzyme catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340.
PANTAROTTO, E., FANELLI, R., BIDOLI, F., MORAZZONI, P., SALMONA, M., AND SZCZAWINSKA, K.
(1978). Arene oxides in styrene metabolism, a new perspective in styrene toxicity? Stand. J. Work Environ. Health 4, Suppl. 2, 67-77. PFKFFLI, P., VAINIO, H., AND H~sso, A. (1979). Styrene and styrene oxide concentrations in the air during the lamination process in the reinforced plastics industry. Stand. J. Work Environ. Health 5, 158-161. SOVISH, R. C. (1959). Preparation and polymerization of p-vinylphenol. J. Org. Chem. 24, 1345-1347. VAINIO, H. (1978). Vinyl chloride and vinyl benzene (styrene)-metabolism, mutagenicity and carcinogenicity. Chem. -Bio/. Interact. 22, 117- 124. VAINIO, H., PKAKK~NEN, R., R~NNHOLM, RAUNIO, V., AND PELKONEN, 0. (1976). A
K.,
study on the mutagenic activity of styrene and styrene oxide.
Stand. J. Work Environ. Health 3, 147-151. WATABE, T., ISOBE, M., SAWAHATA,T., YOSHIKAWA, K., YAMADA, S., AND TAKABATAKE, E. (1978). Metabolism and mutagenicity of styrene. Stand. J. Work Environ. Health 4, Suppl. 2, 142-155.
AND
APPLIED
4-Vinylphenol
PIRKKO Institute
60,85-90(1981)
PHARMACOLOGY
Excretion Suggestive Workers Occupationally
PFAFFLI,
of Occupational
ANTTI
HESSO, HARRI VAINIO.
Health, Department *Municipal Health
Received
of Arene Oxide Formation Exposed to Styrene
November
AND MARTTI
of Industrial Hygiene and Toxicology, Center, Lo&a, Finland
28. 1980; accepted
March
in
HYV~NEN* Helsinki:
and
24, 1981
4-Vinylphenol Excretion Suggestive of Arene Oxide Formation in Workers Occupationally Exposed to Styrene. FWFFLI, P., HESSO, A., VAINIO, H., AND HYVBNEN, M. (1981). Toxicol. Appl. Pharmacol. 60, 85-90. The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,Coxide is easily converted to corresponding phenols. In this study the presence of Cvinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both Cvinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandehc acid and 4-vinylphenol was fairly good (r = 0.93): increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4vinylphenoVmandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.
The metabolism of styrene has been extensively studied both in experimental animals and in man (cf. Leibman, 1975; Bardodej, 1978; Vainio, 1978). The major metabolic route is styrene oxidation to styrene glycol, mandelic acid, phenylglyoxylic acid, benzoic acid, and hippuric acid. The toxicity of styrene in most cases has been attributed to the formation of reactive epoxide intermediate, styrene-7,8oxide (Vainio, 1978; Norppa et al., 1980). On the other hand. it has been suggested that also arene oxides will be formed in styrene metabolism (Pantarotto et al., 1978). Alkylated arene oxides are easily converted to phenols (Kasperek and Bruice, 1972; Kaubisch et nl., 1972). Bakke and Scheline (1969) and Pantarotto et N/. (1978) have 85
identified 4-vinylphenol as a metabolite of styrene in the rat. Particular interest in arene oxide toxicity has led us to study the presence of 4-vinylphenol in the urine of workers occupationally exposed to styrene and the relation of 4vinylphenol to mandelic acid excretion. METHODS Subject.v
The urine samples for this study were obtained from workers in two reinforced plastics factories producing boats, car parts, and building materials. The workers were engaged in rolling polyester plastics by hand or by spray application. The average air concentration of styrene in these factories during the routine lamination process was around 130 ppm (Pfaffli et (I/.. 1979). 0041-008X/81/100085-06$02.00/0 Copyright All rights
‘0 1981 by Academic Press, Inc. of reproduction m any form recerved
86
PFAFFLI
ET AL.
B lb I 0
4 JLL-4
8 5
I1 15
10
min
C
4
3
4
il.J.i
0
5
10
15 min
FIG. 1. Gas chromatographic separation of acetylated Cvinylphenol with glass capillary column coated with OV-225. Peak 4 indicates Cacetoxystyrene (retention time, 13.5 min).
FIG. 2. Gas chromatographic separation 4-vinylphenol from urine extracts (A and analysis of a reference compound (B) using glass capillary column. Peak 3 indicatesp-cresol tion time, 9.5 min) and peak 4 4-vinylphenol tion time, 13.9 min).
The subjects provided urine samples at the end of the 8hr work shift. The urine samples were preserved at +4”C and analyzed within a week, with the exception of cases where the preservation of 4-vinylphenol in the urine was studied.
tion of 800, the accelerating voltage being 820 V and the electron beam energy 70 eV. The ion source and interface temperatures were 200 and 220°C respec-
Qualitative
Analysis
of Urinary
of free C) and OV-210 (reten(reten-
I-Vinylphenol
The 4-vinylphenol in the urine samples was identified by gclms in comparison to the retention times and mass spectra with a reference substance. The identification was made both as phenol direct and as its acetylated derivative (see later). The reference substance, I-vinylphenol, was synthesized fromp-hydroxybenzaldehyde as described by Sovish (1959). The identity and purity (more than 95%) were confirmed by gcims and ‘H NMR analysis. The low-resolution electron impact mass spectra were obtained with a Varian MAT 112S-SS 166 mass spectrometer-data system. The mass spectrometer was integrated with a Varian 1400 gas chromatograph equipped with a glass capillary column with open coupling to the mass spectrometer. Analyses were carried out at a helium flow of 2 mYmin and with a temperature program from 80 to 200°C rising 6Wmin. The mass spectrometer was operated under the resolu-
FIG. 3. A part of the reconstructed gc/ms trace of the urine sample of a worker exposed to styrene analyzed by OV-210 glass capillary column. Mass spectrum taken from peak 4 (vinylphenol) is presented in Fig. 4.
ARENE OXIDE
FORMATION
AND CVINYLPHENOL
87
120
OH
FIG. 4. Mass spectrum of 4-vinylphenol reference substance (B).
(A) taken from peak 4 in Fig. 3 and mass spectrum of the
tively High-resolution measurements and data acquisition were carried out on a Jeol 01 SG-2 mass spectrometer using photoplate detection. Operating conditions were: electron energy, 75 eV; accelerating voltage, 7.8 kV; resolution, 6000; and ion source temperature, 260°C. The capillaries used in gc/ms were coated with OV-210. The length was 30 m and the inner diameter 0.32 mm. The ‘H NMR spectra were obtained on a Jeol PFI-100 spectrometer (100 MHz) using tetramethylsilane as an internal standard. The synthesis and the ‘H NMR analysis of Cvinylphenol were performed in the Department of Chemistry of the University of Helsinki.
Quantitative Mandelic
analysis Acid
of Urinary
4-Viny/phenol
and
Enzymatic hydrolysis of phenol conjugates (Bakke and Scheline. 1969) was effected by adjusting a lo-ml portion of urine to pH 5 adding 2 ml 0.2 M acetate buffer (pH 5.0), 0.5 ml neomycin sulfate solution (1.5 mg/ml), and 12,500 units of fi-glucuronidase,’ and incubating the mixture for 20 hr at 37°C. AAer hydrolysis the samples were acidified to pH 1 by adding a few drops of sulfuric acid (diluted 1: 1 with water) and extracted three times with 25-ml portions of dichloromethane. The combined extracts were shaken three times with SO-ml portions of 5% NaHCO, solution to remove acids. In case of emulsion formation the layers were separated by centrifugation. The dichloromethane extract was dried over Na,S04, evaporated in a rotating evaporator until dry and dissolved in 1 ml of dichloromethane. These dichloromethane solutions ’ Sigma Chemical Co., St. Louis, MO., No. G-0876; activity, 120,000 units/ml; containing sulfatase.
(0.25 ml) were acetylated in room temperature by adding 0.05 ml acetic anhydride and 0.02 ml pyridine. The acetylated derivatives were assayed by capillary gas chromatography using external standards prepared from synthesized Cvinylphenol in dichloromethane. A gas chromatograph* was equipped for analysis with a glass capillary (length, 29 m; inner diameter, 0.2 mm) which was coated with OV-225. Helium was used as the carrier gas (1 mkmin). The l-w1 injection was split 1 to 10. The oven temperature was programmed from 140 to 180°C rising lO”C/min and finally holding at 180°C for 5 min. The detection was made by flame ionization. Urinary mandelic acid concentrations were analyzed as silyl derivatives by gas chromatography according to Engstrom and Rantanen (1974). Both urinary 4-vinylphenol and mandelic acid concentrations were corrected for excretion volume by creatinine determination.
RESULTS The presence of 4-vinylphenol in the urine samples of workers exposed to styrene was proved by gas chromatographic and by gc/ms analysis. Figure 1 shows the gas chromatographic separation of acetylated 4-vinylphenol (Cacetoxystyrene) on OV-225 glass capillary and Fig. 2 the separation of free 4-vinylphenol on OV-210 glass capillary column. The detection was made by flame ionization. Figure 3 shows a part of the reconstructed gc/ms trace separated on OV* Perkin-Elmer
Sigma 2 gas chromatograph.
PFAFFLI
88 ‘F
ET AL.
A
12C
I
OCOCHO
r 50 m/e 162
0
FIG. 5. Mass spectrum of 4-acetoxystyrene:
210 glass capillary from the same urine extract before acetylation. The mass spectrum of peak 4 in Fig. 3, which indicates 4-vinylphenol, is presented in Fig. 4A. The mass spectrum for the acetylated derivative is presented in Fig. 5. 4-Acetoxystyrene gives a molecular ion with a 15% intensity at rnle 162 and a base peak at mle 120. 4Vinylphenol gives molecular ion as the base peak at m/e 120. The spectra of these two compounds are otherwise quite similar. The elemental composition of the most important ions of 4-vinylphenol are presented in Table 1. The proposed fragmentation pathways for 4-acetoxystyrene are shown in the Scheme 1. The expulsion of ketene from the radical cation of acetylated 4-vinylphenol gives a rearrangement ion at m/e 120 which probably has the same structure as the molecular ion of 4-vinylphenol. The rearrangement reaction TABLE HIGH-RESOLUTION
DATA
(A) sample, (B) reference compound.
may involve a six-centered transition state (Scheme 2, II) in which hydrogen is transferred to carbon on the ortho position or a four-centered transition state (Scheme 2, I) where hydrogen is transferred to oxygen. Gamble et af. (1971) have suggested that the four-centered transition state provides the preferred pathway in the fragmentation of some substituted phenylacetates. The elimination of acetylene from the molecular ion of 4-vinylphenol gives phenol at m/e 94 and the loss of formyl radical gives an ion at mle 91 and a further ion at mle 65. Both mandelic acid and 4-vinylphenol could be detected in all the urine samples studied. No 4-vinylphenol could be detected in urine of unexposed individuals. The lowest detectable concentration in the analysis of added samples was 0.001 mmol/liter. The coefficient of variation was 0.02 at the concentration of 0.01 mmol/liter (n = 9).
1 FOR~VINYLPHENOL
m/e
Measured mass
Diff.” Onmu)
120 119 94 91 89
120.0576 119.0484 94.0421 91.0549 89.0369
0.1 1.2 0.2 0.1 -2.1
Elemental composition W-W C&O G&O W-L W-L
a Mass measurement error in millimass units (mmu).
CH=CH* -I” -cflzo 1; 0OCOCH3 m/e
t2
CkClq s 0:IOH
+
C7”?
m/e 91
m/e 120 -CH=CH
1
' : l" Q OH m/e 94
1 C5";1' m/e 65
SCHEME 1. Proposed fragmentation pathways of 4acetoxystyrene.
ARENE
OXIDE
FORMATION
Figure 6 shows the correlation between two styrene metabolites, mandelic acid, and 4-vinylphenol. The correlation was fairly good (Y = 0.93). Increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation in the 4vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3% (Table 2). DISCUSSION
CH=CH2”’
o+H”2 (1)
x9
4-VINYLPHENOL
-12.0
I /,”
T p -10.0 : .c 2 - 8.0 g Ps
. . / . .
6.0 /
Mandelic
The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that a fraction of styrene is metabolized via arene oxidation, styrene-3,4-oxide functioning as an intermediate. The metabolic route via the oxidation of the vinyl group is, however, at least quantitatively the most important pathway, because the amount of 4-vinylphenol is only about 0.3% of that of mandelic acid. There is, in addition, a possibility that pathways to 4-vinylphenol which do not involve arene oxides are also present to a certain extent in liver (Jerina, 1974). Mandelic acid is formed by the oxidation of styrene glycol or by the dehydrogenation of phenylglyoxylic acid (Leibman, 1975). Styrene glycol is formed from the epoxide intermediate styrene-7,8-oxide by the action of the epoxide hydrolase (Oesch, 1973). The importance of arene oxide intermediates in the toxicity of styrene has been suggested (Pantarotto et al., 1978). In light of the present data, provided that the urinary concentration of 4-vinylphenol gives any hint of flow in the arene oxide pathway, the impact of arene oxidation
1; 0
AND
CH=CHJ*’ : 8
’ ,.j 432 ‘F 0 (II)
SCHEME 2. Possible transition states during the rearrangement reaction.
.
// y= 0.31x * 0.17 r - 0.93 N=29
.
2p acid
2B (mM/g
3p 35 creatinine)
40
FI,G. 6. The correlation between 4-vinylphenol and mandelic acid in the urine samples of occupationally exposed workers.
appears less significant. It may, however, also be possible that the cellular “targetsite” concentrations of styrene-3,4-oxide are greater than what can be inferred on the basis of urinary 4-vinylphenol. Styrene-3.4oxide, in addition to isomerizing to corresponding phenols, will also form glutathione conjugates as well as react readily with proteins and nucleic acids. Styrene-7,8-oxide has recently been shown to be accumulated in human lymphocytes when incubated in vitro in the presence of styrene (Norppa et al., 1980). Styrene7,8-oxide as well as styrene-3,4-oxide are both known to possess mutagenic activity (Vainio et al., 1976; Watabe et al., 1978) as well as to be able to bind covalently to macromolecules (Marniemi et al., 1977; Pantarotto et (ll., 1978; Watabe et nl., 1978). The steady-state concentrations of intermediate epoxides or arene oxides are related to the rates at which they are formed, their ability to isomerize to phenols, to react with nucleophiles such as glutathione, and to undergo enzymatic hydration. However, when the arene oxidation of styrene is compared to vinyl group oxidation, the latter appears at least quantitatively far more important. The major metabolic pathway certainly proceeds via the formation of styrene7,8-oxide. On the basis of the present data. the at-me
90
PFAFFLI
ET AL.
TABLE URINARY
CONCENTRATION
OF 4-VINYLPHENOL TO THAT
Work place
Number of urine samples
A Bl” B*”
9 7 13
2
IN STYRENE-EXPOSED OF MANDELIC ACID
Mandelic acid (nmohg creatinine) 19.5 2 10.4” 8.0 r 5.7 7.5 + 5.2
WORKERS
AND ITS COMPARISON
4-Vinylphenol (nmol/g creatinine)
4-Vinylphenol/ mandelic acid (%)
0.060 f 0.028” 0.027 2 0.023 0.024 f 0.017
0.31 0.34 0.32
D Mean + SD. * Urine samples from factory B were obtained twice.
oxide intermediate in styrene metabolism in humans appears evident and its role in the toxicity of styrene remains a possibility. ACKNOWLEDGMENTS We wish to thank Ms. Kaija Pekari, MSc., and Ms. Helena Kivisto, M.Sc., for the analysis of mandelic acid and creatinine in the urine samples. We want also to thank Ms. Raija Vaaranrinta and Mr. Reijo Kuronen for their skillful technical assistance.
R. S. (1969). Analysis of simple phenols of interest in metabolism. Anal. 0. M.,
Biochem. BARDODEJ,
AND SCHELINE,
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A.,
GILBERT,
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H.
(1980). Styrene and styrene oxide induce SCEs and are metabolised in human lymphocyte cultures. Carcinogenesis 1, 357-361. OESCH, F. (1973). Mammalian
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