Chem. Res. Toxicol. 2001, 14, 1393-1400
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Polymorphism of Xenobiotic-Metabolizing Enzymes and Excretion of Styrene-Specific Mercapturic Acids Giuseppe De Palma, Paola Manini, Paola Mozzoni, Roberta Andreoli, Enrico Bergamaschi, Stefania Cavazzini, Innocente Franchini, and Antonio Mutti* Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy Received March 26, 2001
The role of polymorphic xenobiotic-metabolizing enzymes in the interindividual variability of phenylhydroxyethyl mercapturic acids (PHEMAs) was investigated in 56 styrene-exposed workers. Ambient monitoring was carried out using passive personal samplers (geometric mean, 157 mg/m3 8-h time-weighted average; geometric standard deviation, 2.90). Biomonitoring was based on mandelic acid and phenylglyoxylic acid in urine spot samples collected at the end of the work shift (“end-of-shift”) and prior to the subsequent shift (“next morning”). Four PHEMA diastereoisomers, namely (R,R)-M1, (S,R)-M1, (S,R)-M2, and (R,R)-M2, were determined by HPLC/tandem mass spectrometry. The genotypes of glutathione S-transferases M1-1 (GSTM1), T1-1 (GSTT1) and P1-1 (GSTP1), and microsomal epoxide hydrolase (EPHX) were characterized by PCR-based methods. Workers bearing the GSTM1pos genotype showed PHEMA concentrations five and six times higher (in end-of-shift and next-morning samples, respectively) as compared to GSTM1null people. In GSTM1pos subjects, (R,R)-M1 was the main mercapturate affected by the GSTM1 status, accounting for 54 and 68% of total PHEMAs in end-of-shift and next-morning samples, respectively. Compared to GSTM1null, GSTM1pos subjects excreted more -M1 than -M2 and more (R,R)-M1 and (S,R)-M2 than (S,R)-M1 and (R,R)-M2 diastereoisomers. Thus, GSTM1-1 is the main isoenzyme catalyzing GSH-conjugation of styrene-7,8oxide in humans and it seems to act in a regio- and stereoselective way. PHEMAs cannot be recommended as biomarkers of exposure to styrene, unless the GSTM1 genotype is considered in data interpretation. Their role as biomarkers of susceptibility deserves further studies.
Introduction Styrene monomer remains one of the most important chemicals produced and used worldwide in many applications, including plastics, latex paints and coatings, synthetic rubbers, and polyester resins. Although in most western countries, occupational exposure to styrene is usually low [ Val105) and exon 6 (Ala114 > Val114), giving rise to four different alleles: GSTP1*A (Ile105-Ala114), GSTP1*B (Val105-Ala114),GSTP1*C(Val105-Val114),andGSTP1*D (Ile105-Val114). Compared with the wild-type protein encoded by GSTP1*A allele, variant proteins show in vitro either a reduced half-life or a different catalytic efficiency toward different substrates (16, 17). Two polymorphisms have been identified, respectively, in exon 3 and in exon 4 (His139 > Arg139) of the mEH coding gene, EPHX, with a prevalence among caucasians, respectively, of 36 and 23%. An in vitro expression study has demonstrated that the exon 3 polymorphism (Tyr113 > His113) implies a mEH content, and hence enzyme activity, reduced by 50%, whereas the polymorphism in exon 4 leads to an increase in cellular mEH content, and hence activity, by 25% (18).
The present field study on occupationally exposed workers was aimed at assessing the influence of polymorphic xenobiotic-metabolizing enzymes on the urinary pattern of PHEMAs.
Experimental Section Chemicals. DL-Mandelic acid (99% pure) and phenylglyoxylic acid (98% pure) were obtained from Fluka (Buchs, Switzerland). Standards of mercapturic acids, (R,R)- and (S,R)-N-acetyl-S-(1phenyl-2-hydroxyethyl)-L-cysteine [(R,R)-M1 and (S,R)-M1)] and (R,R)- and (S,R)-N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine [(R,R)-M2 and (S,R)-M2)], were a kind gift from Dr. Luciano Maestri (Fondazione Maugeri, Pavia, Italy). HPLC-grade water and methanol were purchased from LabScan (Dublin, Ireland). Analytical-grade formic acid and ammonium hydroxide were supplied by Fluka. Subjects. The study group consisted of 56 healthy caucasian workers (45 male and 11 female) aged 37.02 ( 9.44 years who had been exposed to styrene for 8.6 years, on average (range 1-28 years). They were recruited on a voluntary basis among the workforce of three factories, one producing polyester resins and two manufacturing glass-fiber reinforced plastics. None of them had history of liver or metabolic dysfunction. The protocol of the study was approved by the local ethical committee. Informed consent was obtained from all workers. Molecular Biology. The genotypes of polymorphic enzymes relevant to styrene metabolism, namely GSTM1, GSTT1, GSTP1, and EPHX, were characterized in DNA extracted from 1 mL of peripheral whole blood by the Nucleon BACC1 commercial kit (Amersham Life Science, Little Chalfont, U.K.). Genetic polymorphisms were characterized, according to already published methods, either using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) protocols (EPHX and GSTP1) (19, 20) or by a multiplex PCR, using albumin as internal control (GSTM1 and GSTT1) (21). The
GSTM1-1 Polymorphism and Styrene Mercapturates Table 1. Distribution of GSTM1, GSTT1, GSTP1, and EPHX Genotypes in the Group of 56 Workers Occupationally Exposed to Styrene genotypes GSTM1 GSTT1 GSTP1a
EPHX 113b EPHX 139c
null positive null positive *A/*A *A/*B *A/*C *B/*B *B/*C *C/*C Tyr/Tyr Tyr/His His/His His/His His/Arg Arg/Arg
no.
(%)
31 25 15 41 26 17 2 9 1 1 25 19 12 39 15 2
55 45 27 73 46 30 4 16 2 2 45 34 21 70 27 3
a GSTP1*A is the wild-type allele, whereas GSTP1*B and *C are the variant alleles. b EPHX genotypes for the polymorphism in exon 3 (Tyr113 > His113) of the EPHX gene: Tyr indicates the wild-type allele whereas His specifies the variant allele. c EPHX genotypes for the polymorphism in exon 4 (His139 > Arg139) of the EPHX gene: His defines the wild-type allele, whereas Arg is used to indicate the variant allele.
observed genotype frequencies are reported in Table 1. In no case could the rare GSTP1*D allele be identified. Observed genotype frequencies did not differ significantly from the expected, thus the study cohort was in Hardy-Weinberg equilibrium (data not shown). Exposure Characterization. To assess the 8-h timeweighted average (TWA) concentrations of airborne styrene, workers wore passive-diffusive samplers (Radiello, Fondazione Maugeri, Padua, Italy) each containing 530 mg of charcoal, during the whole duration of their work-shift. Styrene was desorbed with 2 mL of CS2, added with 20 µL of Internal Standard (I.S.) (n-octane, 10 g/L in CS2) and then agitated for 30 min, according to the instructions given by the manufacturer. Analyses were performed by gas chromatography/mass spectrometry (GC/MS) using a Hewlett-Packard HP 6890 gas chromatograph coupled with a HP 5973 Mass Selective Detector (Hewlett-Packard, Palo Alto, CA). Hydrogen, produced by a Whatman 75-32 Hydrogen generator (Maidstone, Kent, England), was used as the carrier gas at a flow-rate of 1.1 mL/ min. A volume of 1 µL of the extract was injected on a HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness) from Hewlett-Packard. The temperature program was 40 °C, hold 3.5 min, 5 °C/min to 90 °C, 10 °C/min to 150 °C, hold 3 min; the temperatures of injector, transfer line, and detector were set to 250, 280, and 230 °C, respectively. Mass spectra were recorded in the EI mode at 70 eV. Quantitation was performed in selected reaction monitoring (SIM) mode, by acquiring the signal of the following ions m/z 78, 103, and 104 for styrene and m/z 43, 57, 71, 85, and 114 for the I. S. Personal passive monitors revealed a geometric mean (GM) of airborne styrene exposure (as 8-h TWA concentration) of 157 mg/m3 and a geometric standard deviation (GSD) of 2.90, with values ranging from 5.10 to 492 mg/m3. Since a high proportion (>30%) of workers were using individual protective equipment, making ambient monitoring unreliable for exposure assessment, the internal dose was characterized according to the American Conference of Governmental Industrial Hygienists (ACGIH) recommended biomarkers of exposure, namely the urinary excretion of MA and PGA (see below) (22). Biological Monitoring. Spot urine samples (about 10 mL) were collected in dark vials at the end of the work shift (“endof-shift”) and before the work shift, i.e., at least 15 h after the last exposure (“next morning”), and stored at -20 °C until analysis. The urinary excretion of the main styrene metabolites, MA and PGA, was determined by HPLC-UV (23) in a single
Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1395 Table 2. Excretion of Phenylhydroxyethyl Mercapturic Acids (PHEMAs) in “End-of-Shift” and “Next Morning” Urine Spot Samples end-of-shift
next morning
PHEMAs
GMa
GSDb
GMa
GSDb
total (µg/g creatinine) (R,R)-M1 (µg/g creatinine) (S,R)-M1 (µg/g creatinine) (S,R)-M2 (µg/g creatinine) (R,R)-M2 (µg/g creatinine)
592 210 14.7 267 49.4
3.22 4.14 3.10 3.21 2.44
274 139 4.10 91.3 16.6
3.42 4.25 3.05 2.86 2.32
a
Geometric means. b Geometric standard deviations.
laboratory participating in the program for Quality Assurance for organic solvent metabolites run by the Finnish Institute of Occupational Health (FIOH, Helsinki, Finland). Concentrations of analytes in spot urine samples were normalized, expressing data as a function of creatinine concentration. The GM of the sum of MA and PGA (MA + PGA) in end-of-shift and next morning urine spot samples were, respectively, 205 mg/g creatinine (GSD, 2.4; range, 31.6-1412) and 60.8 mg/g creatinine (GSD, 1.85; range, 15.8-199). Owing to the use of masks by a high proportion of workers, urinary metabolites were poorly related to airborne styrene concentrations in the whole population (r ) 0.093, n.s.). In subgroups of workers stratified according to the use of personal protective devices, such correlation coefficients were 0.157 (n.s.) and 0.581 (p < 0.001) in subjects wearing or not the mask, respectively. The four diastereoisomers of PHEMAs were determined by LC/MS/MS on a PE-Sciex API 365 triple-quadrupole mass spectrometer (Sciex, Thornhill, Canada) equipped with an ASPEC XL autosampler (Gilson, Viliers-le-Bel, France) and an ionspray interface for pneumatically assisted electrospray. LC/ MS/MS analyses were done on 0.25 mL of filtered urine samples, and a volume of 5 µL was injected onto the LC column after acidification with formic acid (0.1 M). The four diastereoisomers were separated on a Supelcosil C18-DB column, 75 × 3.0 mm i.d., 3 µm (Supelco, Bellefonte, PA) using variable proportions of 20 mM aqueous formic acid (pH 3.0) and methanol at a flow rate of 0.5 mL/min. Gradient program: 5% methanol, hold for 12 min; from 5 to 14% methanol in 14 min (linear gradient) and then hold for 4 min; from 14 to 90% methanol in 3 min (linear gradient) and then hold for 2 min. The analytes were ionized by electrospray, in negative-ion mode (ionspray voltage, -3800 V; orifice voltage, -30 V) and detected in selected-reaction monitoring (SRM) mode, following the reaction m/z 282.1f153.0, characteristic of all mercapturates (collision energy, 17 eV) (12). Table 2 reports the excretion values of PHEMAs, whereas Table 3 summarizes the excretion values of the metabolites of styrene in subgroups of workers bearing different metabolic genotypes. Statistics. Statistical analysis was carried out by using the SPSS/PC+ software. Parametric statistical tests were applied to log-transformed values, when necessary to obtain a normal distribution, which was assessed by using the one sample Kolmogorov-Smirnov test. Differences between group means were primarily assessed using the Student’s t-test for independent samples. Linear regression analysis was carried out using the least-squares method (Pearson’s correlation). Analysis of variance (ANOVA) models were applied to assess the contribution of genotypes and other covariates (sex, age, smoking habits, exposure levels and duration, protective devices) to the interindividual variability of biomarkers.
Results Excretion Pattern of PHEMAs. Table 2 summarizes the urinary excretion of PHEMAs in end-of-shift and next-morning spot samples. PHEMAs displayed a great variability, ranging from 58.9 µg/g creatinine to 9.3 mg/g creatinine in end-of-shift samples and from 26.3 µg/g creatinine to 4.6 mg/g creatinine in next-morning speci-
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Table 3. Urinary Excretion of Metabolitesa of Styrene in Subgroups of Workers Bearing Different Metabolic Genotypes
genotypes GSTM1 GSTT1 GSTP1 EPHX
positive null positive null *A/*A elsed His113e elsef
no.
end-of-shift MA + PGAb (mg/g creatinine)
next morning MA + PGAb (mg/g creatinine)
end-of-shift PHEMAsc (µg/g creatinine)
next morning PHEMAsc (µg/g creatinine)
25 31 41 15 26 30 31 25
228 (2.17) 190 (2.55) 227 (2.34) 158 (2.43) 212 (2.40) 200 (2.40) 219 (2.58) 189 (2.15)
63.9 (2.07) 58.3 (1.66) 67.6 (1.79)h 45.4 (1.84) 63.2 (1.50) 58.7 (2.10) 62.1 (1.78) 59.1 (1.91)
1438 (2.44)g 312 (2.47) 602 (3.09) 576 (3.55) 541 (2.71) 635 (3.70) 599 (2.71) 584 (3.93)
757 (2.62)g 122 (2.13) 283 (3.05) 254 (4.93) 237 (2.40) 313 (4.50) 257 (3.20) 300 (3.92)
a Values are given as geometric means and geometric standard deviations. b Sum of mandelic (MA) and phenylglyoxylic (PGA) acids. Total phenylhydroxyethyl mercapturic acids. d Genotypes including at least one variant allele at the GSTP1 locus. e Haplotypes at the EPHX locus including at least one variant allele (Tyr113>His113) at exon 3. f All the remaining haplotypes at the EPHX locus. g p < 0.0001 vs GSTM1null genotype. h p < 0.05 vs GSTT1null genotype.
c
Table 4. Distribution of the Excretion Values of Diastereoisomers of Phenylhydroxyethyl Mercapturic Acids (PHEMAs) in GSTM1positive (n ) 25) and GSTM1null (n ) 31) Workers Measured at the “End-of-Shift” and in “Next Morning” end-of -shift GSTM1 null (n ) 31)
next morning
GSTM1 pos (n ) 25)
GSTM1 null (n ) 31)
GSTM1 pos (n ) 25)
PHEMAs
GMa
GSDb
GMa
GSDb
t
p
GMa
GSDb
GMa
GSDb
t
p
(R,R)-M1 (µg/g creatinine) (S,R)-M1 (µg/g creatinine) (S,R)-M2 (µg/g creatinine) (R,R)-M2 (µg/g creatinine)
82.4 8.1 155 51.7
2.42 2.35 2.75 2.65
772 33.7 564 46.4
2.46 2.55 2.56 2.20
8.13 5.19 4.24 0.39
