1 Occupational Studies Branch, National Cancer Institute, Bethesda, MD, USA,
2 School of Public Health, University of California, Berkeley, CA, USA,
3 Chinese Academy of Preventive Medicine, Beijing, China,
4 School of Public Health and Medicine, University of North Carolina, Chapel Hill, NC, USA,
5 School of Medicine, University of California San Francisco, San Francisco, CA, USA,
6 Inhalation Toxicology Research Institute, Albuquerque, NM, USA,
7 California State Health Department, Berkeley, CA, USA,
8 Genetics Laboratory, University of Vermont, Burlington, VT, USA and
9 Institute of Occupational Medicine, Yanshan Petrochemical Products Corporation, Yanshan, People's Republic of China
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Abstract |
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Abbreviations: BD, 1,3-butadiene; B-diol, 3-butene-1,2-diol; BDO, 3,4-epoxy-1-butene; BDO2, 1,2,3,4-diepoxybutane; BDO-diol, 3,4-epoxy-1,2-butanediol; CE, cloning efficiencies; DMF, dimethyl formamide; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; GPA, glycophorin A; M-1, mercapturic acid butanediol; M-2, mercapturic acid butenol; PFPTH, pentafluorophenyl thiohydantoin; SCE, sister chromatid exchange; THBVal, N-(2,3,4-trihydroxybutyl)valine; WBC, total leukocyte count.
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Introduction |
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BD forms three major electrophiles: 3,4-epoxy-1-butene (BDO), 1,2,3,4-diepoxybutane (BDO2) and 3,4-epoxy-1,2-butanediol (BDO-diol). BDO-diol, BDO and BDO2 vary by ~200-fold in increasing genotoxicity as measured by mutagenicity at the hprt and tk loci in human TK6 lymphoblastoid cells (4) and by micronuclei induction in mouse erythrocytes (5). BD is carcinogenic in rodents, but mice are substantially more sensitive than rats (6,7). Following exposure to BD, mice show greater levels of genotoxic BD metabolites (including BDO, BDO2 and their derivatives; 812) than rats and more frequent genotoxic events (1316), suggesting that species differences in metabolism contribute to differential susceptibility to BD-induced cancer.
In a large study of workers in styrene/BD rubber plants, leukemias increased with increasing exposure to BD (1719). Leukemia, however, was not in excess among workers in BD monomer production, whereas excesses were found for lymphosarcoma and reticulosarcoma, but a doseresponse relationship was not evident (20,21). Excesses of lymphohematopoietic malignancies have also been reported among tire manufacturers, but a direct link with BD was not assessed (22,23). BD was recently classified by the International Agency for Research on Cancer as a probable human carcinogen (Group 2A), due to limited evidence in humans but sufficient evidence in animals (24).
Genotoxicity has been evaluated in studies of workers exposed to BD, however, the results have not been consistent (2532). In vitro studies suggest that genetic polymorphisms in glutathione S-transferase enzymes predict genotoxic effects (3342), but few human studies have been done (31,43). In workers at a Chinese BD polymerization facility, we recently demonstrated elevated levels of N-(2,3,4-trihydroxybutyl)valine (THBVal) adducts, which are formed from BDO-diol in its reaction with an N-terminal valine (44). Here, we assess the genotoxic effects of BD in these workers and relate genetic variation in GSTT1 and GSTM1 to these outcomes in vivo.
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Materials and methods |
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Study subjects
On an initial visit to the facility, three groups of workers with high potential exposure were identified. DMF process analysts sample process lines and analyze the product by gas chromatography in the DMF unit, while polymer process analysts carry out these tasks in the recovery and polymerization units. All analysts in these operations who were on duty during the days allocated for sample collection were eligible for study. A third group of exposed workers selected for study were process operators at the recovery facility who carry out routine minor maintenance and, as needed, major repair operations. All subjects in this work unit who would be involved in these activities during the study period were eligible for study.
After the purposes of the study and procedures were explained and informed consent was obtained, 41 of 42 exposed workers were included for study. For comparison, 40 unexposed subjects were selected for study from non-exposed work units. The unexposed subjects were age (5 year) and gender matched in groups to the exposed. Upon review of occupational histories, two controls, who were determined to have worked with BD in the past, were excluded from the analysis. The study groups are shown in Table I.
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A post-shift blood sample (8 ml) was collected, from which mononuclear cells were isolated in a LeucoPREP® tube with Ficoll density gradient liquid and polyester gel (Becton and Dickinson, Lincoln Park, NJ). Mononuclear cells were washed in RPMI medium (UCSF, San Francisco, CA) with 10% fetal bovine serum (FBS) (Gemini Bioproducts), frozen (cells:cryosolution 1:1) in RPMI with 42% FBS and 8% dimethylsulfoxide under rate-controlled (1°C/min) conditions and stored as viable cells in the gas phase above liquid N2. Blood samples (19 ml) were fractionated (serum, plasma, red blood cells and buffy coat) and stored. Whole blood cultures were established for cytogenetic studies. Lymphocytes were stimulated with phytohemagglutinin (PHA) and harvested at 72 h after culture initiation. Within 0.5 h of phlebotomy the MN blood type was determined using rabbit typing serum (Ortho Diagnostics). MN heterozygous blood was kept for 12 h at 4°C until formalin fixation. Spherical, formalin-fixed erythrocytes were prepared according to the method of Langlois et al. (45). The fixed specimens were stored at 4°C until analysis. A differential blood count was carried out with a Coulter blood counter on fresh whole blood within 2 h of collection. Absolute numbers (x103/µl blood) of granulocytes and lymphocytes were derived from a total leukocyte (WBC) count and the lymphocyte percentage. Urine samples were collected during work (03 and 46 h of a 6 h shift). During the work shift, urine samples were kept on ice. Within 2 h of the end of the work shift, the urine samples were aliquoted and frozen.
Air measurements
BD in air was collected by personal samplers and analyzed at the CAPM using an adaptation of NIOSH method 1024 (46). The analyte was desorbed with methylene chloride and analyzed by GC/FID, using a 12 footx1/8 inch packed column (17%) of dibutylphthalate and 8.5% oxidipropionitrile on chromosorb 6201. Breathing zone air samples were analyzed on site within 30 min of collection with a Photovac 10S Plus (photoionization detector) using a 10 mx0.54 mm CP-Sil capillary column, calibrated with a 215 p.p.m. BD standard. Canister air samples were analyzed following the USEPA TO-14 guidelines. The instrument used was an HP 5890/5970 GC/MSD fitted with a flame ionization detector. The thermal desorption apparatus was a Tekmar 5010. The column eluant was split to flame ionization and mass selective detectors. The mass selective detector was operated in the selective ion monitoring mode to quantify 42 targeted organic compounds with re-analysis in the total ion chromatogram monitoring mode to confirm the identity of species. The level of detection for these compounds was estimated to be 12 p.p.b.
Urine measurements
Mercapturic acid butanediol (M-1) and mercapturic acid butenol (M-2) metabolites of BD were measured in urine samples by GC/GC/MS as previously described (47). Briefly, 5 µl injections of the sample were made into a 15 mx0.53 mm i.d. Restek Rtx-1 (1.0 mm film) column and the peaks were captured in a liquid nitrogen cooled loop and analyzed on a Restek Rtx-200 30 mx0.25 mm i.d. (0.25 mm film) capillary column. The MS was operated in selected ion monitoring mode, with monitoring for ions 129/132, 228/232, 377/382, 452 and 457 for M-1 and its deuterated analog, while ions 362/368 and 287/292 were monitored for M-2 and its deuterated analog. Standard curves were used to calculate absolute amounts of analytes in urine. Values were reported relative to mg creatinine, using standard methods.
Hemoglobin adducts
THBVal hemoglobin adducts were determined as previously described (48). In brief, globin was isolated from the red blood cell fraction (49) and derivatized with pentafluorophenyl isothiocyanate (Fluka, Buchs, Switzerland) to the pentafluorophenyl thiohydantoin (PFPTH) based on Törnqvist's modified Edman degradation for specific cleavage of N-alkylated terminal valines of the four chains in hemoglobin (50). A synthesized, derivatized external standard, THB(13C5)Val-PFPTH was added to the sample. Samples were further processed by ultrafiltration with Centricon 30s (Amicon, Beverly, MA). The filtrate was extracted with diethylether (Fluka), washed on C18 columns (Alltech, Deerfield, IL) and eluted with acetonitrile (HPLC grade; Mallinckrodt, Paris, KY). The eluate was acetylated with 25% triethylamine (Aldrich, Milwaukee, WI) in acetonitrile (v/v) and 25% acetic anhydride (Mallinckrodt) in acetonitrile (v/v), dried, redissolved in pentane (Aldrich), washed with 40% aqueous methanol (HPLC grade; J.T. Baker, Phillipsburg, NJ), followed by GC/HRMS quantitation for THBVal-PFPTH at m/z 534.1084 for the analyte and 539.1254 for the external standard. Quantitation was based on the ratio of the peak area of the analyte to the peak area of the external standard.
Somatic mutation assays
For the glycophorin A (GPA) assay, formalin-fixed spherocytes of individuals heterozygous (MN) for GPA were analyzed to determine NN and NØ variant cell frequencies (Vf) (45). Sphered erythrocytes were incubated with anti-M (biotinylated 6A7) and anti-N (fluoresceinated BRIC 157) antibodies and prepared with avidinphycoerythrin for flow cytometry. Singlet erythrocytes were selected for analysis using a polygonal gate based on forward scatter versus side scatter plots. The NØ and NN windows were 24 channels wide out of 256 channels and were individually adjusted using the MN mean peak for each specimen.
For the hprt mutations, mutation frequency (Mf) was determined by the T cell cloning assay (51). Briefly, viable cells were thawed and incubated in medium containing 1 µg/ml PHA (HA17; Wellcome Diagnostics) for 3640 h to achieve mitogen stimulation. Washed cells were then placed in growth medium (RPMI 1640 containing 20% nutrient medium HL-1, 5% defined supplemented bovine calf serum, 1020% LAK supernatant containing 0.125 µg/ml PHA and 1x104 irradiated human lymphoblastoid feeder cells/well. After 1016 days incubation, growing colonies were determined by use of an inverted phase contract microscope. The cloning efficiencies (CE) were calculated by the Poisson relationship CE = ln P0/x, where P0 is the fraction of wells negative for colony growth and x is the average number of cells originally inoculated per well. The thioguanine-selected CE divided by the mean unselected CE yields the Mf.
Cytogenetic analysis
Sister chromatid exchange (SCE) was assessed in cultures, with and without exposure to BDO2. Whole blood (0.5 ml) was cultured for 72 h at 37°C in 5% CO2 with 98% humidity, in growth medium (4.5 ml), in 1 oz glass prescription bottles. For BDO2-treated samples, treatment was carried out for 21 h, to a final concentration of 6 µM, and all samples were treated with 50 µM bromodeoxyuridine for 24 h. BDO2 (Aldrich) was diluted in sterile water and a fresh stock solution was prepared for each experiment. Two hours before fixation, colcemid (2x107 M, final concentration; CIBA Pharmaceuticals, Summit, NJ) was added. Upon harvesting the cultured lymphocytes, microscopic slides were prepared and differentially stained, as described earlier (52). To estimate baseline SCE frequencies, 50 second division metaphases were scored per point; for BDO2-treated cultures, 50 second division metaphases were scored. SCE frequency is expressed as the mean SCE/cell. For each subject, the mean BDO2-induced SCE/cell frequency was calculated by subtracting the mean value for BDO2-induced SCEs from the SCE culture result without BDO2 treatment for that individual.
For fluorescence in situ hybridization (FISH), a total of four chromosomes were examined using two different types of probes purchased from Oncor Inc. (Gaithersburg, MD) and Vysis Inc. (Downers Grove, IL). The centromeres of chromosomes 1 and 7 were targeted by -satellite DNA probes and chromosomes 8 and 12 were painted along their whole lengths with painting probes. The signals on chromosomes 1 and 8 were detected as green and those on 7 and 12 as red. A simplified denaturation and hybridization procedure was performed automatically by the HyBrite Denaturation/Hybridization system from Vysis Inc. The centromere and painting probes were mixed well, then applied to slides and coverslipped. The denaturation temperature was set at 72°C and the time at 7 min. Slides remained in the moist environment of the system at 37°C for 4568 h in order to obtain optimal signals. Slides were then post-washed in 1x SSC at 70°C for 5 min and in phosphate buffer three times at room temperature. After detection and amplification of the hybridization signals, the signals were viewed under a Zeiss fluorescence microscope equipped with epifluorescent illumination, a 100x oil immersion lens and a triple bandpass filter for DAPI/FITC/Texas red. Here, we examine percent aneuploidy as a marker of genotoxic damage. Detailed analyses, including separate examination of hypoploidy and hyperploidy, will be described elsewhere (manuscript in preparation).
Classic karyotype analyses by G-banding were carried out to identify chromosomal abnormalities. Fifty metaphase spreads per subject were examined for structural and numerical changes. Detailed banding analyses will be described elsewhere (manuscript in preparation).
PCR analysis of GSTM1 and GSTT1 deletions
Target DNA (50100 ng) was obtained from heparinized whole blood (Puregene; Gentra, Research Triangle Park, NC). PCR reactions were carried out in 50 µl volume containing 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1 mM 2-mercaptoethanol, 1% glycerol, 1 mM MgCl2, 0.2 mM dNTPs and 2.5 U Amplitaq. For GSTM1 the primers were 5'-GTGCCCTACTTGATTGATGGG-3' and 5'-CTGGATTGTAGCAGATCATGC-3'. The primers for GSTT1 were 5'-TTCCTTACTGGTCCTCACATCTC-3' and 5'-TCACCGGATCATGGCCAGCA-3'. PCR products were electrophoresed on 2% agarose gels and the diagnostic bands were visualized using ethidium bromide staining. Control amplifications were run in all lanes using universal primers for actin.
Statistical analysis
Non-parametric procedures were used for statistical analysis, including the Spearman correlation test, the Wilcoxon test for independent samples and the 2 test. For multivariate analyses of studied markers, linear regression analyses were carried after transformation to the natural log (ln). Analyses were carried out using the SPSS statistical package (53).
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Results |
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THBVal hemoglobin adducts were significantly more common in BD-exposed workers than controls (Table III). BD-exposed workers had greater absolute lymphocyte counts and greater lymphocytes as a proportion of total WBC. Platelet counts also tended to be greater in BD-exposed than unexposed workers. However, hprt mutations (Mf) (as previously reported; 28), erythrocyte GPA mutations (NØ and NN), SCEs (with and without BDO2 induction) and the frequency of total aneuploidy of chromosomes 1, 7, 8 and 12 did not differ significantly between BD-exposed and unexposed workers. As measured by classical cytogenetics, the frequency of structural (
2 test, P = 0.39) and numerical abnormalities (
2 test, P = 0.36) also did not differ between exposed and unexposed (data not shown). Tests of statistical significance for these comparisons were similar when calculated by linear regression, with adjustment for age and sex.
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Among BD-exposed workers, levels of exposure did not differ substantially with respect to GSTT1 (Table V) or GSTM1 status (Table VI
). Neither genotype predicted urinary M-1 or THBVal adduct formation, hprt or GPA mutations or SCEs among exposed workers. Aneuploidy as measured by FISH (Tables V and VI
) and chromosomal aberrations, measured by classical cytogenetics (data not shown), were also not associated with genotype. Granulocyte levels were greater and lymphocytes as a percentage of total WBCs were less in BD-exposed workers with the GSTT1 null genotype. After adjustment for age, sex and THBVal level, weaker associations remained for GSTT1 status with granulocytes (P = 0.06) and lymphocyte percent (P = 0.05) (data not shown).
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Discussion |
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BD-exposed workers did not have increased levels of somatic mutations as measured by the GPA and hprt mutation frequency assays. SCE frequency and chromosomal abnormalities were also not increased in BD-exposed workers. The assays chosen for this study encompassed measures of genotoxicity in erythrocyte precursors (45) and T cells (51) and included outcomes found after exposure to BD metabolites in vitro (4,33,42,52) and in animal studies (16). Our negative results are relevant, however, only for exposures in the BD exposure range studied. Also, the relatively small size of the study sample limits our ability to detect modest effects.
Other studies have shown genotoxic outcomes in workers exposed to BD, but the results have not been consistent. Among US BD/styrene workers, dicentrics were significantly correlated with urinary M-1 and there was evidence of deficiencies in DNA repair by the CAT-host cell reactivation assay (25). Increased frequency of mutations in hprt were also observed in US BD workers (26,27) but excesses were not found in our study, as reported earlier (28), or in the Czech Republic (32). Increased frequency of chromosomal aberrations and SCE were reported in the Czech Republic (31,32); an earlier investigation of these subjects and workers in Portugal showed no excesses (29,30). We found increases in lymphocytes in BD-exposed workers with levels tending to increase with increasing BD exposure, suggesting a role for BD in lymphocyte proliferation and increased cell turnover. However, the mechanism and significance of this modest increase are unknown and earlier studies showed no hematological effects (54,55).
Investigations in the USA (26,27), Europe (30) and China report similar average exposure levels of ~12 p.p.m. BD in air, but individual exposures vary and the approaches to exposure assessment have been limited. Because BD production is a closed operation, exposures are generally low. As we found in China, however, certain tasks are associated with strikingly high, short-term exposures (exceeding 1000 p.p.m.), while full-day high level exposures were found only among workers involved in major repair operations. When peak exposures are brief and intense, air measurements may not accurately reflect biological dose because exposure avoidance may limit the actual dose received, absorbtion and retention may be decreased (56) and technical limitations of the pump sampling methodology may bias results. Further limiting comparisons, historical episodes of high level exposure (e.g. accidents) were not recorded. Also, measurements of exposures have generally been over a limited number of days, while the genotoxic effects being assessed are probably related to exposures occurring over several months.
Assessment of hemoglobin adducts in our study provided an independent approach to BD exposure assessment that obviated some of the limitations of earlier studies. Because erythrocytes are relatively long lived (average ~120 days), adducts in hemoglobin tend to integrate biological dose over an extended period of time. Hemoglobin adducts were also moderately correlated with 1 day air and urine exposure measurements, suggesting that the 1 day measurements tended to reflect an ordinal ranking of exposures in the longer term. Further detailed calibration studies will be needed, however, to determine the quantitative relationship between level of BD exposure and THBVal adduct formation in occupational settings. It should also be noted that measurable levels of THBVal and urinary M-1 are found in subjects unexposed to BD. The sources for this are presently not understood.
In vitro exposure of human lymphocytes to BDO2 characterizes individuals as `sensitive' or `resistant' to BDO2-induced SCE (34), a phenotype that we and others have shown to correlate closely with the GSTT1 genotype (3537). Exposure in vitro of human lymphocytes to BDO also characterizes a `sensitive' phenotype, which appears to be linked to GSTM1 as well as GSTT1 deficiency (3840). BDO-diol also induces SCE in lymphocyte cultures, but results were not dependent upon GSTM1 or GSTT1 genotype (41). Although in vitro exposures may point to inter-individual differences in susceptibility to BD carcinogenicity, extrapolation to studies of humans occupationally exposed may be limited. The in vitro exposures are much higher than occur in humans and this may lead to saturation of this detoxification pathway. It appears that the GSTT1 genotype does not confer an increased susceptibility under the conditions of this study. This lack of genotoxicity supports BDO-diol being the major metabolite of BD in humans (see below).
We found no evidence in our study in China that GSTT1 or GSTM1 genotype was related to BD genotoxicity. Other studies have not shown consistent relationships. In the Czech and Portuguese BD facilities, chromosomal aberration frequency tended to be greater among workers in monomer and polymer production who had the null GSTT1 genotype, while the GSTM1 genotype had no effect among the BD-exposed workers (43). A more recent investigation of BD monomer workers in the Czech facility found no association with GSTT1 genotype, however, decreased chromosomal aberrations were associated with the null genotype of GSTM1 (31). Our cytogenetic analyses showed no effects due to these genetic variants. An added complexity is that weak associations between genotype and chromosome aberrations were also found among non-exposed controls in these studies (31,43).
THBVal adducts can form from the highly genotoxic BDO2 or BDO-diol, an epoxide with 1/200th the mutagenicity (4,5). Although the major pathway for THBVal formation may be through the BDO-diol (57,58), the relative ratio of these precursors is not directly determinable (48). In rodents, the relative contribution of BDO-diol to THBguanine DNA adducts is estimated to be 9598% (59). Thus, elevated THBVal levels indicate exposure to BD, but may not be a good marker for individual genotoxic risk. Glutathione transferases are involved in the detoxification of BDO2 and its precursor (BDO) as well as in the detoxification of the BDO-diol precursors [BDO and 3-butene-1,2-diol (B-diol)], however, in our study THBVal adduct levels did not vary by GSTT1 or GSTM1 status among BD-exposed workers. Similarly, M-1, which is a glutathione conjugation product of B-diol, did not vary with respect to variants in these genes.
In summary, this investigation in China demonstrated exposure to BD, by a variety of short-term and long-term measures, but did not show specific genotoxic effects related to that exposure. Modest perturbations in blood count profiles were found. In contrast to in vitro investigations, studies of potential susceptibility groups also revealed no genotoxic effects.
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Notes |
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References |
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