Human sensitivity to 1,3-butadiene: role of microsomal epoxide hydrolase polymorphisms

Sherif Z. Abdel-Rahman,1, Marinel M. Ammenheuser and Jonathan B. Ward, Jr

Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555-1110, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1,3-Butadiene (BD) is a major commodity chemical used in the manufacture of synthetic rubber and various plastics and has been shown to be a potent animal carcinogen and a probable human carcinogen. The bioactivation of BD to reactive epoxides, and the balance between activation and detoxication of these reactive metabolites, is thought to play a critical role in the genotoxic and carcinogenic effects of BD. The detoxication of reactive BD metabolites involves enzymatic conjugation with glutathione by glutathione S-transferases (GSTs) and by hydrolysis, a reaction mediated by microsomal epoxide hydrolase (mEH). Since polymorphisms in genes of xenobiotic-metabolizing enzymes such as mEH may influence individual susceptibility to adverse health effects from BD exposure, we tested the hypothesis that the mEH Tyr113His polymorphism increases sensitivity to the genotoxic effects of BD in exposed workers. We used the autoradiographic hprt mutant lymphocyte assay as a biomarker of effect to identify genotoxicity associated with BD exposure in 49 workers from two styrene/butadiene polymer plants in Southeast Texas. Exposure to BD was assessed by collecting breathing zone air samples using passive badge dosimeters for three full 12 h work shifts 25, 20 and 14 days before blood was collected for genotyping and for the hprt assay. We genotyped the study participants for the Tyr113His polymorphism in the mEH gene and also for deletion polymorphisms in the glutathione S-transferase genes, GSTM1 and GSTT1, as potential biomarkers of susceptibility to BD. Our data indicate that the majority of the study subjects (67%) were exposed to very low levels of BD of <150 parts per billion (p.p.b.) time-weighted average (TWA). In some workers, however, we found levels of BD exposures that exceeded a TWA of 2000 p.p.b. Our data indicate a significant (P < 0.05) 2-fold increase in frequencies of hprt variant (mutant) lymphocytes (Vf) in workers exposed to >150 p.p.b. BD, compared with workers exposed to <150 p.p.b. There was no significant effect from individual GSTM1, GSTT1 or mEH genotypes in workers exposed to <150 p.p.b. BD. In workers exposed to >150 p.p.b., individuals with at least one polymorphic mEH His allele (His/His or His/Tyr genotypes) had a significant (P < 0.001) 3-fold increase in Vf (mean Vfx10–6 ± SE = 13.25 ± 1.78) compared with individuals with the Tyr/Tyr genotype (mean Vfx10–6 ± SE = 4.02 ± 0.72). There was no significant effect from individual GSTM1 or GSTT1 polymorphisms, but combined polymorphism analysis showed that the genetic damage was highest in individuals who had at least one mEH His allele and either the GSTM1 and/or GSTT1 null genotypes (hprt Vf = 14.19 ± 2.30 x10–6). In contrast, this response was not observed in individuals exposed to levels of BD < 150 p.p.b. These results indicate that polymorphisms in the mEH gene may play a significant role in human sensitivity to the genotoxic effects of BD exposure, and that the hprt mutant lymphocyte assay can serve as a sensitive biomarker of genotoxicity for monitoring occupational exposure to BD in industrial settings. Additional investigations in larger populations of workers are needed to confirm our results and to characterize the possible role of additional mEH polymorphisms in the induction of genetic damage associated with occupational exposure to butadiene.

Abbreviations: BD, 1,3-butadiene; DEB, 1,2,3,4-diepoxybutane; EB, 3,4-epoxy-1-butene; EBD, 3,4-epoxy-1,2-butanediol; GM, growth medium; GSTs, glutahione S-transferases; IARC, International Agency for Reseach on Cancer; LI, labeling index; mEH, microsomal epoxide hydrolase; OSHA, Occupational Safety and Health Administration; PEL, permissible exposure limit; SBR, styrene-butadiene rubber; SCE, sister chromatid exchanges; SEM, standard error of the mean; TG, 6-thioguanine; TWA, tame-weighted average; UTMB, University of Texas Medical Branch.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1,3-Butadiene (BD) is a major commodity chemical used in the manufacture of synthetic rubber and various plastics. Global consumption of BD was 6.1 million metric tons in 1995, with consumption expected to rise to more than 7.5 million metric tons in the year 2000 (1). BD ranked 36th in production volume in the United States in 1995 (over 3.6 billion pounds), with the United States producing approximately one fourth of the world's total industrial butadiene output of more than 5 million metric tons per year. It is estimated that at least 65 000 US workers may be exposed annually to BD (2). While BD is mainly an industrial chemical, it is also a common air contaminant found in auto emissions and cigarette smoke. It is a component of automotive exhaust and of the vapor phase of environmental tobacco smoke (~400 µg/cigarette) (3). Due to the potential for serious health effects associated with BD exposure, it was classified as one of 33 priority air toxics listed in the EPA's Integrated Urban Air Toxics Strategy (EPA FRL-6157-2 Docket No. A-97-44).

BD is carcinogenic in rodent bioassays (4), and exposure of mice to BD at 20 parts per million (p.p.m.) for 4 days induced mutations in spleen lymphocytes at the hprt locus (5). In several epidemiological studies, excess mortality from lymphatic and hematopoietic cancer was associated with occupational exposure to BD (6,7). Differences in carcinogenic responses between rats and mice, and weaknesses in some of the epidemiological studies, have resulted in controversies regarding the probable carcinogenic risks of BD to humans (8,9). In 1992, BD was classified by the International Agency for Research on Cancer (IARC) as a class 2A carcinogen (probable human carcinogen) (3). Risk assessments reported by the Occupational Safety and Health Administration (OSHA) based on both animal data and human data came to similar conclusions, placing the risk of BD-induced leukemia deaths, after a 45-year occupational exposure, at 1 to 8 per 1000 individuals (10). In 1996, OSHA set a new exposure standard for BD, reducing the permissible exposure limit (PEL) from a time-weighted average (TWA) for 8 h of 1000 p.p.m. to 1.0 p.p.m. (10). This dramatic restriction of the PEL was based on both animal carcinogenicity data (11) and human epidemiological data (12,13). Current occupational levels of BD are usually <2 p.p.m. (2).

The balance between the bioactivation of the parent compound, butadiene, and the detoxication of the resulting toxic metabolites most likely mediates the genotoxic and carcinogenic effects of BD. Therefore, characterization of the factors affecting this balance is critical in understanding the risks of adverse health effects from BD exposure. The bioactivation of butadiene produces at least three genotoxic metabolites: 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB) and 3,4-epoxy-1,2-butanediol (EBD). The genotoxic activity of EB and DEB has been reviewed by Jacobson-Kram and Rosenthal (14) and the IARC (3). EBD and EB are direct acting mutagens in human cells, but are relatively weak compared with DEB (15). DEB is a more potent genotoxic metabolite of butadiene than EB (16). In in-vitro studies with human lymphocytes, DEB was mutagenic at the hprt locus (15) and induced sister chromatid exchanges (16,17). Based on the ratio of urinary metabolites, humans appear to detoxify butadiene epoxides predominantly by hydrolysis, a reaction mediated by the microsomal epoxide hydrolase (mEH) enzyme. In mice, both hydrolysis of BD epoxides and glutathione conjugation by glutathione S-transferases (GSTs) contribute about equally (18). Mice have lower levels of mEH activity than humans or rats (8), but the conjugation pathway of detoxication is more active. In rats, the hydrolytic pathway appears to be more important. This difference has been cited as an important factor in the apparent difference in susceptibility of humans and mice to the carcinogenic effects of butadiene (8).

Despite the recent dramatic decrease in the PEL for BD, there is no guarantee that current levels of occupational exposure are safe for all workers. Our continuing research evaluating occupational exposures to BD, using biomarkers of exposure and genotoxic effects, has demonstrated that TWA exposure of workers to about 1–3 p.p.m. BD is associated with increases in frequencies of hprt mutant lymphocytes. This increase is about 3-fold in comparison with the mutant frequencies of workers in lower exposure areas or non-exposed subjects (19,20).

Although the establishment of appropriate safety limits for human exposure is crucial, it is limited by several factors. For example, there are apparent differences in response to BD between humans and laboratory animals. Thus, direct extrapolation of toxic effects from laboratory animals to humans would not be appropriate. More importantly, the metabolism of BD in humans is still poorly defined, partly because humans are very heterogeneous in their ability to metabolize and detoxify BD and other environmental agents. This variability in response can be largely attributed to the inheritance of polymorphisms (allelic variants with frequencies >1%) in genes of key enzymes regulating the metabolism of many environmental chemicals. In this respect, in vitro studies have shown that sensitivity to the reactive butadiene metabolites EB and DEB is increased in cultured human lymphocytes from individuals with the deletion polymorphisms for the GSTM1 and GSTT1 genes (2123). However, in the few human studies that have been conducted so far (2426), the association between polymorphisms in these genes and sensitivity to the genotoxic effects of BD exposure has been equivocal.

These equivocal results with polymorphisms in GSTs are not surprising given that hydrolysis by the epoxide hydrolase enzyme can also play a key role in detoxication of BD-reactive intermediates in humans. Several polymorphisms associated with alteration in enzyme activity have recently been described in the human microsomal epoxide hydrolase (mEH) gene, and it is possible that these polymorphisms may play a significant role in human sensitivity to butadiene (27,28). One of these polymorphisms was identified in the coding region of the mEH gene at exon 3. This polymorphism results in substitution of the amino acids tyrosine by histidine at residue 113 (Tyr113His), and in vitro expression studies of cDNA have indicated that, with this polymorphism, the corresponding mEH enzymatic activity is decreased by approximately 40% (27). This low activity His113 allele was shown to be associated with an increased risk of ovarian cancer (29), obstructive pulmonary disease and emphysema (30), hepatocellular carcinoma that developed following aflatoxin exposure (31) and increased risk of colorectal cancer (32).

The role of the mEH polymorphisms in BD-induced genetic damage in occupational settings has not previously been characterized. In the current study, we tested the hypothesis that the Tyr113His polymorphism increases sensitivity to the genotoxic effects of butadiene in exposed workers. We used personal badge dosimeters to quantitate the exposure to BD, and used the autoradiographic hprt mutant lymphocyte assay as a biomarker of effect to identify the genotoxicity associated with the BD exposure. The hprt variant (mutant) frequency (Vf) was determined in non-smoking workers from two styrene-butadiene rubber (SBR) plants who worked in areas of relatively high and low exposure to BD. These individuals were genotyped for polymorphisms in mEH and the glutathione S-transferases, GSTM1 and GSTT1, as potential biomarkers of susceptibility to BD. Here we describe our results indicating that individuals with the mEH 113 His allele are likely to be more susceptible to the genotoxic effects associated with exposure to BD.


    Materials and methods
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 Materials and methods
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Study population and sample collection
Forty-nine non-smoking subjects recruited from two SBR plants in Southeast Texas, as part of an ongoing biomonitoring study of BD-exposed workers, were included in our study. The subjects were recruited with the assistance of the Health and Safety Committees of the local unions. Exclusion criteria for participation in the study included recent treatment with potentially mutagenic agents, such as radiation or cancer chemotherapeutic drugs; chronic conditions such as autoimmune diseases; recent acute infections; and any exposure that could potentially cause elevations in hprt mutation rate such as previous employment involving chronic exposure to organic chemicals, radiation or heavy metals.

All study subjects were asked to sign an informed consent approved by the Institutional Review Board at the University of Texas Medical Branch (UTMB) prior to enrollment in the study. They were also asked to complete a brief questionnaire that requested information on gender, age, employment history, chemical exposure and history of tobacco use, alcohol and caffeine consumption, recent illnesses, general health and medications. Most of the study subjects were men and were a mixture of non-Hispanic white, African–American and Hispanic individuals. There were very few female workers in either facility.

Workers in both facilities work 12 h shifts with a 28 day cycle of rotating shifts. Exposure to BD was assessed by collecting breathing zone air samples using 3M 3520 passive badge dosimeters for three full 12 h work shifts. Air sampling was done 25, 20 and 14 days before blood collection, so that both days and nights were covered. This allowed estimates of exposure to be based on more than one measurement. In addition, each worker kept a simple diary during the shift when air samples were collected. This diary recorded the beginning and end of the exposure period for the badge, the badge identification number and activities for the day. The form asked the participants to list, for each hour of the shift, their work location, and whether their exposure to butadiene was low, average or high for their usual activities. They were also asked to describe any activities that produced above average exposures. These diaries had proven very useful in interpreting exposure values in our previous studies (20,33). In addition, each participant was given a calendar for the study period on which to record any unusual exposures that they experienced on days when air samples were not collected. At the end of the shift, the badges were capped and sealed, as instructed by the manufacturer, and sent to an outside contractor for GC/MS analysis of BD concentration. The detection limit for the 3M 3520 passive badge dosimeter in a 12 h shift for butadiene was typically 2.5 parts per billion (p.p.b.) (33).

From each study subject, a blood sample (approximately 65 ml) was obtained at the end of the 28 day cycle by a Registered Nurse from the UTMB Clinical Research Center (GCRC). A code number was assigned to each sample. The lymphocytes and plasma were isolated from the whole blood by density gradient centrifugation using Histopaque (Sigma). A 2 ml aliquot of plasma was frozen for subsequent determination of cotinine concentration by a radioimmunoassay method (34). The remaining plasma was stored at –20°C for later use in the hprt assay. The lymphocytes were washed, and resuspended at 107 cells/ml in RPMI 1640 medium (Gibco) with 10% dimethyl sulfoxide and 50% fetal bovine serum. The cells were then frozen in 1.2 ml cryotubes (Nunc) and stored in liquid nitrogen. Some of these lymphocytes were later thawed and cultured for the determination of exposure-associated genetic damage using the hprt mutant lymphocyte assay (35,36). About 10x106 cells were used for isolation of DNA, using a non-organic extraction procedure (37) and subsequent genotype analysis. The overall investigation was done using samples that were coded so that the identity or workplace activity of the subjects was not revealed.

Determination of exposure-associated genetic damage
The autoradiographic hprt mutant lymphocyte assay was used for the determination of BD exposure-associated genetic damage. The method for the autoradiographic hprt mutant lymphocyte assay is described in detail in Ammenheuser et al. (35,38). Briefly, the cryopreserved lymphocytes were thawed, washed and resuspended at a density of 1.1x106 cells/ml in growth medium (GM) prepared with RPMI 1640 with added L-glutamine and antibiotics (Gibco). Final volume of the GM contained 20% HL-1 medium supplement (Biowhittaker), 2% reagent grade phytohemagglutinin (Murex) and 25% autologous plasma. The cell suspension was pipetted in 4.5 ml volumes of 5x106 cells each into 50 ml flasks. Flasks for mutant selection received 0.5 ml 2x10–3 M 6-thioguanine (TG) and one labeling index flask received 0.5 ml RPMI 1640 medium adjusted to the same pH as the TG solution. All flasks were incubated in an upright position at 37°C for 24 h, labeled with 25 µCi [3H]thymidine and incubated for an additional 18 h. After the 42 h incubation period, 9 ml chilled 0.1 M citric acid per flask was added to produce a suspension of free nuclei. The nuclei were washed in fixative [7:1.5 (vol/vol) methanol:acetic acid] combined into one or two centrifuge tubes and resuspended in 0.25 ml fixative. A 20 µl aliquot of the cells was counted with a particle counter (Coulter). The remaining nuclei were added to 18x18 mm coverslips, previously affixed to microscope slides and were stained with aceto-orcein (Gurr). The slides were dipped in NTB-2 emulsion (Kodak), stored for 2 days at 4°C in light-tight boxes and developed with Kodak D-19. Slides were coded (so that readers were unaware of their origin), and then scanned with a light microscope and a count was made of all of the rare labeled (mutant) nuclei on slides made with cells from the TG-containing cultures. The slide from the labeling index (LI) culture was prepared with about 0.2x106 nuclei so that a random differential count could be made of the labeled and unlabeled cells. This count provides an estimate of the proportion of normal (non-mutant) lymphocytes that are able to grow in culture.

Mutant frequency calculation
The proportion of mutant lymphocytes (Vf) is calculated by dividing the total number of labeled (mutant) cells, derived from the TG-containing cultures (M), by the number of evaluatable cells (N). The number of evaluatable cells is determined by multiplying the Li by the total number of nuclei recovered from the mutant-selection cultures. (For most assays we obtain at least 1.5 million evaluatable cells). The Vf calculation is M/N.

Genotype analysis
mEH genotyping
A modification of the PCR–RFLP method described by Lancaster et al. (29) was used to identify the Tyr113His polymorphism in the mEH gene. The primer sequences and the PCR conditions described by Lancaster et al. (29) were used to create a Tth111I restriction site for the His113 allele using a reverse primer containing a mismatched C residue, 4 nucleotides from the 3'-end. The digested product was separated on high-resolution Metaphor® agarose gels that provided a better resolution of the digested fragments than conventional agarose. Tth111I restriction of the PCR product produces fragments of 209 and 22 bp for the His113 allele, whereas the Tyr113 allele remains undigested yielding a 231 bp fragment.

GSTM1 and GSTT1 genotyping
The deletion polymorphisms for GSTM1 and GSTT1 were determined simultaneously in a single assay using a multiplex PCR protocol developed in our laboratory (39). The presence or absence of GSTM1 and GSTT1 is detected by the presence or absence of a band at 480 bp (corresponding with GSTT1) and a band at 215 bp (corresponding with GSTM1). A band at 312 bp (corresponding with the CYP1A1 gene) is used as an internal control to ensure successful PCR amplification.

Statistical analysis
Analyses were performed using the PRISM computer software program. All variables were expressed as mean ± standard error of the mean (SEM). Statistical significance was determined by analysis of variance (ANOVA). Independent t-tests were used to assess the mean differences between the groups. A two-sided probability level of less than 0.05 was used as the criterion of significance.


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Only non-smokers were included in this study. These were individuals identified from their questionnaires as being non-smokers, and whose status as non-smokers was confirmed by analysis of plasma cotinine levels (34). Only individuals with plasma cotinine levels of <20 ng/ml were included in the investigation. The influence of the mEH Tyr/His113 polymorphism on the relationship between hprt Vf and exposure was examined. Visual examination of the data (Figure 1Go) revealed that individuals who were heterozygous or homozygous for the His113 allele had increased hprt Vf with increasing average exposure to BD. Individuals with the homozygous wild-type Tyr113 genotype had a different response, where very little increase in hprt Vf was observed with increasing average exposure to BD. The dose–response curves began to diverge at an average BD exposure of about 150 p.p.b. Most individuals above this level had exposures exceeding 200 p.p.b. For statistical comparison, the population was divided into two groups with the upper group having average exposures over 150 p.p.b. and the lower group having BD exposures <150 p.p.b. Sixteen individuals had exposures below the detection limit (2.5 p.p.b.).



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Fig. 1. Relationship between BD exposure and genetic damage as determined by the hprt mutant lymphocyte assay in SBR workers. •, Individuals with the Tyr/His or His/His genotype for the mEH gene; {diamond}, individuals with the wild-type Tyr/Tyr genotype for the mEH gene. Individuals with BD exposure below the detection limit (~2.5 p.p.b.) were assigned an exposure value of 1.25 p.p.b.

 
Characteristics of the study population are presented in Table IGo. The mean age for workers in the upper BD exposure group was 47.6 years and the mean age was 44.3 years for the lower group. In the study population, 63.3% of the workers were non-Hispanic whites, 30.6% were African–Americans and 6.1% were Hispanics. The mean (± SEM) levels of BD exposures were 2244.2 (± 749.1) and 18.4 (± 5.5) p.p.b. in the upper and lower groups, respectively. The frequencies of hprt mutant lymphocytes in exposed individuals (Vf) correlated well with the levels of exposure to BD. A significant 2-fold increase in Vf was detected (P = 0.02) in the upper exposure group (n = 16) compared with the lower group (n = 33) (Table IGo).


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Table I. Characteristics of the study population
 
Single polymorphism analysis
Genotypic analysis indicated that among the upper group exposed to BD levels higher than 150 p.p.b. (n = 16), two individuals had the GSTT1 homozygous deletion polymorphism (GSTT1 null), while 14 had the gene present. Of these 16 individuals, six were homozygous GSTM1 null, while 10 had the gene present. The genotypic analysis of the mEH Tyr113His polymorphism revealed that among the 16 individuals in the upper exposure group, seven were homozygous for the wild-type Tyr allele (Tyr/Tyr genotype), two were homozygous for the mutant 113His allele (His/His genotype) and seven had the heterozygous Tyr/His genotype.

In the lower exposure group, exposed to <150 p.p.b. BD (n = 33), genotypic analysis indicated that five individuals had the GSTT1 null genotype, while 27 had the gene present. Ten individuals had the GSTM1 null genotype, while 22 had the gene present. Data on both GSTM1 and GSTT1 polymorphisms were not available on one individual. The genotypic analysis of the mEH Tyr113His polymorphism revealed that among the 33 individuals in the lower exposure group, 12 were homozygous for the wild-type Tyr allele (Tyr/Tyr genotype), five were homozygous for the mutant 113His allele (His/His genotype) and 16 had the Tyr/His genotype.

Combined polymorphism analysis
Combined polymorphism analysis indicated that among the 16 individuals in the upper exposure group (exposed to BD >150 p.p.b.), four had the mEH Tyr/Tyr genotype and had both the GSTM1 and GSTT1 genes present, three had the mEH Tyr/Tyr genotype and were GSTM1 and/or GSTT1 null, four had at least one mutant His allele for mEH (His/His or His/Tyr genotype) and both GSTM1 and GSTT1 present, and five had at least one mutant His allele for mEH (His/His or His/Tyr genotype) and were GSTM1 and/or GSTT1 null.

Similarly, combined polymorphism analysis indicated that among 33 individuals in the lower exposure group (exposed to BD <150 p.p.b.), seven had the mEH Tyr/Tyr genotype and had both the GSTM1 and GSTT1 genes present, five had the mEH Tyr/Tyr genotype and were GSTM1 and/or GSTT1 null, 12 had at least one mutant His allele for mEH (His/His or His/Tyr genotype) and both GSTM1 and GSTT1 present, and five had at least one mutant His allele for mEH (His/His or His/Tyr genotype) and were GSTM1 and/or GSTT1 null. Combined polymorphism data on one individual in the lower exposure group were not available.

The effect of the individual genotypes on the level of genetic damage associated with BD exposure, as determined by the frequencies of hprt mutant lymphocytes in exposed individuals (Vf), was investigated. The data presented in Table IIGo indicate that in individuals in the upper exposure group (exposed to >150 p.p.b. BD), the mEH polymorphism seems to play a significant role in the sensitivity to BD exposure. In this group (n = 16), the His/His genotype was included with the Tyr/His genotype since only two individuals were homozygous for the mEH His/His genotype, and no significant difference was observed in the frequencies of hprt mutant lymphocytes between these two individuals and the seven individuals with the heterozygous Tyr/His genotype (data not shown). Our results indicate that individuals with at least one His allele (His/His or His/Tyr genotypes) had more than a 3-fold higher Vf (mean Vfx10–6 ± SEM = 13.25 ± 1.78) compared with individuals with the Tyr/Tyr genotype (4.02 ± 0.72). This difference in Vf between the two groups was highly significant (P < 0.001). The six individuals with the GSTM1 deletion polymorphism had a higher hprt Vf (12.05 ± 2.76) than the 10 individuals with the GSTM1 gene present (7.57 ± 1.77). The difference between these groups, however, was not significant (P = 0.16). The role of the GSTT1 polymorphism could not be evaluated since only two individuals had the GSTT1 deletion polymorphism.


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Table II. Effect of GSTT1, GSTM1 and mEH genotypes on BD-induced genetic damagea
 
Although the same observation was true with respect to the mEH polymorphism in the lower group (exposed to <150 p.p.b. BD), the difference in the frequencies of hprt mutant lymphocytes between individuals with at least one His allele (His/His or His/Tyr genotypes) (mean V f x10–6 ± SE = 5.32 ± 1.07) compared with individuals with the Tyr/Tyr genotype (3.95 ± 0.70) was not statistically significant. Similarly, no significant differences in Vf were detected with regards to the GSTM1 and GSTT1 polymorphisms in this group. Vf values were 5.06 (± 0.84) for GSTT1 positive individuals compared with 3.88 (± 1.21) for GSTT1 null individuals, and were 5.24 (± 1.01) for GSTM1 positive individuals compared with 4.05 (± 0.94) for GSTM1 null individuals (Table IIGo).

The combined effect of the polymorphisms in the GSTM1, GSTT1 and mEH genes on BD-induced genetic damage was also investigated. We conducted a case series analysis where we classified the studied population into four groups according to their genotypes. The first group (Group A) included individuals with the genetic combination mEH Tyr/Tyr and both GSTM1 and GSTT1 positive genotypes. The second group (Group B), included individuals with the mEH Tyr/Tyr and either GSTM1 and/or GSTT1 null. The third group (Group C) included individuals with the mEH Tyr/His and/or His/His and both GSTM1 and GSTT1 positive genotypes, and the fourth group (Group D), included individuals with mEH Tyr/His and/or His/His and either GSTM1 and/or GSTT1 null genotypes. As shown in Figure 2Go, the results indicate that in the upper group (individuals exposed to >150 p.p.b. BD), a statistically significant difference (P = 0.01) was observed between the mean Vf values of the four groups studied. These results indicate that, regardless of their GSTM1 or GSTT1 genotypes, individuals who had the polymorphic mEH His allele (Groups C and D) had significantly (P < 0.05) higher frequencies of mutant lymphocytes (mean V fx 10–6 ± SEM = 12.08 ± 3.08 for Group C and 14.19 ± 2.3 for Group D) than individuals with the homozygous Tyr/Tyr genotype (mean V f x10–6 ± SEM = 4.14 ± 1.09 for Group A and 3.87 ± 1.16 for Group B). The level of genetic damage was highest in individuals who had at least one mEH His allele and either the GSTM1 and/or GSTT1 null genotypes (Group D). In contrast, there were no statistically significant differences in mean Vf values between the four groups of individuals in the lower group exposed to BD < 150 p.p.b. (Figure 2Go).



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Fig. 2. Effect of combined genetic polymorphisms in GSTT1, GSTM1 and mEH on BD-associated genetic damage. Genotoxicity was determined by frequencies of hprt mutant lymphocytes (Vf) in exposed individuals using the autoradiographic assay. Group A, individuals with the genotypic combination: mEH Tyr/Tyr and both GSTM1 and GSTT1 positive; group B, individuals with mEH Tyr/Tyr and either GSTM1 and/or GSTT1 null; group C, individuals with mEH Tyr/His and/or His/His and both GSTM1 and GSTT1 positive and group D, individuals with mEH Tyr/His and/or His/His and either GSTM1 and/or GSTT1 null. *Significantly different from group A (P < 0.05).

 

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Although humans are frequently exposed to very low concentrations of BD, the most intense and potentially hazardous exposures occur in occupational settings. As industry has responded to the recognized health hazards from butadiene exposure with tighter exposure standards, occupational exposures have declined. In the current investigation, we studied a population of workers in two SBR plants in Southeast Texas to determine whether current levels of BD exposure in industrial settings in the United States are associated with genotoxicity. This report addresses one aspect of a larger study of the effect of occupational exposure to BD on the frequency of mutations at the hprt gene. The details of the study are described elsewhere (Ward et al., submitted). The focus of this report is to evaluate the effects of polymorphisms in genes of biotransformation enzymes on sensitivity to BD exposure as measured by hprt Vf.

As is typical of petrochemical workers in Southeast Texas, the subjects who volunteered for our study were predominantly middle-aged males, with a few older individuals and a few individuals under the age of 30. Automation of the chemical industry has resulted in reductions in total employment, with proportionally more of the senior employees remaining and fewer new hires. The demographic make-up of the study population was largely a reflection of the make-up of the union-affiliated worker population.

One of the goals of the present study was to obtain information regarding BD exposure in industrial settings integrated over several weeks. Therefore, repeated air measurements were performed in the current study to provide a better estimate of each worker's usual average daily exposure. The hprt mutant lymphocyte assay, the biomarker of effect used in our studies, responds to cumulative exposures over several months but also may require as long as 2 weeks, following exposure to a mutagen, for expression of the mutant phenotype (38,40). In both of the SBR plants studied, we collected air samples on three occasions: 25, 20 and 14 days prior to collection of the blood sample to be used for hprt analysis. Using this exposure monitoring approach, we found that the majority of the study subjects (67%) were exposed to very low levels of BD (TWA <150 p.p.b.). However, in certain areas of the plants, such as the reactor, recovery, tank farm, laboratory and the polymerization areas, we found levels of BD exposures in some of the workers that averaged >2 p.p.m., which is twice the PEL set by OSHA (10).

Despite the reduction in current exposure levels compared with a few years ago, we could still detect genetic damage associated with BD exposure. Our results show a significant 2-fold increase in frequencies of hprt mutant lymphocytes in the group of workers with exposure above our criterion level of >150 p.p.b. BD compared with the group of workers exposed to <150 p.p.b. These results are consistent with our earlier observations that have established that occupational exposure to BD, in either monomer or SBR polymer production, is associated with an increase in the frequency of somatic cell mutations in lymphocytes (19,20,33,51, Ammenheuser et al., manuscript submitted). These findings indicate that there still may be potential health risks to some workers under current exposure conditions. It is interesting to note that although there was a dose-related increase in Vf in SBR workers starting about 150 p.p.b., there were also a number of high Vf values for individuals in whom BD exposure was not detectable (five subjects, whose Vf values ranged from 8.7x10–6 to 18.9x10–6; Figure 1Go). Although the reason for this elevation is not fully understood, we believe that it may be due to the fact that these workers have had higher exposure to BD in the past, when the permitted limits were much higher. Consistent with this hypothesis, these five individuals (four of whom had the 113His low activity allele for the mEH gene) were long-term employees with work-place longevity that ranged from 28.5 to 37 years in the SBR industry. The extent of persistence of hprt mutant lymphocytes, following periods of chronic, low-level exposure to mutagenic agents, is not known. In a recent study, however, Thomas et al. (53) reported elevated frequencies of hprt mutant lymphocytes in Russian liquidators 6–10 years after exposure to radiation from the Chernobyl nuclear power plant accident. Our data are consistent with this observation, indicating that some hprt mutant cells have long life spans. This opens the door for future studies to clarify the relationship between past exposures and the possible persistence of genotoxic damage. The combination of past chronic exposure and a low activity mEH genotype could have contributed synergistically to the high Vf values observed in several SBR workers who seem to have no current BD exposure.

Occupational exposure to butadiene has been evaluated with biomarkers in several studies in our laboratory (19,20,33,51,52) and by other investigators (21,25,26,4144). Most of the evaluations of worker populations for cytogenetic effects have failed to detect a relationship between BD exposure and genotoxic effect (21,25,26,43,44,52). For example, Sorsa et al. (44) evaluated 70 workers in two manufacturing plants for chromosome aberrations, sister chromatid exchanges (SCE) and micronuclei in lymphocytes. Exposure levels were below 3 p.p.m. and no differences between higher and lower exposure groups were seen in any assay. SCE were also not elevated in one of the populations we have investigated (21). However, Tates et al. (43) in a 1993–94 study of workers exposed to a mean level of about 1.8 p.p.m. BD, detected a small, but significant increase in the percentage of chromosome aberrations in BD-exposed workers compared with low-exposure controls (P < 0.01).

We have consistently observed increased frequencies of mutations in the hprt reporter gene in BD exposed workers (19,20,33,51, Ammenheuser et al., submitted). Two other studies of occupational exposure to BD have been conducted, one in the Czech Republic and one in China. Both of these studies used the cloning version of the hprt assay to assess the mutagenic effects of BD exposure. In the study conducted with workers from a BD production plant near Prague in the Czech Republic, there was no significant difference between exposed and control subjects in the geometric mean hprt Mf, adjusted for cloning efficiency, age and smoking (43). A multi-national collaborative study of the same facility in the Czech Republic was recently completed and reports are now in preparation (Albertini et al., submitted). In this study a very detailed evaluation of occupational exposure to BD was conducted, and biological exposure was assessed by both urine metabolite analysis and measurement of hemoglobin adducts. Effects of exposure were measured by analyses for chromosome damage and by assays for hprt mutation frequencies. While a significant correlation between exposure to BD and the exposure biomarkers was observed, the genetic biomarkers did not correlate with exposure (Albertini et al., submitted; Tates et al., submitted). The mean level of BD in the polymer production area in this study was only 0.81 p.p.m., which is considerably lower than mean BD levels in our studies of workers in a SBR facility in Texas (33,51, Ammenheuser et al., submitted).

A more recent study conducted in China indicated that BD-exposed workers had no increase in hprt mutation frequencies (Mfs) in lymphocytes studied with the cloning assay compared with unexposed controls (26). In contrast with these findings, using the cloning assay, we have observed a significant elevation in hprt mutation frequency (P < 0.05) in lymphocytes of BD-exposed workers (mean Mf = 17.63x10–6) compared with unexposed control subjects (Mf = 8.47x10–6) (20). Furthermore, sequence analysis of hprt cDNA from 175 independent mutant clones indicated that the distribution of mutation types was different between workers and controls. The mutational spectral analysis revealed a significant increase in exon deletions (P < 0.05) in BD-exposed workers compared with controls (20). The reason for the lack of correlation between exposure and hprt mutation frequency reported in the study conducted in China is not clear. A possible explanation would be the high background level in mutation frequency observed in unexposed controls (Mf = 17.2x10–6), which could have masked a possible increase in Mf due to BD exposure (Mf = 16.8x10–6 in the exposed group) (26). No information regarding the backgrounds of the unexposed individuals was provided to allow the identification of other exposures or biological factors that might account for the elevated hprt Mf in this group.

The levels of BD exposure detected in our study may not seem extremely high compared with previous levels of exposure before the new OSHA standards. At these current exposure levels, however, the contribution to health risks from specific genetic traits that could influence the biotransformation and toxicity of BD may become extremely critical. Certain polymorphisms in genes of enzymes that metabolize or detoxify BD may leave a subset of the population of exposed workers at greater risk than the general population. In order to characterize the influence of some of these genetic traits on human sensitivity to BD, several in vitro studies have addressed the role of two key enzymes in butadiene metabolism. These studies suggested that genetic polymorphisms in the GST detoxication enzymes could affect the genotoxicity of BD (21,22,4548). However, in the few human studies that have been conducted so far (2426), no clear association has been observed between polymorphisms in these GST genes and sensitivity to BD exposure. Consistent with these observations, our results indicate that GST polymorphisms seem to play a minor role in human sensitivity to BD, particularly at low exposure levels. Our data suggest that at higher BD exposure levels, GSTM1 may play a small role, as indicated by the non-significant 1.5-fold increase in genetic damage observed in the GSTM1 null individuals compared with the GSTM1 positive individuals in the higher exposure group. The role for GSTT1, with respect to higher exposure levels of BD, could not be assessed in the current study due to the small number of individuals in that group with the GSTT1 null polymorphism (n = 2).

The lack of a clear role for GST polymorphisms prompted us to consider the possibility of the involvement of other genetic susceptibility factors that could influence human sensitivity to BD. In the current investigation, we tested the hypothesis that the Tyr113His polymorphism in the mEH gene may affect human sensitivity to BD. Our data indicate that individuals who were exposed to BD levels of >150 p.p.b., who also had at least one His allele (His/His or His/Tyr genotypes), had more than a 3-fold higher Vf compared with individuals with the Tyr/Tyr genotype (P < 0.001). This observation was also true in the lower exposure group, but the difference between the Tyr113 and the His113 genotypes at low BD exposures was not significant. This is not surprising since the concept is that a reduced rate of detoxication would only be important if exposure to BD has occurred. The effect of the homozygous His/His genotype could not be compared with the heterozygous His/Tyr genotype in this study due to the small sample size. Additional studies are in progress to address this issue.

Since susceptibility to the genotoxicity of BD is likely to be determined by differences in genotypes for a number of metabolizing enzymes, we also conducted a case series analysis where we classified the studied population into four groups according to their genotypes for mEH, GSTT1 and GSTM1. Again, in the presence of BD exposure >150 p.p.b., the mEH polymorphism seemed to be the determinant sensitivity factor. The observation that the highest level of genetic damage was detected in individuals with the 113His allele, who also had a deletion polymorphism in either or both GSTs (Figure 2Go), suggests that the GSTs may still contribute to detoxication of reactive BD metabolites in exposed workers.

Although additional studies are still needed to provide a clear mechanistic explanation for our findings, it seems plausible to assume that individuals with the low-activity His allele might have reduced ability to detoxify the reactive metabolites of BD and, hence, might have increased susceptibility to the genotoxic effects of BD. The bioactivation of butadiene produces at least three genotoxic metabolites: EB, DEB and EBD, and mEH plays a role in the detoxication of all of these reactive metabolites (49). The importance of mEH in BD detoxication is supported by the findings of Bond et al. (8) who indicated that mice, which have lower levels of EH activity than humans or rats (49) are more sensitive to the carcinogenic effects of BD compared with rats and humans. The importance of mEH is also supported by our recent molecular studies that indicated that BD-exposed workers had significantly higher frequencies of large deletions at the hprt locus (20). A high frequency of large deletions is an indication of the possible accumulation of the reactive DEB metabolite, as documented by in vitro studies investigating the changes in the hprt mutation spectra induced by different BD metabolites (15,50).

In summary, our data indicate that polymorphisms in the mEH gene may play a significant role in human sensitivity to the genotoxic effects of BD exposure, and that detection of elevations in the frequency of hprt mutant lymphocytes can serve as a sensitive biomarker of genotoxicity for monitoring occupational exposure to BD in industrial settings. Additional investigations in larger populations of workers are needed to confirm our results, and additional research is needed to provide mechanistic explanations for our findings. Studies are currently in progress in our laboratory to address these issues and to characterize the possible role of additional mEH polymorphisms in genetic damage associated with occupational exposure to butadiene.


    Notes
 
1 To whom correspondence should be addressed Email: sabdelra{at}utmb.edu Back


    Acknowledgments
 
The authors thank Dr Thomas Stock and Dr Maria Morandi of the University of Texas School of Public Health in Houston, TX for analysis of exposure badges. We thank Sharon McConley and Jene Barker for their help with some of the assays. We also thank the Workplace Toxics Foundation, and the NIEHS Environmental Toxicology Center at UTMB, funded by ES 06676. Studies were conducted with assistance of the General Clinical Research Center at UTMB, funded by grant M01 RR-00073 from the National Center of Research Resources, NIH, USPHS. This work was supported by a RO1-ES06015 grant from the National Institute of Environmental Health Sciences.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 6, 2000; revised November 21, 2000; accepted November 27, 2000.