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Carcinogenesis Advance Access originally published online on September 10, 2008
Carcinogenesis 2008 29(12):2325-2329; doi:10.1093/carcin/bgn208
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Polymorphisms in phase I and phase II metabolism genes and risk of chronic benzene poisoning in a Chinese occupational population

Pin Sun1,2,{dagger}, Ji Qian3,{dagger}, Zhong-bin Zhang1,2, Jun-xiang Wan1,2, Fen Wu1,2, Xi-peng Jin1,2, Wei-wei Fan3, Da-ru Lu3, Nai-qing Zhao4, David C. Christiani5 and Zhao-lin Xia1,2,*

1 Department of Occupational Health, School of Public Health, Fudan University, Shanghai 200032, China
2 Key Laboratory of Public Health Safety (Fudan University), Ministry of Education, Shanghai 200032, China
3 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200043, China
4 Department of Epidemiology and Statistics, School of Public Health, Fudan University, Shanghai 200032, China
5 Environmental and Occupational Medicine and Epidemiology Program, School of Public Health, Harvard University, Boston, MA, USA

* To whom correspondence should be addressed. Tel/Fax: +86 21 54237050; Email: zlxia{at}shmu.edu.cn

Correspondence may also be addressed to Da-ru Lu. Tel/Fax: +86 21 65642799; Email: drlu{at}fudan.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
It is widely accepted that the cytotoxicity and genotoxicity of benzene results from the action of reactive metabolites. Therefore, genetic variation in metabolic enzyme genes may contribute to susceptibility to chronic benzene poisoning (CBP) in the exposed population. Using a case–control study that included 268 benzene-poisoned patients and 268 workers occupationally exposed to benzene in South China, we aimed to investigate the association between single-nucleotide polymorphisms in genes with phase I and II of metabolism and risk of CBP. The TaqMan technique was used to detect polymorphisms of CYP1A1, CYP1A2, CYP1B1, ADH1B, EPHX1, EPHX2, NQO1, MPO, GSTP1 and UGT1A6 genes. We also explored potential interactions of these polymorphisms with lifestyle factors such as cigarette smoking and alcohol consumption. A weak positive association was found between glutathione S-transferase pi-1 (GSTP1) rs1695 polymorphism and the risk of CBP (P = 0.046), but this association was not statistically significant (P = 0.117) after adjustment for potential confounders. Further analysis showed that the risk of CBP increased in the subjects with EPHX1 GGAC/GAGT diplotype (P = 0.00057) or AGAC/GAGT diplotype (P = 0.00086). In addition, we found that alcohol drinkers with the EPHX1 rs3738047 GA + AA genotypes and non-alcohol drinkers with the GSTP1 rs1695 AA genotype tended to be more susceptible to benzene toxicity. Our results suggest that genetic polymorphisms in EPHX1 may contribute to risk of CBP in a Chinese occupational population.

Abbreviations: ADH, alcohol dehydrogenase; CBP, chronic benzene poisoning; CI, confidence interval; CYP, cytochrome P450; GSTP1, glutathione S-transferase pi-1; mEH, microsomal epoxide hydrolase; MPO, myeloperoxidase; NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1; OR, odds ratio; sEH, soluble epoxide hydrolase; SNP, single-nucleotide polymorphism; UGT1A6, uridine 5'-diphospho-glucuronosyltransferase 1A6


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Benzene is widely recognized as a prototypical hematotoxic and genotoxic carcinogen. Exposure to benzene can induce chronic benzene poisoning (CBP) characterized by hematotoxicities, leading to pancytopenia, aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia and chronic lymphocytic leukemia (1,2).

It is generally accepted that the metabolites of benzene cause cytotoxicity and genotoxicity (3). Benzene is initially oxidized via cytochrome P450 (CYP) 2E1 in the liver, forming benzene oxide, which can form phenol spontaneously or be hydrolyzed by microsomal epoxide hydrolase (mEH; EPHX1) to form catechol (4). Phenol is further catalyzed by CYP2E1 to form potentially toxic hydroquinone (5). Once in the bone marrow, hydroquinone and catechol are converted by myeloperoxidase (MPO) to benzoquimoes, a potent hematotoxic agent, which can be detoxified by reduction via nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1 (NQO1) to less harmful hydroxybenzenes (6). Catalyzed by phase II metabolic enzymes such as glutathione S-transferase pi-1 (GSTP1) and uridine 5'-diphospho-glucuronosyltransferase 1A6 (UGT1A6) isozymes, these benzene metabolites are easily conjugated with glutathione or glucuronide to form less toxic or non-toxic derivatives (7,8) and being excreted in urine (supplementary Figure S1 is available at Carcinogenesis Online). Other metabolic enzymes, including CYP1A1, CYP1A2, CYP1B1 and soluble epoxide hydrolase (sEH; EPHX2), can convert benzene and its metabolites to other reactive intermediates, which are also thought to contribute to benzene toxicity (9–12). In addition, there exists an interaction between benzene and alcohol (13,14), so that alcohol dehydrogenase (ADH) affecting alcohol metabolism may change the toxicity of benzene. Thus, we hypothesized that the deficient or altered phase I and II metabolic enzymes involved in benzene metabolism, such as CYP1A1, CYP1A2, CYP1B1, ADH1B, mEH, sEH, NQO1, MPO, GSTP1 and UGT1A6, would effect individual susceptibility to benzene toxicity.

Genetic polymorphisms may affect the level of expression, structure or catalytic activity of metabolic enzymes, thereby influencing the risk of cancer. Petersen et al. (15) reported that a MspI polymorphism in CYP1A1 was associated to changes in enzyme inducibility, and Sachse et al. (16) found that a single-nucleotide polymorphism (SNP) in intron 1 at position 734 (rs762551) of CYP1A2 gene can affect the inducibility of the enzyme. An early in vitro study has shown that the ADH1B*2 allele increases ethanol oxidation by 40-fold compared with ADH1B*1 (17). The codon 139 polymorphism (rs2234922) in EPHX1 gene, the codon 287 polymorphism (rs751141) in EPHX2 gene and the codon 105 polymorphism (rs1695) in GSTP1 gene were also reported to modify the catalytic activity of the enzyme (18–20). For UGT1A6 gene, van der Logt et al. (21) found that metabolic rates of phenols by recombinant UGT1A6*2 were lower than those of the most common enzyme. These common SNPs are indicated to be associated with individual cancer susceptibility (22–25). Although no clear association of CYP1B1 polymorphisms with altered enzyme activity has been shown, epidemiological studies suggest that the polymorphism at codon 453 (rs1056836) in this gene may be an important modifier of cancer risk (26,27). However, whether or not these SNPs affect risk of CBP is still unknown.

Recently, Wang et al. (10) reported that the CYP1A1 HincII polymorphism may modify the association between low benzene exposure and shortened gestation, and Lan et al. (28) reported that the MPO –463 G > A polymorphism (rs2333227) and NQO1 465 C > T polymorphism (rs4986998) influence susceptibility to benzene hematotoxicity. However, it is yet unclear whether other polymorphisms in those genes could affect the risk of CBP; among these include the C-to-A transversion in exon 2 (rs7208693) of MPO, resulting in an amino acid substitution from phenylalanine to valine at codon 53, and the C-to-T transversion at codon 187 in exon 2 (rs1800566) for the NQO1 gene, associated with loss of enzyme activity (29).

So far, few published studies have investigated more than a few SNPs in genes involved in benzene metabolism. Thus, a more comprehensive understanding is needed with respect to relevant genes and their potentially functional polymorphisms. Furthermore, most chronic and complex diseases are believed to be caused by interactions among environmental factors, genes and lifestyle. In this study, we conducted a case–control study to explore the associations of SNPs with human susceptibility to CBP, focusing on 10 genes of phase I and II metabolic enzymes. The potential modification effect of lifestyle was also investigated as a confounding factor.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Study subjects
The details of this case–control study have been described elsewhere (30,31). Briefly, 228 of 268 CBP patients in this study came from five factories where clusters of cases were reported in Shanghai, Hangzhou, Maanshan, Wuhu and Guangzhou, China. Another 40 patients who returned to the hospital periodically for health examinations were recruited from 12 other small factories that had been closed down. Benzene poisoning was diagnosed between 1980 and 2004 by the locally authorized Occupational Disease Diagnostic Team. The diagnostic criteria for occupational benzene poisoning, according to the Ministry of Health of China, has also been described previously (30,31). Two hundred and sixty-eight healthy workers who had been occupationally exposed to benzene in the five major factories were selected as controls. Control subjects were frequency matched to cases by gender, age within 5 years, employment duration of the same working environment within 3 years and exposure level at the workplace. The calendar range for the start of employment of the cases and the controls was between 1958 and 2004 and between 1963 and 2004, respectively. All the eligible individuals gave informed consent before entering the study.

The subjects were administered a questionnaire for general information including ethnic background, nutrition, cigarette smoking, alcohol consumption, protective measures, self-reported symptoms, medical history and occupational history such as work unit (department), type of work and exposure duration. The subjects underwent a thorough physical examination at a local occupational disease hospital. Alanine aminotransferase level in serum was also examined for liver function evaluation.

Exposure data
Exposure estimation was based on monitoring data or industrial hygienists and long-term employees’ evaluation, considering historical changes (32). The intensity of benzene exposure (milligrams per cubic meter) for the patients was taken as the benzene level of workplaces while diagnoses were made; the intensity of benzene exposure for the controls was taken as the current level monitored by organic vapor-passive dosimetry badges during collection of the blood samples from controls.

Genotyping
Genomic DNA was isolated by a conventional phenol–chloroform extraction and ethanol precipitation. Genotyping for CYP1A1 (rs4646421, rs4646422, rs1048943 and rs4646903), CYP1A2 (rs2445618, rs762551, rs2472304 and rs2470890), CYP1B1 (rs1056836), ADH1B (rs1229984), EPHX1 (rs2854451, rs3738047, rs2234922 and rs1051741), EPHX2 (rs781141), NQO1 (rs1800566), MPO (rs7208693), GSTP1 (rs1695) and UGT1A6 (rs6786892, rs1105879, rs4124874, rs3755319, rs887829 and rs4148323) was analyzed using TaqMan-MGB probes and primers, designed through the Assay-by-Design or Assay-on-Demand service of Applied Biosystems (Foster City, CA). All polymerase chain reactions were performed in 384-well plates in a 6 µl volume containing 2 µl 2x TaqMan Universal PCR Master Mix (QIAGEN Ltd, Crawley, UK), 0.04 µl 40x Assay-by-Design probe or 0.08 µl 20x Assay-on-Demand probe and ~5 ng genomic DNA. Assays were carried out according to the manufacturer’s recommendations on an ABI 7900HT apparatus. Genotyping error rate was directly determined by regenotyping 10% of randomly chosen samples for each SNP. The overall error rate was <0.005. Haplotypes and diplotypes (haplotype pairs) of CYP1A1, CYP1A2, EXPH1 or UGT1A6 were estimated from genotype data for individual participants by the PHASE 2.0.2 software (University of Washington, Seattle, WA) (33,34).

Statistical analysis
Student’s t-test was used to compare the differences between continuous variables, such as age and exposure duration. Each SNP was tested in controls to ensure conformity with the Hardy–Weinberg equilibrium. Pearson’s chi-squared test or Fisher’s test was used to evaluate the differences in the distribution of genetic polymorphisms between cases and controls. To evaluate whether lifestyle modified the association between genetic polymorphisms and risk of CBP, Pearson’s chi-squared test or Fisher’s test was also applied after stratification according to cigarette smoking or alcohol consumption. The test for homogeneity of odds ratios (ORs) was examined by the Breslow–Day method. The heterogeneity of ORs indicated that there may be interaction (P < 0.05). The OR and 95% confidence interval (CI) for estimating the associations of genetic polymorphisms with the risk of CBP were obtained from unconditional logistic regression models with adjustment for potential confounders including gender, cigarette smoking, alcohol consumption, exposure duration and intensity of benzene exposure. In the haplotype-based and diplotype-based case–control analysis, haplotypes or diplotypes with a frequency <0.01 were excluded. The frequencies of haploype and diplotype in cases and controls were compared by chi-squared statistics. Two-tailed P-value <0.05 was considered statistically significant, and the results were adjusted using the Bonferroni correction method for multiple comparisons. All analyses were performed with SPSS version 10.0 (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Demographics of the cases and controls
The distributions of age, gender, exposure duration, intensity of benzene exposure, cigarette smoking and alcohol consumption in the cases and controls are shown in Table I. The median age and exposure duration was 30.0 (range: 18.0–57.0) and 4.5 (range: 1.0–38.0) in 268 CBP cases, respectively, and 32.0 (range: 17.0–68.0) and 4.0 (range: 1.0–36.0) in 268 controls, respectively. There was no significant difference in the distribution of age (in four groups: <26, 26–35, 36–45 and ≥46 years), exposure duration (<6, 6–10, 11–15, 16–20 and ≥21 years), intensity of benzene exposure (≤40, 41–100 and >100 mg/m3) and gender (P > 0.05 for all), suggesting that frequency matching was adequate. Because the data were in part coming from historical records, some records were incomplete for data on cigarette smoking or alcohol consumption. For those with historical data, the percentage of females was 62.1% in cases and 62.9% in controls for those who had cigarette smoking data and 61.2% in cases and 62.9 % in controls for those who had alcohol consumption data. The range for total white blood cell counts in the controls was from 4500 to 5900/µl; the mean was 4838 ± 259/µl.


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  		Table I. Characteristics of selected demographic and exposure variables in chronic benzene poisoning cases and controls

 
Prevalence of genotypes of CYP1A1, CYP1A2, CYP1B1, ADH1B, EPHX1, EPHX2, NQO1, MPO, GSTP1, ADH1B and UGT1A6 genes
The frequency distributions of genotypes of phase I and II metabolic enzyme genes in this study population are shown in supplementary Table 1 (available at Carcinogenesis Online). Except for CYP1A2 rs2445618 and UGT1A6 rs1105879 and rs887929, the distributions of the genotypes of other SNPs in the controls were all in Hardy–Weinberg equilibrium (P > 0.05, data not shown).

Effect of genetic polymorphisms of metabolic enzyme genes on the risk of CBP
Except for the GSTP1 rs1695 polymorphism, no association between variant genotypes of other genes and risk of CBP was found (P > 0.05). The proportion of individuals carrying GSTP1 rs1695 variant genotypes (AG + GG) was lower in cases (23.6%) than in controls (31.9%; P = 0.046). However, after adjustment for gender, smoking, alcohol consumption, exposure duration and intensity of benzene exposure, the difference was not significant (ORadj = 0.63; 95% CI, 0.36–1.12; P = 0.119) in the distribution of GSTP1 rs1695 genotypes between cases and controls.

Relation of genetic polymorphisms of NQO1, MPO, CYP2E1, GSTM1 and GSTT1 with the risk of CBP modified by lifestyle
Test for homogeneity (H) of ORs indicated a possible interaction between GSTP1 rs1695, EPHX1 rs3738047, EPHX1 rs2234922, EXPH1 rs1051741 and alcohol consumption ({chi}Formula=7.020, P = 0.008; {chi}Formula=4.179, P = 0.011; {chi}Formula=6.713, P = 0.010 and {chi}Formula=5.283, P = 0.022, respectively). The risk of CBP stratified by alcohol consumption indicated a significantly decreased risk associated with GSTP1 rs1695 AG + GG genotypes was confined to non-alcohol drinkers (OR = 0.44; 95% CI, 0.24–0.81; P = 0.007). Adjustment for gender, exposure duration, intensity of benzene exposure and smoking did not substantially affect on the results (ORadj = 0.48; 95% CI, 0.25–0.90; P = 0.018). Compared with subjects carrying the EPHX1 rs2234922 AA genotype or rs1051741 CC genotype among alcohol drinkers, those with EPHX1 rs2234922 AG + GG or rs1051741 CT + TT genotypes had a decreased risk of CBP (Fisher’s exact test, P = 0.008, P = 0.043, respectively). Compared with those of EPHX1 rs3738047 GG genotype among alcohol drinkers, subjects with EPHX1 rs3738047 GA + AA genotypes had a 5-fold increase for CBP (OR = 5.00; 95% CI, 0.89–30.52; Fisher’s exact test, P = 0.073), and this risk increased even higher, to 16.51-fold, after adjustment for potential confounders (ORadj = 16.51; 95% CI, 1.83–148.80; P = 0.012).

The subjects were further stratified by smoking habits, and no significant differences were observed in genotype distributions of these genes between cases and controls before and after the adjustment for gender, exposure duration, intensity of benzene exposure and alcohol consumption (P > 0.05 for all).

Haplotypes of polymorphisms in CYP1A1, CYP1A2, EXPH1 and UGT1A6 and the risk of CBP
Applying the PHASE software, we reconstructed the haplotypes of CYP1A1, CYP1A2, EXPH1 and UGT1A6 genes in this study population (Table II, supplementary Tables 2 and 3 available at Carcinogenesis Online). Since some SNPs in CYP1A2 and UGT1A6 in this present study departed from Hardy–Weinberg disequilibrium, we only investigated the role of CYP1A1 haplotypes and EXPH1 haplotypes on the risk of CBP. For EXPH1, significant differences were observed in the distributions of those haplotypes in cases and those in controls. Using the Bonferroni correction with five tests (requiring P < 0.01), the statistically significant differences were observed in the distribution of haplotype of GAGT and GGGT (P < 0.01 for all). Compared with those carrying the GGAC haplotype, subjects with GAGT haplotype had an increased risk of CBP (OR = 7.08; 95% CI, 2.73–18.38; P = 0.000005), but subjects with GGGT haplotype had a decreased risk of CBP (OR = 0.19; 95% CI, 0.06–0.57; P = 0.001). For CYP1A1, no significant differences were observed.


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  		Table II. Haplotypes of EPHX1 and the risk of CBP

 
In this study population, we also reconstructed diplotypes of CYP1A1, CYP1A2, EXPH1 and UGT1A6 (Table III and supplementary Tables 2, 4 and 5 are available at Carcinogenesis Online). For EPHX1, a statistically significant difference between cases and controls was observed in the distribution of GGAC/GAGT diplotype (P = 0.00057) or GAAC/GAAC diplotype (P = 0.00086), and using the Bonferroni correction with 13 tests (requiring P < 0.0038), the results were still significant. The diplotypes of other genes were not found to affect the risk of CBP in this population.


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  		Table III. Diplotypes of EPHX1 and the risk of CBP

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
It is postulated that oxidation of benzene and resultant metabolites is responsible for benzene toxicity because these metabolites can alkylate proteins and DNA (35). Studies have shown that individuals vary in their susceptibility to the adverse effects of benzene exposure (36,37), probably due to differences in metabolic genes and their corresponding enzyme activity. Many case–control studies have focused on genetic polymorphisms that affect benzene metabolism or metabolic enzymes involved in phase I and II of the metabolism of the toxicant (10,28,30,38). However, due to the complexity of benzene metabolism, investigations of multiple SNPs or genes in the relevant metabolic pathways need to be explored to clarify the associations between genetic polymorphisms of candidate metabolic genes and risk of CBP.

In the present study, we assessed EPHX1 polymorphisms that may affect individual susceptibility to CBP and tested four SNPs that are thought to affect enzymatic activities. Although none of these EPHX1 SNPs was significantly different in the genotype frequency between cases and controls, respectively, haplotype analysis using the Bonferroni correction showed that the subjects with GAGT haplotype had a significantly increased risk of CBP, whereas those with GGGT haplotype had a significantly decreased risk of CBP. Further, diplotype analysis showed that the subjects with GGAC/GAGT diplotype (P = 0.00057) or AGAC/GAGT diplotype (P = 0.00086) had a significantly increased risk of CBP. We hypothesized that this observation was due to the diplotypes’ alteration of mEH activity, resulting in increasing benzene toxicity. However, no studies to date have confirmed that these diplotypes actually alter mEH activity. Once confirmed, these EPHX1 risk diplotypes may be useful in future occupational health surveillance as markers to screen and monitor workers who are occupationally exposed to benzene.

The GSTP1 rs1695 A > G polymorphism, which modifies the catalytic activity of the enzyme (20), has been shown to be associated with a higher risk for several cancer types (39–41). In this study, we found a weak, but statistically significant, association between this SNP and the risk of CBP. However, after adjustment for potential confounders, the association was not statistically significant. Considering that the GSTP1 rs1695 GG genotype appeared to be uncommon in this study population and that most subjects who reported smoking or alcohol consumption were females, the role of GSTP1 polymorphism in increasing the risk of CBP should be taken as exploratory until it is validated in a more varied and larger sample.

Since CYP1A1 rs1048943 polymorphism was not found to affect the enzyme activity in vitro (42), it appears reasonable that we did not observe a significant association between this polymorphism and the risk of CBP. However, the rs1048943 polymorphism has been associated with individual cancer susceptibility in various studies (43–45), perhaps because this polymorphism serves as a marker for another casually related polymorphism. Considering that only four SNPs were tested, other untested functional polymorphisms in the promoter and coding region of the CYP1A1 gene need to be identified.

Although some studies indicated that CYP1A2 activity was associated with testicular cancer, hepatacelluar carcinoma and colon cancer (46,47), limited data are available on the potential impact of CYP1A2 polymorphisms on cancer incidence (22). This may explain, in part, why no apparent association of the CYP1A2 polymorphism with the risk of CBP was found in this study. In addition, CYP1A2 is inducible by a large number of common exposures, such as tobacco smoke and alcohol, but women in this study seldom smoked cigarettes or consumed alcohol. Lastly, the sample size is small and thus reduced statistical power in detecting any associations if the gene is of minor effect.

In earlier research, we found no significant effect of UGT1A6 rs2070959 polymorphism on the risk of carcinogenesis development in CBP (48). Since UGT1A6 is one of the most important phase II metabolic enzymes, reducing benzene toxicity by catalyzing the conjugation of glucuronic acid to benzene metabolisms (3), we tested another six SNPs of UGT1A6, two in exon 1 and four in intron 1 to confirm the role of UGT1A6 polymorphisms in susceptibility to the risk of CBP. Consistent with our previous findings, no significant association was observed, suggesting that individuals who bear the UGT1A6 alleles encoding for less active enzymes do not have a higher risk for CBP. However, given that environmental influences and as yet unidentified genetic variants are responsible for the majority of interindividual variability in UGT1A6-mediated glucuronidation (49), further studies are needed to confirm our finding.

For other metabolic enzyme genes including CYP1B1, ADH1B, EPHX2, MPO and NQO1, there were no apparent associations of genetic polymorphisms with risk of CBP. It is probably that this study did not have enough statistical power to detect moderate risk associated with the variant genotypes.

While genetic polymorphisms play an important role in the expression of disease, most chronic and complex illnesses are probably caused by interactions between environmental exposure, genetic polymorphism and lifestyles such as smoking and alcohol consumption, all of which are known risk factors for CBP. Consistent with expectations, we found a reduced risk of CBP for non-alcohol users with GSTP1 rs1695 AG + GG genotypes and for alcohol users with EPHX1 rs3738047 GG genotype. Since no alcohol drinkers with EPHX1 rs2234922 G allele or rs1051741 T allele were observed among cases, further analysis of adjustment for potential confounders was not possible. In addition, the relatively low frequency of alcohol consumption and smoking may have biased the results in this study, as the majority of subjects with information available on alcohol consumption or smoking were females who also were less likely than men to have indulged in alcohol and cigarette use. As such, replication of our findings in other benzene-exposed populations is needed. Furthermore, accurate exposure estimation is quite important for evaluating exposure levels between the case and control groups. Because not all cases and controls experienced the same exposure environment and due to difference in types of work, we were limited to evaluating the intensity of benzene exposure according to the method described by Dosemeci et al. (32). That is, exposure levels were measured differently between cases and controls and therefore may not completely reflect individual variation in exposure. To minimize the potential for bias, more accurate methods for exposure estimation must be used. These methods include personal sampling, which is more accurate than the traditional area sampling used here. Benzene metabolites reflecting the ambient exposure levels are also needed in accurate exposure estimation.

In summary, we found that the GAGT haplotype, the GGAC/GAGT diplotype and the AGAC/GAGT diplotpe of EPHX1 may all contribute to the development of CBP in occupational exposure to benzene in a Chinese population. This finding may have an important implication for the prevention of CBP in susceptible workers. The strengths of this study include a homogenous ethnic background of the subjects, well-documented workplace exposure history to benzene and a frequency-matching design. Nevertheless, the joint effect between genetic polymorphisms and lifestyle risk factors on special diseases such as CBP is complicated, and small studies like the present one do not have enough statistical power to detect gene–environment interactions. Thus, a more comprehensive, large-scale confirmatory study is needed to explore further the effects of gene–environment interaction on genetic susceptibility to CBP.


   Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure S1 and Tables 1–5 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
National Natural Science Foundation of China (30271113); China National Key Basic Research and Development Program (2002CB512902).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Weiwei Liu (Guangzhou Hospital for Occupational Diseases, Guangzhou, Guangdong, China), Duozhi Cao (Institute of Occupational Health attached to Maanshan Steel & Iron Group, Maanshan, Anhui, China), as well as Rong Ye and Jiru Guan (Hangzhou Center for Diseases Control and Prevention, Hangzhou, Zhejiang, China) who are physicians for their contribution to field research and physical examination in this study. We also thank Dr Qingyi Wei of The University of Texas M. D. Anderson Cancer Center for his critical review and scientific editing.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received April 30, 2008; revised September 1, 2008; accepted September 3, 2008.


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P. Sun, Y. Qiu, Z. Zhang, J. Wan, T. Wang, X. Jin, Q. Lan, N. Rothman, and Z.-l. Xia
Association of Genetic Polymorphisms, mRNA Expression of p53 and p21 with Chronic Benzene Poisoning in a Chinese Occupational Population
Cancer Epidemiol. Biomarkers Prev., June 1, 2009; 18(6): 1821 - 1828.
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