Shuguang Leng

Biomarkers measured by cytokinesis-block micronucleus cytome assay for evaluating genetic damages induced by polycyclic aromatic hydrocarbons.

Coke oven workers are regularly exposed to polycyclic aromatic hydrocarbons (PAHs) and have a high risk for lung cancer. Limited evidence has demonstrated a direct link between exposure to PAHs and early genetic damage in exposed workers. The cytokinesis-block micronucleus (CBMN) cytome assay is a comprehensive system for measuring DNA damage and cytotoxicity. In the current study, we investigated different chromosomal damage endpoints including micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs), in 141 PAH-exposed subjects and 66 unexposed controls. The frequencies of MN, NPBs and NBUDs were all significantly higher in PAH-exposed workers than in controls (2.4-, 5-, and 3-fold, respectively). We further classified the PAH-exposed workers into different PAHs exposure groups based on their work positions on the oven and their urinary 1-hydroxypyrene and found that the frequencies of NPBs and NBUDs increased with the increasing level of both external and internal PAHs exposure levels. Similar trend was not found for MN due to the reduced MN frequency in the highest PAHs exposure group compared with the second highest PAHs exposure group. Using principal component analysis, we confirmed that the frequencies of NPBs and NBUDs are more sensitive to reflect the external or internal levels of PAHs exposure. In PAH-exposed subjects, NPB and NBUD frequencies were influenced by gender and females have lower frequencies of NPB and NBUD. Taken together, our observations indicate that NPBs and NBUDs are more sensitive and reliable biomarkers for genetic damages induced by PAHs and could potentially be used for the biomonitoring of genotoxin-exposed populations.

Genomic damages in peripheral blood lymphocytes and association with polymorphisms of three glutathione S-transferases in workers exposed to formaldehyde.

DNA and chromosome damages in peripheral blood lymphocytes were evaluated in 151 workers occupationally exposed to formaldehyde (FA) and 112 non-FA exposed controls. The effects of polymorphisms in three glutathione-S-transferase (GSTs) genes on the DNA and chromosome damages were assessed as well. Alkaline comet assay and cytokinesis-block micronucleus (CBMN) assay were used to determine DNA and chromosome damages, respectively. The genotypes of GSTP1 (Ile105Val), GSTT1, and GSTM1 were assayed. The mean 8-h time-weighted average (TWA) concentrations of FA in two plywood factories were 0.83ppm (range: 0.08-6.30ppm). FA-exposed workers had higher olive tail moment (TM) and CBMN frequency compared with controls (Olive TM, 3.54, 95%CI=3.19-3.93 vs. 0.93, 95%CI=0.78-1.10, P<0.01; CBMN frequency, 5.51+/-3.37 vs. 2.67+/-1.32, P<0.01). Olive TM and the CBMN frequency also had a dose-dependent relation with the personal FA exposure. Significant association between FA exposure history and olive TM and CBMN frequency were also identified. The level of olive TM was slightly higher in FA-exposed workers with GSTM1 null genotype than those with non-null genotype (3.86, 95%CI=3.31-4.50 vs. 3.27, 95%CI=2.83-3.78, P=0.07) with adjustment of covariates. We also found that FA-exposed workers carrying GSTP1 Val allele had a slightly higher CBMN frequency compared with workers carrying only the wild-type allele (6.32+/-3.78 vs. 5.01+/-2.98, P=0.05). Our results suggest that the FA exposure in this occupational population increased DNA and chromosome damages and polymorphisms in GSTs genes may modulate the genotoxic effects of FA exposure.

Genetic polymorphisms of cytokine genes and risk for trichloroethylene-induced severe generalized dermatitis: A case-control study

Trichloroethylene (TCE)-induced severe generalized dermatitis (SGD) is considered to be a contact allergic disease and is dependent on a cell-mediated immune response. Little is known about its pathogenesis. Several lines of evidence suggest that tumour necrosis factor (TNF) and interleukin 4 (IL-4) are involved in the immunological and inflammatory reactions. To investigate the relation between polymorphisms of TNF and the IL-4 gene and the risk of TCE-induced SGD, a case-control study was conducted consisting of 111 patients diagnosed with SGD and 152 TCE-exposed workers without SGD. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the polymorphisms of TNF-α (G-238A, G-308A), TNF-β (intron 1) and IL-4 (C-590T). Logistic regression was applied to calculate the odds ratios (OR) and 95% confidence intervals. The results reveal that the frequency of TNF α-308 wild allele in cases was significantly higher than that in control subjects (p=0.049). Individuals with a heterozygous genotype of TNF α-308 were associated with the decreased risk of TCE-induced SGD relative to the homozygous genotype (OR=0.398, 95% CI=0.164–0.967). No significant differences in the allele and genotype frequencies could be demonstrated at any other polymorphic loci among both groups. The finding of a possible contribution of a TNF-α genetic polymorphism is a primary result because the pathogenesis of TCE-induced SGD is complex and likely to involve the interaction of a number of genes. A further study should be conducted to illustrate the influence of a link between certain relevant alleles in the assessment of genetic susceptibility.

Associations between XRCC1 and ERCC2 polymorphisms and DNA damage in peripheral blood lymphocyte among coke oven workers.

A wide variety of base damages and single-strand breaks formed by reactive oxygen species during metabolic activation of polycyclic aromatic hydrocarbons (PAHs) have been recognized to be involved in PAH carcinogenesis. In this study, alkaline comet assay was used to detect the DNA damage in peripheral blood lymphocytes among 143 coke-oven workers and 50 non-coke-oven workers, and the effects of genetic polymorphisms of XRCC1 and ERCC2 genes on DNA damage were evaluated. The olive tail moment was significantly higher in coke-oven workers than in non-coke-oven workers (2.6, 95% CI=2.1–3.3 versus 1.0, 95% CI=0.8–1.2, p<0.01), and significant correlation between ln-transformed urinary 1-OHP and ln-transformed olive tail moment was found in total population (n=193, Pearsons r=0.393, p<0.001) and in coke-oven workers (n=143, Pearsons r=0.224, p=0.007). The olive tail moment was significantly higher in coke-oven workers with GA genotype of G27466A polymorphism of XRCC1 than those with GG genotype (4.6, 95% CI=2.5–8.7 versus 2.4, 95% CI=1.9–2.9, p<0.01 with adjustment for covariates). No significant associations between C26304T, G28152A and G36189A polymorphisms of XRCC1 and G23591A and A35931C polymorphisms of ERCC2 and olive tail moment were found in both groups. The study showed that the alkaline comet assay is a suitable biomarker in the detection of DNA damage among coke-oven workers and it suggested that the A allele of G27466A polymorphism of XRCC1 may be associated with decreased DNA repair capacity toward PAH-induced base damage and strand breaks.

Suboptimal DNA Repair Capacity Predisposes Coke-Oven Workers To Accumulate More Chromosomal Damages in Peripheral Lymphocytes

DNA repair is an essential mechanism for cells to maintain their genomic integrity under endogenous or exogenous assault. Reduced DNA repair capacity (DRC) is associated with increased risk for several environmentally related cancers. The micronucleus in peripheral lymphocytes has been validated as a biomarker of chromosomal damage, increasing cancer risk in human populations. We hypothesized that suboptimal DRC is associated with the increase in chromosomal damage among 94 coke-oven workers and 64 noncoke-oven controls. DRC was evaluated in isolated lymphocytes by comet assay. Chromosomal damage in peripheral lymphocytes was detected by cytokinesis-block micronucleus assay. Four common coding single nucleotide polymorphisms in the XRCC1 gene were genotyped. Coke-oven workers have significantly increased urinary 1-hydroxypyrene (9.0; 6.8-11.7 μg/L versus 1.5, 1.3-1.7 μg/L; P < 0.01) and micronucleus frequency (7.4‰ ± 4.3‰ versus 3.0‰ ± 3.0‰; P < 0.01), and decreased DRC (55.9% ± 16.4% versus 63.6% ± 18.5%; P < 0.01) compared with controls. Significant correlations between DRC and micronucleus frequency were found in coke-oven workers (r = −0.32; P < 0.01; n = 94) and all study subjects (r = −0.32; P < 0.001; n = 158) but not in controls (r = −0.21; P = 0.11; n = 64). Variants of the Arg399Gln polymorphism were associated with a decreased DRC in both coke-oven workers (51.6% ± 16.1% versus 60.6% ± 15.7%; P < 0.01) and controls (59.1% ± 18.5% versus 68.4% ± 17.5%; P = 0.04). The complicated interrelationship of these multiple biomarkers was also identified by path analysis. These findings should facilitate developing a biomarker-based risk assessment model for lung cancer in this occupational population. (Cancer Epidemiol Biomarkers Prev 2009;18(3):987–93)

Modulation of DNA repair capacity by ataxia telangiectasia mutated gene polymorphisms among polycyclic aromatic hydrocarbons-exposed workers.

The purpose of this study was to address the association between the ataxia telangiectasia mutated (ATM) gene polymorphisms and susceptibility to DNA repair capacity (DRC) among polycyclic aromatic hydrocarbons (PAHs)-exposed workers. Polymorphisms of ATM were genotyped. DRC was determined by comet assay. Chromosomal damage was detected by cytokinesis-block micronucleus (CBMN) assay. Flow cytometry was used to detect the distributions of cell cycle. Expressions of ATM and rH2AX were determined by immunoblotting analysis. Luciferase assays were performed to determine the functional difference of ATM promoter region allele. Subjects carrying T allele of rs228589 had significantly lower DRC compared with those with AA genotype. Subjects carrying G allele of rs652311 had significantly lower DRC than those with zero copy number of haplotype CGGT. SH ataxia telangiectasia mutated (SHATM) cells had significantly lower DRC than SH green fluorescent protein (SHGFP) cells induced by bleomycin and higher CBMN frequencies treated by benzo(a)pyrene [B(a)P] than SHGFP cells. After B(a)P treatment, a decrease in the percentage of G1 phase cells was observed in SHATM cells compared with SHGFP cells, rH2AX expressions were increased in SHATM cells and SHGFP cells, but ATM expressions had no change in 16HBE-SHGFP cells and HEK-SHGFP cells. Luciferase expression was not different between rs228589T and rs228589A plasmid constructs. In conclusions, it is suggested that ATM polymorphisms are associated with DRC among PAHs-exposed workers and ATM plays key roles in repair of chromosomal damage and cell cycle control with the treatment of B(a)P.

Association of Aryl Hydrocarbon Receptor Gene Polymorphisms and Urinary 1-Hydroxypyrene in Polycyclic Aromatic Hydrocarbon–Exposed Workers

Polycyclic aromatic hydrocarbons (PAH) in coke oven emissions could cause lung cancer in human. Individuals genotype of the metabolic enzymes and early biological changes were known to be associated with the susceptibility of cancer development. Knowledge of metabolic gene polymorphisms, which affect on the urinary 1-hydroxypyrene (1-OHP), could benefit us in understanding the interindividual difference in the mechanism of PAH-induced carcinogenesis. In this study, we investigated the association of aryl hydrocarbon receptor (AhR) gene polymorphisms and urinary 1-OHP. One hundred forty-seven workers exposed to PAH and 69 nonexposure workers were recruited. Seven tagging single nucleotide polymorphisms in AhR gene were selected by pariwise r2 method and minor allele frequency cutoff of 0.05 from Chinese genotype data in HapMap project. These seven tagging single nucleotide polymorphisms were genotyped by PCR-based methods. Multivariate analysis of covariance revealed that the levels of 1-OHP in PAH-exposed workers carrying genotype CT were lower than workers carrying wild genotype TT at loci rs10250822 and rs2282885 of AhR gene (P = 0.032 and 0.044, respectively). In PAH-exposed workers, the urinary 1-OHP levels showed a linear correlation (Ptrend = 0.041) with the genotypes at locus rs2282885, especially in low and moderate exposure groups. In contrast, no significant association was found between urinary 1-OHP level and AhR genotypes among nonexposed workers. Our findings indicated that polymorphisms of AhR gene were associated with the level of 1-OHP among PAH-exposed workers, suggesting that AhR-mediated signaling might contribute to individual susceptibility to PAH exposure. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1702–8)

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Abstract Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR = 5.58, 95% CI = 1.32–23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.

Antaxia-telangiectasia mutated gene polymorphisms and susceptibility to chromosomal damage among polycyclic aromatic hydrocarbons exposed workers.

The aim was to explore the relationship between the ataxia-telangiectasia mutated (ATM) gene polymorphisms and susceptibility to cytokinesis-block micronucleus among workers exposed to polycyclic aromatic hydrocarbons (PAH). Blood and urine samples of 140 PAH-exposed workers and 66 non-PAH-exposed workers were collected. Seven tagging single nucleotide polymorphisms in ATM gene were selected by pariwise r(2) method and minor allele frequency cutoff of 0.05 from Chinese genotype data in HapMap project. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze the polymorphisms of ATM rs600931, rs652311, rs227060, rs227292, rs624366, rs189037 and rs228589. The results showed that ATM rs600931 AG and AG+AA genotypes exhibited significantly higher cytokinesis-block micronucleus (CBMN) frequency (11.14+/-6.91 per thousand and 10.57+/-6.82 per thousand) than did the GG genotype (7.66+/-5.69 per thousand, P=0.015 and 0.038, respectively). The subjects with rs189037 GA and GA+AA genotypes exhibited significantly higher CBMN frequency (10.99+/-6.90 per thousand and 10.51+/-6.76 per thousand) than that of the GG genotype (7.72+/-5.82 per thousand, P=0.018 and 0.035, respectively). The PAH-exposed workers with rs624366 GC and GC+CC genotypes exhibited significantly higher CBMN frequency (11.34+/-6.74 per thousand and 10.73+/-6.62 per thousand) than did the GG genotype (7.61+/-6.07 per thousand, P=0.001 and 0.003, respectively). rs227092 GT genotype exhibited significantly higher CBMN frequency (10.78+/-6.60 per thousand) than did the GG genotype (7.91+/-6.30 per thousand, P=0.025) among the PAH-exposed workers. The haplotype pairs GGGGTGC/AAACATT exhibited significantly higher CBMN frequency (12.05+/-7.40 per thousand) than did the GGGGTGC/GGGGTGC (7.51+/-6.19 per thousand, P=0.007) among the PAH-exposed group. In conclusion, it is suggested that the polymorphisms of ATM were associated with the CBMN frequencies among PAH-exposed workers.

Uridine diphosphoglucuronosyltransferase 1A7 gene polymorphism and susceptibility to chromosomal damage among polycyclic aromatic hydrocarbons exposed workers.

Objective: The aim of this study was to explore the relationship between UGT1A7 gene polymorphisms and susceptibility to chromosomal damage among polycyclic aromatic hydrocarbons (PAH)-exposed workers. Methods: Blood and urine samples of 140 PAH-exposed workers and 66 non–PAH-exposed workers were collected. The polymorphisms of UGT1A7, cytokinesis-block micronucleus (CBMN) and 1-hydroxypyrene were analyzed. Results: UGT1A7 codon 208 Arg carriers exhibited significantly higher CBMN frequencies than did the Trp/Trp (P < 0.05). Stratification analysis revealed that the significant association between CBMN frequencies and the polymorphisms of UGT1A7 Asn129Lys, Arg131Lys, and Trp208Arg was most pronounced. Moreover, higher CBMN frequencies were found among subjects with *3 allele than those with *1 allele (P = 0.008) in younger PAH-exposed workers. Conclusion: Polymorphisms of UGT1A7 gene may alter the severity of PAH-induced chromosomal damage among the exposed workers.