Yufei Dai

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.

The Association of XRCC1 Haplotypes and Chromosomal Damage Levels in Peripheral Blood Lymphocyte among Coke-Oven Workers

Theoretically, a haplotype has a higher level of heterozygosity than individual single nucleotide polymorphism (SNP) and the association study based on the haplotype may have an increased power for detecting disease associations compared with SNP-based analysis. In this study, we investigated the effects of four haplotype-tagging SNPs (htSNP) and the inferred haplotype pairs of the X-ray cross-complementing group 1 (XRCC1) gene on chromosome damage detected by the cytokinesis-block micronucleus assay. The study included 141 coke-oven workers with exposure to a high level of polycyclic aromatic hydrocarbons and 66 nonexposed controls. The frequencies of total MN and MNed cells were borderline associated with the Arg194Trp polymorphism (P = 0.053 and P = 0.050, respectively) but not associated with the Arg280His, Arg399Gln and Gln632Gln polymorphisms among coke-oven workers. Five haplotypes, including CGGG, TGGG, CAGG, CGAG, and CGGA, were inferred based on the four htSNPs of XRCC1 gene. The haplotype CGGG was associated with the decreased frequencies of total MN and MNed cells, and the haplotypes TGGG and CGAG were associated with the increased frequencies of total MN and MNed cells with adjustment for covariates among coke-oven workers. This study showed that the haplotypes derived from htSNPs in the XRCC1 gene were more likely than single SNPs to correlate with the polycyclic aromatic hydrocarbon–induced chromosome damage among coke-oven workers.

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.

Cytokine expression in trichloroethylene-induced hypersensitivity dermatitis: An in vivo and in vitro study

The purpose of this study was to address the association between cytokine expression and the hypersensitivity dermatitis induced by trichloroethylene (TCE) exposure. 28 TCE-induced hypersensitivity dermatitis patients, 22 TCE exposed workers and 22 non-exposed controls were enrolled in the study. The serum levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α were analyzed using a magnetic colorbead-based multiplex assay. The patients showed significantly higher levels of serum IL-1β (p=0.033 and p=0.015), IL-6 (p<0.001), IL-8 (p<0.001 and p=0.002) and TNF-α (p=0.009 and p=0.005) than the TCE exposed workers and non-exposed controls. There was a significantly positive correlation among these cytokine concentrations, but no significant correlation was found between these cytokine concentrations and the disease duration in patient group. We further compared the effects of trichloroethanol (TCOH) and trichloroacetic acid (TCA), two major metabolites of TCE, on cytokine expression in keratinocyte cell line (HaCaT). IL-1α, IL-6, IL-8 and TNF-α concentrations were tested using enzyme-linked immunosorbent assay (ELISA) after HaCaT cells were treated with different concentrations of TCOH or TCA for 24h. We found that TCOH, but not TCA, increased the levels of IL-1α and IL-6 in a dose-dependent manner. We also found that TCOH activated the nuclear factor kappa B (NF-κB) pathway. Bay 11-7082 (NF-κB inhibitor) significantly attenuated the TCOH-induced production of IL-6 in HaCaT cells, but IL-1α production was not affected. In conclusions, it is suggested that IL-1β, IL-6, IL-8 and TNF-α were associated with TCE-induced hypersensitivity dermatitis. TCOH induced IL-6 expression through activation of the NF-κB pathway in HaCaT cells and may play an integral role in TCE-induced skin hypersensitivity.

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.

Path Analysis of Biomarkers of Exposure and Early Biological Effects among Coke-Oven Workers Exposed to Polycyclic Aromatic Hydrocarbons

Many host factors or biomarkers are involved in the process of early DNA damage induced by occupational exposure to polycyclic aromatic hydrocarbons (PAH) as seen in coke-oven workers. This paper aimed to identify complicated causal interrelationship of various biomarkers using the path analysis. In this analysis, we included 235 subjects (166 coke-oven workers and 69 nonexposed controls) whose data on the comet assay (e.g., Olive tail moment) and cytogenetic analysis of peripheral blood lymphocytes as well as urinary 1-hydroxypyrene (1-OHP) were available. The path analysis showed that coke-oven exposure and tobacco smoke were both significant predictors of the concentrations of urinary 1-OHP (P < 0.05), with a coefficient of determination of 0.75. The factors having significant influence on the Olive tail moment were in the following order: urinary 1-OHP > XRCC1-exon 9 variant genotype > ERCC2-exon 10 variant genotype > XRCC1-exon 6 variant genotype, with a coefficient of determination of 0.22. The variables of relative importance in influencing on cytokinesis-block micronucleus frequencies were in the following order: coke-oven exposure > urinary 1-OHP > age > mEH3 variant genotype > ERCC2-exon 10 variant genotype > XRCC1-exon 6 variant genotype, with a coefficient of determination of 0.27. These results indicated that exogenous agents, especially the coke-oven exposure, played a more important role than the genotypes in the induction of early genetic damage. In conclusion, the path analysis seemed to be an alternative statistical approach for the ascertainment of complicated association among related biomarkers for the assessment of occupational exposure. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1193–9)

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

Abstract The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers

Long-term exposure to diesel engine exhaust induces primary DNA damage: a population-based study

Objectives Diesel engine exhaust (DEE) is a ubiquitous environmental pollutant and is carcinogenic to humans. To seek early and sensitive biomarkers for prediction of adverse health effects, we analysed the components of DEE particles, and examined the genetic and oxidative damages in DEE-exposed workers. Methods 101 male diesel engine testing workers who were constantly exposed to DEE and 106 matched controls were enrolled in the present study. The components of DEE were analysed, including fine particulate matter (PM2.5), element carbon (EC), nitrogen dioxide (NO2), sulfur dioxide (SO2) and polycyclic aromatic hydrocarbons (PAHs). Postshift urine samples were collected and analysed for 1-hydroxypyrene (1-OHP), an internal exposure marker for DEE. Levels of DNA strand breaks and oxidised purines, defined as formamidopyrimidine-DNA glycosylase (FPG) sites in leucocytes, were measured by medium throughput Comet assay. Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) was also used to determine the level of oxidative stress. Results We found higher levels of PM2.5, EC, NO2, SO2 and PAHs in the diesel engine testing workshop and significantly higher urinary 1-OHP concentrations in exposed subjects (p<0.001). Compared with controls, the levels of parameters in normal Comet and FPG-Comet assay were all significantly higher in DEE-exposed workers (p<0.001), and in a dose-dependent and time-dependent manner. There were no significant differences between DEE-exposed workers and controls in regard to leucocyte FPG sensitive sites and urinary 8-OHdG levels. Conclusions These findings suggest that DEE exposure mainly induces DNA damage, which might be used as an early biomarker for risk assessment of DEE exposure.

Genetic damage induced by organic extract of coke oven emissions on human bronchial epithelial cells

Coke oven emissions are known as human carcinogen, which is a complex mixture of polycyclic aromatic hydrocarbon. In this study, we aimed to clarify the mechanism of action of coke oven emissions induced carcinogenesis and to identify biomarkers of early biological effects in a human bronchial epithelial cell line with CYP1A1 activity (HBE-CYP1A1). Particulate matter was collected in the oven area on glass filter, extracted and analyzed by GC/MS. DNA breaks and oxidative damage were evaluated by alkaline and endonucleases (FPG, hOGG1 and ENDO III)-modified comet assays. Cytotoxicity and chromosomal damage were assessed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The cells were treated with organic extract of coke oven emissions (OE-COE) representing 5, 10, 20, 40μg/mL extract for 24h. We found that there was a dose-effect relationship between the OE-COE and the direct DNA damage presented by tail length, tail intensity and Olive tail moment in the comet assay. The presence of lesion-specific endonucleases in the assays increased DNA migration after OE-COE treatment when compared to those without enzymes, which indicated that OE-COE produced oxidative damage at the level of pyrimidine and purine bases. The dose-dependent increase of micronuclei, nucleoplasmic bridges and nuclear buds in exposed cells was significant, indicating chromosomal and genomic damage induced by OE-COE. Based on the cytotoxic biomarkers in CBMN-Cyt assay, OE-COE may inhibit nuclear division, interfere with apoptosis, or induce cell necrosis. This study indicates that OE-COE exposure can induce DNA breaks/oxidative damage and genomic instability in HBE-CYP1A1 cells. The FPG-comet assay appears more specific for detecting oxidative DNA damage induced by complex mixtures of genotoxic substances.