Huawei Duan

Aberrant Expression of miR-638 Contributes to Benzo(a)pyrene-Induced Human Cell Transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.

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.

CpG Site–Specific Hypermethylation of p16INK4α in Peripheral Blood Lymphocytes of PAH-Exposed Workers

Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAH-associated biologic consequence. Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16INK4α methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results: Compared with the control group, PAH-exposed workers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 μg/L), p16INK4α methylation (7.95% vs. 1.14% for 22 “hot” CpG sites), and CBMN (7.28% vs. 2.92%) in PBLs. p16INK4α hypermethylation in PAH-exposed workers exhibited CpG site specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16INK4α gene. The degree of p16INK4α methylation was associated with the levels of DNA damage and internal exposure. Impact: p16INK4α hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 21(1); 182–90. ©2011 AACR.

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.

Increased Micronucleus, Nucleoplasmic Bridge, and Nuclear Bud Frequencies in the Peripheral Blood Lymphocytes of Diesel Engine Exhaust-Exposed Workers

The International Agency for Research on Cancer has recently reclassified diesel engine exhaust (DEE) as a Group 1 carcinogen. Micronucleus (MN), nucleoplasmic bridge (NPB), and nuclear bud (NBUD) frequencies in peripheral blood lymphocytes (PBLs) are associated with cancer risk. However, the impact of DEE exposure on MN frequency has not been thoroughly elucidated due to mixed exposure and its impact on NPB and NBUD frequencies has never been explored in humans. We recruited 117 diesel engine testing workers with exclusive exposure to DEE and 112 non-DEE-exposed workers, and then we measured urinary levels of 4 mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) using high-performance liquid chromatography-mass spectrometry as well as MN, NPB, and NBUD frequencies in PBLs using cytokinesis-block MN assay. The DEE-exposed workers exhibited significantly higher MN, NPB, and NBUD frequencies than the non-DEE-exposed workers (P < 0.05). Among all study subjects, increasing levels of all 4 urinary OH-PAHs, on both quartile and continuous scales, were associated with increased MN, NPB, and NBUD frequencies (all P < 0.05). When the associations were analyzed separately in DEE-exposed and non-DEE-exposed workers, we found that the association between increasing quartiles of urinary 9-hydroxyphenanthrene (9-OHPh) and MN frequencies persisted in DEE-exposed workers (P = 0.001). The percent of MN frequencies increased, on average, by 23.99% (95% confidential interval, 9.64-39.93) per 1-unit increase in ln-transformed 9-OHPh. Our results clearly show that exposure to DEE can induce increases in MN, NPB, and NBUD frequencies in PBLs and suggest that DEE exposure level is associated with MN frequencies.

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.

Trichloroethanol up-regulates matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in HaCaT cells.

Occupational trichloroethylene (TCE) exposure could induce generalized skin hypersensitivity reactions complicated with severe liver dysfunctions. Active extracellular matrix degradation and remodeling are involved in the skin hypersensitivity reaction induced by chemical exposure. In the present study, we have compared the effects of in vitro exposure to trichloroethanol (TCOH) and trichloroacetic acid (TCA) of a keratinocyte cell line (HaCaT). The modulation of matrix metalloproteinases (MMPs) was selected as marker of sensitization. HaCaT cells were treated with different concentrations of TCOH or TCA up to 6days. The gelatinolyic activities of MMP-2 and MMP-9 were detected by gelatin-zymography. MMP-2, tissue inhibitor of metalloproteinase (TIMP)-2, MMP-9 and TIMP-1 mRNAs were analyzed by real-time PCR and MMP-9 and TIMP-1 proteins were tested by Western blotting. A dose-effect relationship between TCOH treatment and MMP-9 activity, mRNA and protein expression levels was found in HaCaT cells. TCOH also induced up-regulation of TIMP-1 mRNA and protein. We found no such effects in HaCaT cells treated with TCA. Moreover, previously published literatures on patch tests suggested that TCOH could induce moderately positive reactions at low concentrations in hypersensitivity patients caused by occupational TCE exposure. In summary, these observations indicated that TCOH might play an important role in TCE-induced skin hypersensitivity.

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)

Occupational exposure to diesel engine exhaust and alterations in lymphocyte subsets

Background The International Agency for Research on Cancer recently classified diesel engine exhaust (DEE) as a Group I carcinogen based largely on its association with lung cancer. However, the exposure–response relationship is still a subject of debate and the underlying mechanism by which DEE causes lung cancer in humans is not well understood. Methods We conducted a cross-sectional molecular epidemiology study in a diesel engine truck testing facility of 54 workers exposed to a wide range of DEE (ie, elemental carbon air levels, median range: 49.7, 6.1–107.7 µg/m3) and 55 unexposed comparable controls. Results The total lymphocyte count (p=0.00044) and three of the four major lymphocyte subsets (ie, CD4+ T cells (p=0.00019), CD8+ T cells (p=0.0058) and B cells (p=0.017)) were higher in exposed versus control workers and findings were highly consistent when stratified by smoking status. In addition, there was evidence of an exposure–response relationship between elemental carbon and these end points (ptrends<0.05), and CD4+ T cell levels were significantly higher in the lowest tertile of DEE exposed workers compared to controls (p=0.012). Conclusions Our results suggest that DEE exposure is associated with higher levels of cells that play a key role in the inflammatory process, which is increasingly being recognised as contributing to the aetiology of lung cancer. Impact This study provides new insights into the underlying mechanism of DEE carcinogenicity.