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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.

Cadmium (Cd) has been reported to induce kidney damage by triggering oxidative stress and inflammation. The NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated a role in the pathogenesis of inflammation. However, the connection between Cd and NLRP3 inflammasome in the development of renal inflammation remains unknown. In this study, in vitro experiments based on the telomerase-immortalized human renal proximal-tubule epithelial cell line (RPTEC/TERT1) were carried out. Results revealed that CdCl2 (2–8 μM) increased ROS production and activated NLRP3, thereby enhancing secretion of IL-1β and IL-18 (P

To explore the epigenetic alterations in response to DNA damage following polycyclic aromatic hydrocarbons (PAHs) exposure and the crosstalk between different epigenetic regulations, we examined trimethylated Lys 36 of histone H3 (H3K36me3) and methylation of ‘long interspersed element-1 (LINE-1)’ and ‘O 6-methylguanine-DNA methyltransferase (MGMT)’ in peripheral blood lymphocytes (PBLCs) of 173 coke oven workers (PAH-exposed group) and 94 non-exposed workers (control group). The PAH-exposed group showed higher internal PAH exposure level, enhanced DNA damage and increased MGMT expression (all P

Experimental evidence suggests that exposure to cadmium (Cd) could affect immune cells in vivo and in vitro. However, the associations of long-term Cd exposure with white blood cell (WBC) subtype counts and hemogram-derived indices have been rarely investigated. Therefore, we evaluated these relationships in residents of cadmium-polluted areas.This cross-sectional study included 431 participants aged 45-75 years without occupational exposure histories from Cd-contaminated areas of southern China. We detected WBC, neutrophil, lymphocyte, and monocyte counts using routine blood tests and calculated neutrophil-lymphocyte ratio (NLR), systemic inflammation response index (SIRI), and lymphocyte-monocyte ratio (LMR). Urinary Cd (U-Cd) was measured with inductively coupled plasma mass spectrometry and adjusted for creatinine. To evaluate the associations of U-Cd with peripheral WBC subtype counts and indices, we performed multivariate linear regression, logistic regression and subgroup analyses using U-Cd categorized into quartiles.In models adjusted for all potential confounders, U-Cd was negatively associated with WBC, neutrophil, and monocyte counts in Q2, compared with Q1 of U-Cd (p 0.05). A similar relationship was observed between U-Cd and NLR and SIRI, whereas the corresponding association for LMR was positive (p 0.05). In subgroup analyses, U-Cd was negatively associated with neutrophil count, except for never smokers, after full adjustment.U-Cd was negatively associated with WBC count, neutrophil count, monocyte count, NLR, and SIRI, and positively associated with LMR. Therefore, neutrophil count could be a potential indicator of long-term Cd exposure-associated immunosuppressive effect.

Cadmium accumulates in the kidney and causes inflammation. The NLRP3 inflammasome has been linked to the pathogenesis of inflammation. Hyperoside (HYP) possesses potent nephroprotective properties against of kidney injury. This study aimed to research the effects and related mechanism of HYP on Cd-induced kidney damage. Wide-type and NLRP3

Environmental insults can lead to alteration in DNA methylation of specific genes. To address the role of altered DNA methylation in prediction of polycyclic aromatic hydrocarbons (PAHs) exposure-induced genetic damage, we recruited two populations, including diesel engine exhausts (low-level) and coke oven emissions (high-level) exposed subjects. The positive correlation was observed between the internal exposure marker (1-hydroxypyrene) and the extents of DNA damage (P 0.05). The methylation of representative genes, including TRIM36, RASSF1a, and MGMT in peripheral blood lymphocytes was quantitatively examined by bisulfite-pyrosequencing assay. The DNA methylation of these three genes in response to PAHs exposure were changed in a CpG-site-specific manner. The identified hot CpG site-specific methylation of three genes exhibited higher predictive power for DNA damage than the respective single genes in both populations. Furthermore, the dose-response relationship analysis revealed a nonlinear U-shape curve of TRIM36 or RASSF1a methylation in combined population, which led to determination of the threshold of health risk. Furthermore, we established a prediction model for genetic damage based on the unidirectional-alteration MGMT methylation levels. In conclusion, this study provides new insight into the application of multiple epi-biomarkers for health risk assessment upon PAHs exposure.

To explore the epigenetic alterations in response to DNA damage following polycyclic aromatic hydrocarbons (PAHs) exposure and the crosstalk between different epigenetic regulations, we examined trimethylated Lys 36 of histone H3 (H3K36me3) and methylation of ‘long interspersed element-1 (LINE-1)’ and ‘O (6)-methylguanine-DNA methyltransferase (MGMT)’ in peripheral blood lymphocytes (PBLCs) of 173 coke oven workers (PAH-exposed group) and 94 non-exposed workers (control group). The PAH-exposed group showed higher internal PAH exposure level, enhanced DNA damage and increased MGMT expression (all P

Previous studies have demonstrated that oxidative stress is implicated in benzene-induced hematotoxicity. However, the low dose-response effects and the mechanism underlying perturbation of hematopoiesis remain to be defined. This study aims to address the role of Nrf2 pathway in mediating benzene-induced hematotoxicity. Nrf2+/+ (wildtype, Nrf2-WT) and Nrf2−/− (knockout, Nrf2-KO) mice were administrated with benzene at doses of 0.1, 1.0, 10.0, 100.0 mg/kg by oral gavage for a consecutive 4 weeks (6 times/week). As a result, benzene exposure caused a decline of WBC and lymphocyte counts in a dose-dependent manner at a dose range from 1.0 to 100.0 mg/kg, while low dose benzene induced hormesis effects. Interestingly, Nrf2 deficiency seemed to relieve the decline of peripheral blood cell counts upon benzene exposure, indicating the involvement of Nrf2 in regulation of benzene-induced hematotoxicity. The suppression of phase II enzyme expression in Nrf2-KO mice resulted in considerable reduction in detoxification indicated by the decrease of urinary S-phenylmercapturic acid (SPMA), a metabolite of benzene. Ex vivo assay revealed enhanced cytotoxicity and oxidative stress were induced by benzene in Nrf2-KO mice. Notably, the depletion of Nrf2 triggered the proliferation and differentiation of hematopoietic cells, but induced aberrant morphological changes in periphery erythrocytes and bone marrow cells, implicating the compensatory effects carried on at the expense of induction of dysfunctional blood cells. Our findings provide a new insight into a low dose–response towards benzene-induced hematotoxicity and uncover the critical role of Nrf2 pathway in mediating abnormal hematopoiesis in response to oxidative stress.

Cell immortalization is the initial step for cancer development. To identify the differentially expressed genes regulated by DNA methylation over the course of human primary bronchial epithelial cell (HPBECs) immortalization, an immortalized HBE cell line (HBETT) was generated via introduction of an SV40 LT and a catalytic subunit of human telomerase reverse transcriptase (hTERT) into the HPBECs. Microarrays of mRNA and DNA methylation were performed to compare the transcriptomes and DNA methylomes between these two types of cells. The results from the mRNA microarray revealed many genes whose expression changed upon cell immortalization. We identified signatures including global hypomethylation, perturbation of ECM-receptor interaction, focal adhesion, and PI3K-Akt pathways associated with cell immortalization. Moreover, we revealed 155 differentiated methylation regions (DMRs) within the CpG islands (CGIs) of 42 genes and the perturbation of several key pathways that might be involved in HBE cell immortalization. Among these genes, the hypermethylation of the plasma glutamate carboxypeptidase (PGCP) gene appeared specifically in lung cancer tissues. The inhibition of PGCP expression by promoter hypermethylation was observed in both immortal HBETT cells and benzo[a]pyrene (Bap)-transformed HBE cells. In conclusion, these findings provide new insight into the epigenetic modifications that are critical in the transition and maintenance of cell immortalization.

Chronic exposure to aflatoxin B1 (AFB1) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB1-induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB1-transformed L02R (L02RT-AFB1) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3μM AFB1 weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB1) could grow in an anchorage independent fashion and form tumors in immunodeficient mice. qRT-PCR was performed to examine whether gene methylation led to a reduction in gene expression of methylated candidate genes. As a result, the expression of the following seven genes including JUNB, RUNX3, NAV1, CXCR4, RARRES1, INTS1, and POLL was down-regulated in transformed L02RT-AFB1 cells. The reduction of gene expression of these genes could be reversed by treatment of 5-azadeoxycytidine. The methylated CpG sites of RUNX3 genes were verified using bisulfite sequencing PCR (BSP) assay. Furthermore, a dynamic change in RUNX3 methylation was observed over the course of AFB1-induced cell transformation, which was corresponded to the alteration of gene expression and the extent of DNA damage. In vitro study showed that methylation of RUNX3 tended to abate in L02R cells treated with AFB1 for a short-term period of time. Notably, hypermethylation of RUNX3 appeared in 70% (14/20) of human hepatocellular carcinomas. Moreover, LINE-1 hypomethylation and dynamic changes of DNMTs, TETs and MeCP2 expression were also observed during AFB1-induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB1-induced carcinogenesis.

Long term exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with the increasing risk of lung cancer. To identify differentially hypermethylated genes associated with PAHs-induced carcinogenicity, we performed genome-wide DNA methylation analysis in 20 μM benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells at different stages of cell transformation. Several methylated genes (CNGA4, FLT1, GAREM1, SFMBT2, TRIM36) were differentially hypermethylated and their mRNA was suppressed in cells at both pre-transformed and transformed stages. Similar results were observed in HBE cells transformed by 20 μg/mL coke oven emissions (COEs) mixture collected from a coking manufacturing facility. In particular, hypermethylation of TRIM36 and suppression of TRIM36 expression were gradually enhanced over the time of COEs treatment. We developed bisulfite pyrosequencing assay and assessed TRIM36 methylation quantitatively. We found that hypermethylation of TRIM36 and reduced gene expression was prevalent in several types of human cancers. TRIM36 hypermethylation appeared in 90.0% (23/30) of Non-Small Cell Lung Cancer (NSCLCs) tissues compared to their paired adjacent tissues with an average increase of 1.32 fold. Furthermore, an increased methylation rate (5.90% v.s 7.38%) and reduced levels of TRIM36 mRNA were found in peripheral lymphocytes (PBLCs) of 151 COEs-exposed workers. In all subjects, TRIM36 hypermethylation was positively correlated with the level of urinary 1-hydroxypyrene (P < 0.001), an internal exposure marker of PAHs, and the DNA damage (P = 0.013). These findings suggest that aberrant hypermethylation of TRIM36 might be involved in the acquisition of malignant phenotype and could be served as a biomarker for risk assessment of PAHs exposure.

Background Ubiquitously distributed benzene is a known hematotoxin. Increasing evidence has suggested that erythroid-related hematologic parameters may be sensitive to benzene exposure. Fat content, which is also closely associated with erythroid-related hematologic parameters, may affect the distribution and/or metabolism of benzene, and eventually benzene-induced toxicity. Methods To explore the influence of benzene exposure, fat content, and their interactions on erythroid-related hematologic parameters, we recruited 1669 petrochemical workers and measured their urinary S-phenylmercapturic acid (SPMA) concentration and erythroid-related hematological parameters. Indices for fat content included body fat percentage (BF%), plasma total cholesterol (TC) and triglycerides (TG), and occurrence of fatty liver. Results The dose-response curve revealed U-shaped nonlinear relationships of SPMA with hematocrit (HCT) and mean corpuscular hemoglobin concentration (MCHC) (P-overall P-nonlinear P-overall ≤0.005). We also observed modification effects of fat content on the associations between benzene exposure and erythroid-related hematological parameters, with workers of lower or higher BF% and TG more sensitive to benzene-induced elevation of MCHC (Pinteraction = 0.021) and benzene-induced decrease of HCT (Pinteraction = 0.050), respectively. We also found that some erythroid-related hematologic parameters differed between subgroups of workers with different SPMA levels and fat content combination. Conclusions Our study suggested that benzene exposure, fat content, and their interactions may affect erythroid-related hematological parameters in petrochemical workers in a complex manner that are worthy of further investigation.

Identification of aberrant histone H3 phosphorylation during chemical carcinogenesis will lead to a better understanding of the substantial roles of histone modifications in cancer development. To explore whether aberrant H3 phosphorylation contributes to chemical carcinogenesis, we examined the dynamic changes of H3 phosphorylation at various residues in chemical carcinogen-induced transformed human cells and human cancers. We found that histone H3 phosphorylation at Ser10 (p-H3S10) and Ser28 (p-H3S28) was upregulated by 1.5-4.8 folds and 2.1-4.3 folds, respectively in aflatoxin B1 -transformed hepatocytes L02 cells (L02RT-AFB1 ), benzo(a)pyrene-transformed HBE cells (HBERT-BaP), and coke oven emissions-transformed HBE cells (HBERT-COE). The ectopic expression of histone H3 mutant (H3S10A or H3S28A) in L02 cells led to the suppression of an anchorage-independent cell growth as well as tumor formation in immunodeficient mice. In addition, an enhanced p-H3S10 was found in 70.6% (24/34) of hepatocellular carcinoma (HCC), and 70.0% (21/30) of primary lung cancer, respectively. Notably, we found that expression of H3 carrying a mutant H3S10A or H3S28A conferred to cells the ability to maintain a denser chromatin and resistance to induction of DNA damage and carcinogen-induced cell transformation. Particularly, we showed that introduction of a mutant H3S10A abolished the bindings of p-H3S10 to the promoter of DNA repair genes, PARP1 and MLH1 upon AFB1 treatment. Furthermore, we revealed that PP2A was responsible for dephosphorylation of p-H3S10. Taken together, these results reveal a key role of persistent H3S10 or H3S28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response (DDR) genes.

Objective: To define whether aberrant methylation of DNA repair genes is associated with chronic arsenic poisoning.Methods: Hundred and two endemic arsenicosis patients and 36 healthy subjects were recruited. Methylight and bisulfite sequencing (BSP) assays were used to examine the methylation status of ERCC1, ERCC2 and XPC genes in peripheral blood lymphocytes (PBLs) and skin lesions of arsenicosis patients and NaAsO2-treated HaCaT cells.Results: Hypermethylation of ERCC1 and ERCC2 and suppressed gene expression were found in PBLs and skin lesions of arsenicosis patients and was correlated with the level of arsenic exposure. Particularly, the expression of ERCC1 and ERCC2 was associated with the severity of skin lesions. In vitro studies revealed an induction of ERCC2 hypermethylation and decreased mRNA expression in response to NaAsO2 treatment.Conclusion: Hypermethylation of ERCC1 and ERCC2 and concomitant suppression of gene expression might be served as the epigenetic marks associated with ar...

Coke oven emissions (COEs) are common particle pollutants in occupational environment and the major constituents of COEs are polycyclic aromatic hydrocarbons (PAHs). Previously, we identified aberrant methylation of the fms related tyrosine kinase 1 (FLT1) gene over the course of benzo(a)pyrene (BaP)-induced cell transformation via genome-wide methylation array. To quantify FLT1 methylation, we established a bisulfite pyrosequencing assay and examined the FLT1 hypermethylation in several human cancers. The results revealed that 70.0% (21/30 pairs) of lung cancers harbored hypermethylated FLT1 and concomitant suppression of gene expression compared to the adjacent tissues. This implies that FLT1 hypermethylation might play a role in malignant cell transformation. In addition, FLT1 hypermethylation and gene suppression appeared in primary human lymphocytes in a dose-response manner following COEs treatment. To explore whether FLT1 methylation is correlated with COEs exposure and DNA damage, we recruited 144 male subjects who had been exposed to high levels of COEs and 84 male control subjects. Notably, the FLT1 methylation in peripheral blood lymphocytes (PBLCs) of the COEs-exposed group (19.8 ± 3.2%) was enhanced by 17.9% compared to that of the control group (16.8 ± 2.8%) (P 0.001). The FLT1 methylation status was positively correlated with urinary 1-hydroxypyrene (1-OHP) levels, an internal exposure marker of PAHs (β = 0.029, 95% CI = 0.010-0.048, P = 0.003) and positively correlated with DNA damage (β

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A–B56α suppression in HepG2 cells resulted in persistent β-catenin phosphorylation at Ser(33)-Ser(37)-Thr(41) in response to CYP2E1 agonists. In parallel, nuclear translocation of β-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A–B56α, β-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.

Primary mouse hepatocyte cultures are widely used in toxicological and pharmacological studies. However, the strain differences in alterations of metabolic enzymes and the regulation of gene expression in response to different stimuli remains unclear. To address this issue, we examined the expression of metabolic enzymes and the regulatory role of DNA methylation in the primary hepatocytes of two mouse strains, CD-1 and C57BL/6. Primary culture of mouse hepatocytes was established using collagen sandwich configuration. Analysis of gene expression of 24 phase I, 18 phase II, and 6 phase III metabolic enzymes on 4 consecutive days after cell seeding revealed that the basal levels of most enzymes in primary cultured hepatocytes differed greatly between the two mouse strains. However, the dynamic changes in most genes were identical between the two strains. In addition, treatment with 3-methylcholanthrene, phenobarbital, and rifampin led to the induction of cytochrome P-450 (cyp) 1a1 and cyp1a2, cyp2b10, cyp3a11. However, induction varied in degree between the two types of primary hepatocytes. The dynamic changes in global DNA methylation and the expression of DNA methylation regulatory factors of the two mouse strains were similar. Of the genes down-regulated over the culture period, hypermethylation of cyp2e1 gene appeared in both mouse strains and led to a suppression of gene expression. Taken together, these results demonstrate that the expression of metabolic enzymes and the response to agonists in primary hepatocytes differ between CD-1 and C57BL/6 mouse strains. Epigenetic regulation might be involved in the suppression of cyp 450s' expression.

Previous studies have shown an etiologic link between exposure to polycyclic aromatic hydrocarbons (PAHs) and lung cancer development. While the tumor suppressor gene RASSF1a is mostly silenced by DNA methylation in various tumors, it is unclear whether aberrant methylation of RASSF1a is involved in the process of PAH-induced biological consequences. To address this issue, 69 coke-oven workers (exposure group) and 46 steel rolling workers (control group) were recruited in this study. Bisulfite sequencing (BSP) was performed to examine the methylation status of RASSF1a promoter in peripheral blood lymphocytes (PBLs) from PAH-exposed and control workers. The DNA fragment examined lies across −282 bp to +638 bp from the transcription start site, containing 966 bp and 87 CpG sites across the RASSF1a promoter. Of the 87 CpG sites we analyzed, 5 were significantly hypermethylated in the PAH-exposed workers compared to the control (2.5% vs. 0%, P < 0.001). We defined these 5 CpG sites as “Hot CpG sites”. The levels of methylation from Hot CpG sites were positively correlated with the concentration of urinary 1-OHP (β = 0.98, P = 0.001) and the frequency of cytokinesis-block micronucleus (CBMN) (β = 1.29, P = 0.019) in PBLs, indicating that PAH exposure induced CpG site-specific hypermethylation of RASSF1a was associated with the levels of internal exposure and the degree of DNA damage. Moreover, the Hot CpG site hypermethylation and the corresponding down-regulation of RASSF1a expression were also found in COE (coke-oven emission)-treated human primary lymphocytes and HBE cells. Taken together, these observations revealed that RASSF1a Hot CpG site hypermethylation could be a promising biomarker for the PAH exposure and DNA damage.

An aryl hydrocarbon receptor (AhR) is a transcription factor mediating the responses to polycyclic aromatic hydrocarbon (PAH) compounds. To investigate the epigenetic mechanism involved in the regulation of AhR, we treated human bronchial epithelial cells (16HBE) with benzo(a)pyrene (BaP) and found a transcriptional suppression of AhR in a dose- and time-dependent manner. Suppression of AhR significantly attenuated the extent of BaP-induced CYP1A1 expression and the cell growth arrest, and conferred 16HBE cells insensitive to DNA damage. In addition, we found that the mRNA level of AhR was elevated more than twice in 16HBE cells treated with histone deacetylase inhibitor trichostatin A (TSA), indicating that AhR expression might be regulated via histone modification. Moreover, we showed that BaP or 3-MC treatment led to a reduction of acetylation at residues H3K9, H3K18 and H3K27, suggesting that histone modifications are associated with chemical exposure. Using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we further demonstrate that the reduction of histones H3K18ac and H3K27ac is correlated with the decreased binding affinity with the AhR promoter in HBE cells treated with BaP or 3-MC. In addition, we identified that specific regions located at the transcriptional start site (TSS) of the AhR gene were responsible for H3K18ac- and H3K27ac-related transcriptional activity of the AhR promoter. Taken together, we identified that two specific histone modifications, H3K18ac and H3K27ac, were involved in regulation of the transcriptional activation of AhR, which might contribute to BaP-induced toxicity and the response to DNA damage.

Approaches for assessing the carcinogenic potential of complex mixtures of environmental pollutants are still under development. In this study, a human cell transformation assay was used to evaluate the carcinogenic potential of organic extracts of water pollutants collected from the Jialu River (S1), one of the main tributaries of the Huaihe River in Henan province, China. The incidence of digestive cancers in the basin has risen dramatically within the past three decades. In addition, we collected water samples from the local wells in two liver cancer patients’ homes. The distance of these two wells is 1 km (S2) and 20 km (S3) from the site of S1. Organic chemicals were extracted using hydrophilic–lipophilic balance solid phase cartridges and the fraction was dissolved in DMSO. Human hepatic immortal cells (HL-7702) were treated with each extract and the cytotoxicity was measured. The cells were treated with each extract and the efficiency of cell transformation was examined periodically. The subcutaneous injection of treated cells in immune-deficient mice was performed to confirm the malignant cell transformation. The latency of malignant transformation for samples S1, S2, and S3 was 14, 14, and 16 weeks, respectively, at lowest concentrations of 1.0, 0.5, and 2.0 microlitres (μL) of extract per millilitre medium (mL), enriched from 10, 50, and 200 mL source water, respectively. Moreover, we analyzed the organic extracts for 16 polycyclic aromatic hydrocarbons (PAHs) using GC-MS analysis and found 13 components appearing in all water samples. Our study indicates that human cell transformation assays can potentially be used for assessing the carcinogenic potential of mixtures of environmental pollutants.

In this study, we explore whether altered global histone modifications respond to low-level benzene exposure as well as their association with the hematotoxicity. We recruited 147 low-level benzene-exposed workers and 122 control workers from a petrochemical factory in Maoming City, Guangdong Province, China. The internal exposure marker level, urinary S-phenylmercapturic acid (SPMA), in benzene-exposed workers was 1.81-fold higher than that of the controls (P 0.001). ELISA method was established to examine the specific histone modifications in human peripheral blood lymphocytes (PBLCs) of workers. A decrease in the counts of white blood cells (WBC), neutrophils, lymphocytes, and monocytes appeared in the benzene-exposed group (all P 0.05) compared to the control group. Global trimethylated histone 3 lysine 4 (H3K4me3) modification was enhanced in the benzene-exposed group (P 0.05) and was positively associated with the concentration of urinary SPMA (β = 0.103, P = 0.045) and the extent of DNA damage (% Tail DNA: β = 0.181, P = 0.022), but was negatively associated with the leukocyte count (WBC: β = -0.038, P = 0.023). The in vitro study revealed that H3K4me3 mark was enriched in the promoters of several DNA damage responsive (DDR) genes including CRY1, ERCC2, and TP53 in primary human lymphocytes treated with hydroquinone. Particularly, H3K4me3 modification was positively correlated with the expression of CRY1 in the PBLCs of benzene-exposed workers. These observations indicate that H3K4me3 modification might mediate the transcriptional regulation of DDR genes in response to low-dose benzene exposure.

Induction of metallothionein (MT) expression is involved in metal homeostasis and detoxification. To identify the key pathways that regulate metal-induced cytotoxicity, we investigate how phosphorylated metal-responsive transcription factor-1 (MTF-1) contributed to induction of MT expression. Immortal human embryonic kidney cells (HEK cells) were treated with seven kinds of metals including cadmium chloride (CdCl2), zinc sulfate (ZnSO4), copper sulfate(CuSO4), lead acetate (PbAc), nickel sulfate (NiSO4), sodium arsenite (NaAsO2), and potassium bichromate (K2Cr2O7). The MT expression was induced in a dose-response and time-dependent manner upon various metal treatments. A cycle of phosphorylation and dephosphorylation was required for translocation of MTF-1 from cytoplasm to nucleus, leading to the up-regulation of MTs expression. Protein phosphatase 2A (PP2A) participated in regulating MT expression through dephosphorylation of MTF-1. A loss-of-function screen revealed that the specific PP2A complexes containing PR110 were involved in metal-induced MT expression. Suppression of PP2A PR110 in HEK cells resulted in the persistent MTF-1 phosphorylation and the disturbance of MTF-1 nuclear translocation, which was concomitant with a significant decrease of MT expression and enhanced cytotoxicity in HEK cells. Notably, MTF-1 was found in complex with specific PP2A complexes containing the PR110 subunit upon metal exposure. Furthermore, we identify that the dephosphorylation of MTF-1 at residue Thr-254 is directly regulated by PP2A PR110 complexes and responsible for MTF-1 activation. Taken together, these findings delineate a novel pathway that determines cytotoxicity in response to metal treatments and provide new insight into the role of PP2A in cellular stress response.

Chronic exposure to aflatoxin B

This study aims to assess the effects of low-dose benzene on DNA damage and OWe recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay.The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p 0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage.MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.

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.

To investigate whether polycyclic aromatic hydrocarbon (PAH) exposure is associated with specific histone modifications and whether DNA damage triggers epigenetic alterations, we recruited 190 male workers with occupational exposure to PAHs and 100 male control workers from Benxi Steel Plant, Liaoning province, China. Urinary 1-hydroxypyrene (1-OHP), DNA damage, specific histone modification levels and the expression of selected DNA damage response (DDR) genes were measured in peripheral blood lymphocytes (PBLCs) of the subjects. The results showed that trimethylated Lys 27 of histone H3 (H3K27me3) and trimethylated Lys 36 of histone H3 (H3K36me3) were elevated in the PAH-exposed group (both P < 0.001), while trimethylated Lys H3 of histone H3 (H3K4me3) was decreased compared to the unexposed group (P < 0.001). Notably, H3K36me3 was positively associated with the level of internal exposure marker 1-OHP (β = 0.197; P < 0.001) and the degree of DNA damage (β = 0.175; P < 0.001) in all subjects, indicating that the PAH-induced DNA damage response might be mediated by H3K36me3 and/or H3K4me3 modifications. Particularly, the ChIP-qPCR assay revealed that the modifications of H3K36me3 were enriched in the gene body of DDR genes, MGMT and MLH1. The up-regulation of MGMT and MLH1 was correlated with the elevated H3K36me3 in the PAH-exposed workers (P < 0.001). Collectively, we revealed that H3K36me3 could be an indicator of PAH exposure and might be involved in the transcriptional regulation of DNA repair genes in response to DNA damage.

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P

Recently, diesel engine exhaust (DEE) was reclassified as a known carcinogen to humans. DNA methylation alterations in DNA damage response (DDR)-related genes have the potential to affect DEE exposure-related cancer risk. However, the evidence regarding the association between DEE exposure and methylation alterations in DDR-related genes is limited. In 117 DEE-exposed workers and 112 non-DEE-exposed workers, we measured urinary concentrations of six mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs). We also determined the methylation levels of three DDR-related genes (p16, RASSF1A, and MGMT) and LINE-1 by bisulfite-pyrosequencing assay. We found that DEE-exposed workers exhibited significantly lower mean promoter methylation levels of p16, RASSF1A, and MGMT than non-DEE-exposed workers (all p

It is reported that Ca-doping can improve the weak links between grain boundaries and increase the critical current density J c of YBa 2 Cu 3 O 7− δ polycrystalline films [Phys. Rev. B 58 (1998) 9543; Nature 407 (2000) 162]. In this paper, we investigated the preparation and superconducting properties of YBa 2 Cu 3 O 7− δ (YBCO) and Y 1− x Ca x Ba 2 Cu 3 O 7− δ ( x = 0.05, 0.1, and 0.2) (YCBCO) coatings on yttrium stabilized zirconia (YSZ) single crystal substrates by powder melting growth process. The X-ray diffraction (XRD) indicated that: (1) the coatings were of YBa 2 Cu 3 O 7− δ (Y123) phase with c -orientation; (2) the Y 2 BaCuO 5 (Y211) phase remained clearly in the YBCO coating, but much less in the YCBCO coatings. The effect of Ca-doping on the formation of the Y211 phase was analyzed with the help of the differential thermal analysis (DTA). The results of R – T and I – V relationship at 77 K demonstrated that the zero resistance temperature T c0 decreased with the Ca content, while the critical current density J c (77 K) was improved for x = 0.05.

The Ca-doped YBa2Cu3O7−δ/Y2BaCuO5 bulk-textured superconductors were prepared by using the top-seeding melt-textured growth process. Composition analyses were made for such prepared samples. We found that Ca concentration in the YBa2Cu3O7−δ grains decreased along the growing direction; Ca preferred to substitute Y in the YBa2Cu3O7−δ grains, rather than to stay at the grain boundary. The accumulation of extra CuO at the growth front was considered as one of the causes of the inhomogeneous distribution of Ca.

By using top seeding melt-textured growth (TSMTG) process, large single domain YBa2Cu3O7−δ/Y2BaCuO5 (YBCO) samples were prepared with addition of nanometric SnO2 and normal CeO2. Polarized light microscope studies at each step of the TSMTG process showed that before YBa2Cu3O7−δ (123) melting, the Sn-based reacted products were uniformly distributed in micrometer scale; after solidification, the trapped Y2BaCuO5 (211) and Sn-based inclusions were more homogeneously distributed and with mean sizes of about one micrometer. Composition images of scanning electronic microscope for the textured samples showed that, on the whole, the Sn-based inclusions were smaller than the 211. They must be on average below one micrometer in size. Although evidence was found that very fine Sn-based inclusions were uniformly trapped in 123 matrix, the fraction of them was small in comparison to that of 211. No significant enhancement of superconducting critical current density (Jc) was evidenced for the nanometric SnO2-doped samples at a low magnetic field ( B T ). However, values of Jc were decreased at intermediate magnetic field ( 1 T ).

Ultrafine SnO 2 powder was made with grain size down to 20 nm. Such powder was introduced into the textured growth of Y 1+ x Ba 2 Cu 3 O 7+ δ (YBCO) superconductors. Crystalline inclusions, which were cubic in shape and from micrometer to submicrometer in size, were found trapped inside such grown YBCO superconductors. Determined by energy dispersive spectroscopy (EDS), and further confirmed by X-ray diffraction, the composition of the inclusions was Y 2 Ba 5 (Sn 3− y − z Cu y Pt z )O x . This Sn-based phase belongs to double perovskite structures. They were not trapped uniformly inside the YBa 2 Cu 3 O 7− δ (123), possibly caused by the agglomeration of the SnO 2 powder at the very beginning. Nevertheless, it is possible for fine Sn-based inclusions down to a few hundred nanometers to be trapped inside the 123. The addition of the ultrafine SnO 2 to YBCO textured superconductors without Pt did not seem to affect the size of Y 2 BaCuO 5 (211) particles. For comparison, Y 2 Sn 2 O 7 was also introduced to YBCO textured superconductor. In this case, the so-called φ phase YBa 3 Sn 2− x Cu x O 9− δ was found.

Background Aberrant methylation of the global genome has been investigated as a prognostic indicator in various cancers, but the results are controversial and ambiguous. Methods and Findings This meta-analysis presents pooled estimates of the evidence to elucidate this issue. We searched the electronic databases: PubMed, Embase, ISI Web of Science and the Cochrane library (up to August 2013) to identify all of the relevant studies. The association between the level of surrogates' indexes of genome-wide hypomethylation (LINE-1, Alu and Sat–α) and the overall survival (OS) of cancer patients was examined. In addition, the pooled hazard ratios (HRs) with their 95% confidence interval (95%CI) were calculated to estimate the influences through fixed-effects and random-effects model. Finally, twenty studies with total population of 5447 met the inclusion criteria. The results indicate that the summary HRs for the studies employing LINE-1, Alu, and Sat-α repetitive elements also show that the global DNA hypomethylation have significant desirable effects on the tumour prognostic value. The pooled HRs (and CIs) of LINE-1, Alu and Sat-α were 1.83 (1.38–2.44), 2.00 (1.16–3.45), and 2.92 (1.04–8.25), with a heterogeneity measure index of I2 (and p-value) shows of 66.6% (p = 0.001), 57.1% (p = 0.053) and 68.2% (p = 0.076) respectively. The meta-regression and subgroup analysis indicated that the percentage of hypomethylated sample of cancer patients is one source of heterogeneity. Conclusion Our meta-analysis findings support the hypothesis that the global DNA hypomethylation is associated with a detrimental prognosis in tumour patients.

The formation of γ-H2AX in response to DNA double-strand breaks (DSBs) marks damaged regions for recognition and repair. Dephosphorylation of γ-H2AX is required for cells to resume cell cycle. However, the mechanisms of γ-H2AX dephosphorylation remain underexplored. Using a loss of function screen, we identified PP2A specific subunits, B56ε and α4, involved in elimination of γ-H2AX during DSBs repair process. In the early stage of DSBs repair the inhibitory subunit α4 binds and renders PP2Ac inactive. As DNA is repaired, α4 releases PP2Ac and triggers the assembly of an active PP2A B56ε holoenzyme. PP2A B56ε, which translocates from cytoplasm into the nucleus upon DNA damage, is responsible for a direct dephosphorylation of γ-H2AX. Suppression of both B56ε and α4 leads to persistence of γ-H2AX and defects in DNA repair. In contrast, the rapid clearance of γ-H2AX in human hepatocarcinoma is correlated with the over-expression of both B56ε and α4. Functional analysis reveals that PP2A B56ε coordinates with α4 in accelerating HR repair upon DNA damage. Together, these observations gain insight of how γ-H2AX dephosphorylation is kinetically regulated during DNA repair response. ispartof: Journal of Cell Science vol:128 issue:2 pages:421- status: published

Metals such as cadmium and arsenic are ubiquitous toxicants that cause a variety of adverse health effects. Heat shock proteins (HSPs) response to metal-induced stress and protect cells from further damage. However, the intracellular signalling pathways responsible for activation of HSPs expression are not fully understood. Here, we demonstrate that protein phosphatase 2A (PP2A) regulates expression of HSP70 and HSP27 via dephosphorylation of an AMP-activated protein kinase α subunit (AMPKα) at Thr172. Dephosphorylated AMPKα phosphorylates heat shock factor 1 (HSF1) at Ser303, leading to significant transcriptional suppression of HSP70 and HSP27 in CdCl2- or NaAsO2-treated cells. Suppression of PP2A regulatory B56δ subunit resulted in the sustained phosphorylation of AMPKα upon CdCl2 treatment, subsequent reduction in expression of HSP70 and HSP27, and thereby dramatic reduction of reactive oxygen species (ROS) clearance. We further revealed that PP2A B56δ physically interacted with AMPKα, providing evidence that PP2A B56δ-AMPKα-HSF1 signalling pathway participated in regulating the inducible expression of HSPs and ROS clearance. Taken together, we identified a novel PP2A-dependent signalling pathway involved in regulation of HSPs expression in response to metal stress.