A panel study of occupational exposure to fine particulate matter and changes in DNA methylation over a single workday and years worked in boilermaker welders

Background: Exposure to pollutants including metals and particulate air pollution can alter DNA methylation. Yet little is known about intra-individual changes in DNA methylation over time in relationship to environmental exposures. Therefore, we evaluated the effects of acute- and chronic metal-rich PM2.5 exposures on DNA methylation. Methods: Thirty-eight male boilermaker welders participated in a panel study for a total of 54 person days. Whole blood was collected prior to any welding activities (pre-shift) and immediately after the exposure period (post-shift). The percentage of methylated cytosines (%mC) in LINE-1, Alu, and inducible nitric oxide synthase gene (iNOS) were quantified using pyrosequencing. Personal PM2.5 (particulate matter with an aerodynamic diameter ≤ 2.5 μm) was measured over the work-shift. A questionnaire assessed job history and years worked as a boilermaker. Linear mixed models with repeated measures evaluated associations between DNA methylation, PM2.5 concentration (acute exposure), and years worked as a boilermaker (chronic exposure). Results: PM2.5 exposure was associated with increased methylation in the promoter region of the iNOS gene (β = 0.25, SE: 0.11, p-value = 0.04). Additionally, the number of years worked as a boilermaker was associated with increased iNOS methylation (β = 0.03, SE: 0.01, p-value = 0.03). No associations were observed for Alu or LINE-1. Conclusions: Acute and chronic exposure to PM2.5 generated from welding activities was associated with a modest change in DNA methylation of the iNOS gene. Future studies are needed to confirm this association and determine if the observed small increase in iNOS methylation are associated with changes in NO production or any adverse health effect

Epigenetic Influences on Associations between Air Pollutants and Lung Function in Elderly Men: The Normative Aging Study

Background: Few studies have been performed on pulmonary effects of air pollution in the elderly—a vulnerable population with low reserve capacity—and mechanisms and susceptibility factors for potential effects are unclear. Objectives: We evaluated the lag structure of air pollutant associations with lung function and potential effect modification by DNA methylation (< or ≥ median) at 26 individual CpG sites in nine candidate genes in a well-characterized cohort of elderly men. Methods: We measured forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), and blood DNA methylation one to four times between 1999 and 2009 in 776 men from the Normative Aging Study. Air pollution was measured at fixed monitors 4 hr to 28 days before lung function tests. We used linear mixed-effects models to estimate the main effects of air pollutants and effect modification by DNA methylation. Results: An interquartile range (IQR) increase in subchronic exposure (3 to 28 days cumulated), but not in acute exposure (during the previous 4 hr, or the current or previous day), to black carbon, total and nontraffic particles with aerodynamic diameter ≤ 2.5 μm (PM2.5), carbon monoxide, and nitrogen dioxide was associated with a 1–5% decrease in FVC and FEV1 (p < 0.05). Slope estimates were greater for FVC than FEV1, and increased with cumulative exposure. The estimates slopes for air pollutants (28 days cumulated) were higher in participants with low (< median) methylation in TLR2 at position 2 and position 5 and high (≥ median) methylation in GCR. Conclusions: Subchronic exposure to traffic-related pollutants was associated with significantly reduced lung function in the elderly; nontraffic pollutants (particles, ozone) had weaker associations. Epigenetic mechanisms related to inflammation and immunity may influence these associations. Citation: Lepeule J, Bind MAC, Baccarelli AA, Koutrakis P, Tarantini L, Litonjua A, Sparrow D, Vokonas P, Schwartz JD. 2014. Epigenetic influences on associations between air pollutants and lung function in elderly men: the Normative Aging Study. Environ Health Perspect 122:566–572; http://dx.doi.org/10.1289/ehp.120645

Evaluation of DNA methylation of inflammatory genes following treatment of chronic periodontitis: A pilot case–control study

ObjectiveTo evaluate the influence of periodontal therapy on DNA methylation in patients with chronic periodontitis as compared to healthy individuals.Material and MethodsTwenty patients were enrolled into two groups: (i) 10 diagnosed as clinically healthy; and (ii) 10 diagnosed with chronic periodontitis. Clinical measures were recorded and gingival biopsies were harvested at baseline (both patient groups) and at 2 and 8 weeks post‐baseline for diseased individuals. Molecular DNA methylation analysis was performed by pyrosequencing for the putative inflammation‐associated genes LINE‐1, COX‐2, IFN‐γ and TNF‐α. Random‐intercept linear regression models were applied to evaluate methylation levels across groups at baseline and the methylation changes over time in the diseased and normal tissues.ResultsPeriodontal therapy did not influence gene expression methylation of TNF‐α, IFN‐γ and LINE‐1 levels at normal and periodontitis sites over time. However, it significantly reduced COX‐2 methylation levels comparable to healthy individuals at both 2 and 8 weeks post‐treatment (p < .05).ConclusionsPeriodontal therapy resets the DNA methylation status of inflammatory gene for COX‐2 in patients with periodontal disease. DNA methylation levels of TNF‐α, IFN‐γ and LINE‐1 were sustained in periodontitis sites despite therapy. Future studies should consider an expanded panel of inflammatory genes over time. (ClinicalTrials.gov NCT02835898).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138219/1/jcpe12783.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138219/2/jcpe12783_am.pd

Ambient PM exposure and DNA methylation in tumor suppressor genes: a cross-sectional study

Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (β = 0.27, 95% CI: 0.13-0.40), and PM1 (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

Background: Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined.Objectives We aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10). Methods: We measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3. Results: Global methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02). Conclusions: We observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM

Alu and LINE-1 methylation and lung function in the normative ageing study

Objectives: To investigate the association between methylation of transposable elements Alu and long-interspersed nuclear elements (LINE-1) and lung function. Design: Cohort study. Setting: Outpatient Veterans Administration facilities in greater Boston, Massachusetts, USA. Participants: Individuals from the Veterans Administration Normative Aging Study, a longitudinal study of aging in men, evaluated between 1999 and 2007. The majority (97%) were white. Primary and secondary outcome measures: Primary predictor was methylation, assessed using PCR-pyrosequencing after bisulphite treatment. Primary outcome was lung function as assessed by spirometry, performed according to American Thoracic Society/European Respiratory Society guidelines at the same visit as the blood draws. Results: In multivariable models adjusted for age, height, body mass index (BMI), pack-years of smoking, current smoking and race, Alu hypomethylation was associated with lower forced expiratory volume in 1 s (FEV1) (β=28 ml per 1% change in Alu methylation, p=0.017) and showed a trend towards association with a lower forced vital capacity (FVC) (β=27 ml, p=0.06) and lower FEV1/FVC (β=0.3%, p=0.058). In multivariable models adjusted for age, height, BMI, pack-years of smoking, current smoking, per cent lymphocytes, race and baseline lung function, LINE-1 hypomethylation was associated with more rapid decline of FEV1 (β=6.9 ml/year per 1% change in LINE-1 methylation, p=0.005) and of FVC (β=9.6 ml/year, p=0.002). Conclusions: In multiple regression analysis, Alu hypomethylation was associated with lower lung function, and LINE-1 hypomethylation was associated with more rapid lung function decline in a cohort of older and primarily white men from North America. Future studies should aim to replicate these findings and determine if Alu or LINE-1 hypomethylation may be due to specific and modifiable environmental exposures

Prenatal Arsenic Exposure and DNA Methylation in Maternal and Umbilical Cord Blood Leukocytes

Background: Arsenic is an epigenetic toxicant and could influence fetal developmental programming. Objectives: We evaluated the association between arsenic exposure and DNA methylation in maternal and umbilical cord leukocytes. Methods: Drinking-water and urine samples were collected when women were at ≤ 28 weeks gestation; the samples were analyzed for arsenic using inductively coupled plasma mass spectrometry. DNA methylation at CpG sites in p16 (n = 7) and p53 (n = 4), and in LINE-1 and Alu repetitive elements (3 CpG sites in each), was quantified using pyrosequencing in 113 pairs of maternal and umbilical blood samples. We used general linear models to evaluate the relationship between DNA methylation and tertiles of arsenic exposure. Results: Mean (± SD) drinking-water arsenic concentration was 14.8 ± 36.2 μg/L (range: < 1–230 μg/L). Methylation in LINE-1 increased by 1.36% [95% confidence interval (CI): 0.52, 2.21%] and 1.08% (95% CI: 0.07, 2.10%) in umbilical cord and maternal leukocytes, respectively, in association with the highest versus lowest tertile of total urinary arsenic per gram creatinine. Arsenic exposure was also associated with higher methylation of some of the tested CpG sites in the promoter region of p16 in umbilical cord and maternal leukocytes. No associations were observed for Alu or p53 methylation. Conclusions: Exposure to higher levels of arsenic was positively associated with DNA methylation in LINE-1 repeated elements, and to a lesser degree at CpG sites within the promoter region of the tumor suppressor gene :p16. Associations were observed in both maternal and fetal leukocytes. Future research is needed to confirm these results and determine if these small increases in methylation are associated with any health effects

Exposure to Metal-rich Particulate Matter Modifies the Expression of Candidate Micrornas in Peripheral Blood Leukocytes

Background: Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. MicroRNAs (miRNAs) are highly conserved, noncoding small RNAs that regulate the expression of broad gene networks at the posttranscriptional level. Objectives: We evaluated the effects of exposure to PM and PM metal components on candidate miRNAs (miR-222, miR-21, and miR-146a) related with oxidative stress and inflammatory processes in 63 workers at an electric-furnace steel plant. Methods: We measured miR-222, miR-21, and miR-146a expression in blood leukocyte RNA on the first day of a workweek (baseline) and after 3 days of work (postexposure). Relative expression of miRNAs was measured by real-time polymerase chain reaction. We measured blood oxidative stress (8-hydroxyguanine) and estimated individual exposures to PM$_{1}$PM$_{1}$ (< 1 μm in aerodynamic diameter), PM$_{10}$ (< 10 μm in aerodynamic diameter), coarse PM (PM$_{10}$ minus PM$_{1}$), and PM metal components (chromium, lead, cadmium, arsenic, nickel, manganese) between the baseline and postexposure measurements. Results: Expression of miR-222 and miR-21 (using the 2$^{−ΔΔCT}$ method) was significantly increased in postexposure samples (miR-222: baseline = 0.68 ± 3.41, postexposure = 2.16 ± 2.25, p = 0.002; miR-21: baseline = 4.10 ± 3.04, postexposure = 4.66 ± 2.63, p = 0.05). In postexposure samples, miR-222 expression was positively correlated with lead exposure (β = 0.41, p = 0.02), whereas miR-21 expression was associated with blood 8-hydroxyguanine (β = 0.11, p = 0.03) but not with individual PM size fractions or metal components. Postexposure expression of miR-146a was not significantly different from baseline (baseline = 0.61 ± 2.42, postexposure = 1.90 ± 3.94, p = 0.19) but was negatively correlated with exposure to lead (β = −0.51, p = 0.011) and cadmium (β = −0.42, p = 0.04). Conclusions: Changes in miRNA expression may represent a novel mechanism mediating responses to PM and its metal components

Prolonged Exposure to Particulate Pollution, Genes Associated with Glutathione Pathways, and DNA Methylation in a Cohort of Older Men

Background: DNA methylation is a potential pathway linking environmental exposures to disease. Exposure to particulate air pollution has been associated with increased cardiovascular morbidity and mortality, and lower blood DNA methylation has been found in processes related to cardiovascular morbidity

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies