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1.  Integrative Analysis of miRNA and Inflammatory Gene Expression After Acute Particulate Matter Exposure 
Toxicological Sciences  2013;132(2):307-316.
MicroRNAs (miRNAs) are environmentally sensitive inhibitors of gene expression that may mediate the effects of metal-rich particulate matter (PM) and toxic metals on human individuals. Previous environmental miRNA studies have investigated a limited number of candidate miRNAs and have not yet evaluated the functional effects on gene expression. In this study, we wanted to identify PM-sensitive miRNAs using microarray profiling on matched baseline and postexposure RNA from foundry workers with well-characterized exposure to metal-rich PM and to characterize miRNA relations with expression of candidate inflammatory genes. We applied microarray analysis of 847 human miRNAs and real-time PCR analysis of 18 candidate inflammatory genes on matched blood samples collected from foundry workers at baseline and after 3 days of work (postexposure). We identified differentially expressed miRNAs (fold change [FC] > 2 and p < 0.05) and correlated their expression with the inflammatory associated genes. We performed in silico network analysis in MetaCore v6.9 to characterize the biological pathways connecting miRNA-mRNA pairs. Microarray analysis identified four miRNAs that were differentially expressed in postexposure compared with baseline samples, including miR-421 (FC = 2.81, p < 0.001), miR-146a (FC = 2.62, p = 0.007), miR-29a (FC = 2.91, p < 0.001), and let-7g (FC = 2.73, p = 0.019). Using false discovery date adjustment for multiple comparisons, we found 11 miRNA-mRNA correlated pairs involving the 4 differentially expressed miRNAs and candidate inflammatory genes. In silico network analysis with MetaCore database identified biological interactions for all the 11 miRNA-mRNA pairs, which ranged from direct mRNA targeting to complex interactions with multiple intermediates. Acute PM exposure may affect gene regulation through PM-responsive miRNAs that directly or indirectly control inflammatory gene expression.
doi:10.1093/toxsci/kft013
PMCID: PMC3595525  PMID: 23358196
miRNA expression; integrative analysis; mRNA expression; inflammation; metal-rich particulate matter; microarray.
2.  Association between blood pressure and DNA methylation of retrotransposons and pro-inflammatory genes 
Background Methylation of deoxyribonucleic acid (DNA) is an epigenetic regulator of gene expression that changes with age, but its contribution to aging-related disorders, including high blood pressure (BP), is still largely unknown. We examined the relation of BP to the methylation of retrotransposon sequences of DNA and of selected candidate genes.
Methods This investigation included 789 elderly participants in the Normative Aging Study, ranging in age from 55 to 100 years, who had longitudinal measurements of DNA methylation. In these subjects’ DNA we measured the proportion of methylated sites in retrotransposable sequences and in pro-inflammatory genes, expressed as the percent of 5-methylated cytosines (%5mC) among all cytosines. From one to four methylation measurements were made for each subject between 1999 and 2009. We fit mixed-effects models, using repeated measures of BP as the outcome and DNA methylation as the explanatory variable, adjusting for confounding variables. We also fit a Bayesian mixed-effects structural equation model to account for heterogeneity in the effects of methylation sites within each gene.
Results An increase in inter-quartile range (IQR) in the methylation of Alu elements was associated with an increase of 0.97 mm Hg in diastolic blood pressure (DBP) (95% CI 0.32–1.57), but no such association was observed for long interspersed nuclear element-1 (LINE-1). We also found positive associations between DBP and methylation of the genes for toll-like receptor 2 (TLR2) and inducible nitric oxide synthase (iNOS), and a negative association between DBP and methylation of the gene for interferon-γ (IFN-γ). Associations between methylation and systolic blood pressure (SBP) were weaker than those between methylation and DBP. Bayesian mixed-effects structural equation model results were similar for both DBP and SBP models.
Conclusions The results of our study suggest that changes in DNA methylation of some pro-inflammatory genes and retrotransposable elements are related to small changes in BP.
doi:10.1093/ije/dys220
PMCID: PMC3600626  PMID: 23508416
Epigenetics; DNA methylation; blood pressure; inflammation; Bayesian model
3.  Allergen sensitization is associated with increased DNA methylation in older men 
Background
Variation in epigenetic modifications, arising from either environmental exposures or internal physiological changes, can influence gene expression, and may ultimately contribute to complex diseases such as asthma and allergies. We examined the association of asthma and allergic phenotypes with DNA methylation levels of retrotransposon-derived elements.
Methods
We used data from 704 men (mean age 73) in the longitudinal Normative Aging Study to assess the relationship between asthma, allergic phenotypes and DNA methylation levels of the retrotransposon derived elements Alu and LINE-1. Retrotransposons represent a large fraction of the genome (> 30%), and are heavily methylated to prevent expression. Percent methylation of Alu and LINE-1 elements in peripheral white blood cells was quantified using PCR pyrosequencing. Data on sensitization to common allergens by skin prick testing, asthma, and methacholine responsiveness was gathered approximately 8 years prior to DNA methylation analysis.
Results
Prior allergen sensitization was associated with increased methylation of Alu (β=0.32 [sensitized vs. non-sensitized], p value 0.003), in models adjusted for pack-years, BMI, smoking, air pollutants, percent eosinophils, white blood cell count and age. Of the men interviewed, 5 % of subjects reported diagnosis of asthma. Neither Alu, nor LINE-1 methylation was associated with asthma.
Conclusions
These data suggest that increased DNA methylation of repetitive elements may be associated with allergen sensitization, but does not appear to be associated with asthma. Future work is needed to identify potential underlying mechanisms for these relationships.
doi:10.1159/000343004
PMCID: PMC3730837  PMID: 23257623
allergen sensitization; DNA methylation; Alu; and LINE-1
4.  DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand 
Background Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored.
Methods The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts.
Results In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M1dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05).
Conclusions Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage.
doi:10.1093/ije/dys129
PMCID: PMC3535756  PMID: 23064502
Air pollution; Ma Ta Phut; blood leucocytes; DNA methylation; DNA damage
5.  Arsenic exposure and DNA methylation among elderly men 
Epidemiology (Cambridge, Mass.)  2012;23(5):668-676.
BACKGROUND
Arsenic exposure has been linked to epigenetic modifications such as DNA methylation in in vitro and animal studies. This association has also been explored in highly exposed human populations, but studies among populations environmentally exposed to low arsenic levels are lacking.
METHODS
We evaluated the association between exposure to arsenic, measured in toenails, and blood DNA methylation in Alu and Long Interspersed Nucleotide Element-1 (LINE-1) repetitive elements in elderly men environmentally exposed to low levels of arsenic. We also explored potential effect modification by plasma folate, cobalamin (vitamin B12), and pyridoxine (vitamin B6). The study population was 581 participants from the Normative Aging Study in Boston, of whom 434, 140, and 7 had 1, 2, and 3 visits, respectively, between 1999-2002 and 2006-2007. We used mixed-effects models and included interaction terms to assess potential effect modification by nutritional factors.
RESULTS
There was a trend of increasing Alu and decreasing LINE-1 DNA methylation as arsenic exposure increased. In subjects with plasma folate below the median (< 14.1 ng/ml), arsenic was positively associated with Alu DNA methylation (β=0.08 [95% confidence interval = 0.03 to 0.13] for one interquartile range [0.06μg/g] increase in arsenic) while a negative association was observed in subjects with plasma folate above the median (β=-0.08 [-0.17 to 0.01]).
CONCLUSIONS
We found an association between arsenic exposure and DNA methylation in Alu repetitive elements that varied by folate level. This suggests a potential role for nutritional factors in arsenic toxicity.
doi:10.1097/EDE.0b013e31825afb0b
PMCID: PMC3448132  PMID: 22833016
6.  Repetitive element hypomethylation in blood leukocyte DNA and cancer incidence, prevalence and mortality in elderly individuals: the Normative Aging Study 
Cancer causes & control : CCC  2010;22(3):437-447.
Background
Global genomic hypomethylation is a common epigenetic event in cancer that mostly results from hypomethylation of repetitive DNA elements. Case-control studies have associated blood leukocyte DNA hypomethylation with several cancers. Because samples in case-control studies are collected after disease development, whether DNA hypomethylation is causal or just associated with cancer development is still unclear.
Methods
In 722 elderly subjects from the Normative Aging Study cohort, we examined whether DNA methylation in repetitive elements (Alu, LINE-1) was associated with cancer incidence (30 new cases, median follow-up: 89 months), prevalence (205 baseline cases), and mortality (28 deaths, median follow-up: 85 months). DNA methylation was measured by bisulfite pyrosequencing.
Results
Individuals with low LINE-1 methylation (
Conclusion
These findings suggest that individuals with lower repetitive element methylation are at high risk of developing and dying from cancer.
doi:10.1007/s10552-010-9715-2
PMCID: PMC3752839  PMID: 21188491
Repetitive elements; DNA methylation; Epigenetics; Blood; Cancer risk
Epigenomics  2011;3(3):267-277.
The concept of “lifestyle” includes different factors such as nutrition, behavior, stress, physical activity, working habits, smoking and alcohol consumption. Increasing evidence shows that environmental and lifestyle factors may influence epigenetic mechanisms, such as DNA methylation, histone acetylation and microRNA expression.
Several lifestyle factors have been identified that might modify epigenetic patterns, such as diet, obesity, physical activity, tobacco smoking, alcohol consumption, environmental pollutants, psychological stress, and working on night shifts.
Most studies conducted so far have been centered on DNA methylation, whereas only a few investigations have studied lifestyle factors in relation to histone modifications and miRNAs.
Here, we review current evidence indicating that lifestyle factors might affect human health via epigenetic mechanisms.
doi:10.2217/epi.11.22
PMCID: PMC3752894  PMID: 22122337
Epigenetics; DNA methylation; Histone modifications; Environmental exposures; Lifestyle
Epidemiology (Cambridge, Mass.)  2010;21(6):819-828.
Background
Epigenetic features such as DNA hypomethylation have been associated with conditions related to cardiovascular risk. We evaluated whether lower blood DNA methylation in heavily methylated repetitive sequences predicts the risk of ischemic heart disease and stroke.
Methods
We quantified blood DNA methylation of LINE-1 repetitive elements through PCR-pyrosequencing in 712 elderly individuals from the Boston-area Normative Aging Study. We estimated risk-factor adjusted relative risks (RRs) for ischemic heart disease and stroke at baseline (242 prevalent cases); as well as in incidence (44 new cases; median follow-up, 63 months); and subsequent mortality from ischemic heart disease (86 deaths; median follow-up, 75 months).
Results
Blood LINE-1 hypomethylation was associated with baseline ischemic heart disease (RR=2.1 [95% confidence interval = 1.2 to 4.0] for lowest vs. highest methylation quartile) and for stroke (2.5 [0.9 to 7.5]). Among participants free of baseline disease, individuals with methylation below the median also had higher risk of developing ischemic heart disease (4.0 [1.8 to 8.9]) or stroke (5.7 [0.8 to 39.5]). In the entire cohort, persons with methylation below the median had higher mortality from ischemic heart disease (3.3 [1.3 to 8.4]) and stroke (2.8 [0.6 to 14.3]). Total mortality was also increased (2.0 [1.2 to 3.3]). These results were confirmed in additional regression models using LINE-1 methylation as a continuous variable.
Conclusions
Subjects with prevalent IHD and stroke exhibited lower LINE-1 methylation. In longitudinal analyses, persons with lower LINE-1 methylation were at higher risk for incident ischemic heart disease and stroke, and for total mortality.
doi:10.1097/EDE.0b013e3181f20457
PMCID: PMC3690659  PMID: 20805753
Background Estimates of global DNA methylation from repetitive DNA elements, such as Alu and LINE-1, have been increasingly used in epidemiological investigations because of their relative low-cost, high-throughput and quantitative results. Nevertheless, determinants of these methylation measures in healthy individuals are still largely unknown. The aim of this study was to examine whether age, gender, smoking habits, alcohol drinking and body mass index (BMI) are associated with Alu or LINE-1 methylation levels in blood leucocyte DNA of healthy individuals.
Methods Individual data from five studies including a total of 1465 healthy subjects were combined. DNA methylation was quantified by PCR-pyrosequencing.
Results Age [β = −0.011% of 5-methyl-cytosine (%5mC)/year, 95% confidence interval (CI) −0.020 to −0.001%5mC/year] and alcohol drinking (β = −0.214, 95% CI −0.415 to −0.013) were inversely associated with Alu methylation. Compared with females, males had lower Alu methylation (β = −0.385, 95% CI −0.665 to −0.104) and higher LINE-1 methylation (β = 0.796, 95% CI 0.261 to 1.330). No associations were found with smoking or BMI. Percent neutrophils and lymphocytes in blood counts exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = −0.038, 95% CI −0.065 to −0.012) association with LINE-1 methylation, respectively.
Conclusions Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.
doi:10.1093/ije/dyq154
PMCID: PMC3304518  PMID: 20846947
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
PLoS ONE  2012;7(12):e52466.
The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.
doi:10.1371/journal.pone.0052466
PMCID: PMC3531488  PMID: 23300679
PLoS ONE  2012;7(12):e50471.
Chronic occupational exposure to benzene is associated with an increased risk of hematological malignancies such as acute myeloid leukemia (AML), but the underlying mechanisms are still unclear. The main objective of this study was to investigate the association between benzene exposure and DNA methylation, both in repeated elements and candidate genes, in a population of 158 Bulgarian petrochemical workers and 50 unexposed office workers. Exposure assessment included personal monitoring of airborne benzene at work and urinary biomarkers of benzene metabolism (S-phenylmercapturic acid [SPMA] and trans,trans-muconic acid [t,t-MA]) at the end of the work-shift. The median levels of airborne benzene, SPMA and t,t-MA in workers were 0.46 ppm, 15.5 µg/L and 711 µg/L respectively, and exposure levels were significantly lower in the controls. Repeated-element DNA methylation was measured in Alu and LINE-1, and gene-specific methylation in MAGE and p15. DNA methylation levels were not significantly different between exposed workers and controls (P>0.05). Both ordinary least squares (OLS) and beta-regression models were used to estimate benzene-methylation associations. Beta-regression showed better model specification, as reflected in improved coefficient of determination (pseudo R2) and Akaike’s information criterion (AIC). In beta-regression, we found statistically significant reductions in LINE-1 (−0.15%, P<0.01) and p15 (−0.096%, P<0.01) mean methylation levels with each interquartile range (IQR) increase in SPMA. This study showed statistically significant but weak associations of LINE-1 and p15 hypomethylation with SPMA in Bulgarian petrochemical workers. We showed that beta-regression is more appropriate than OLS regression for fitting methylation data.
doi:10.1371/journal.pone.0050471
PMCID: PMC3515615  PMID: 23227177
Epigenomics  2012;4(1):91-100.
Aims
DNA methylation is increasingly proposed as a mechanism for underlying asthma-related inflammation. However, epigenetic studies are constrained by uncertainties on whether samples that can be easily collected in human individuals can provide informative results.
Methods
Two nasal cell DNA samples were collected on different days by nasal brushings from 35 asthmatic children aged between 8 and 11 years old. We correlated DNA methylation of IL-6, iNOS, Alu and LINE-1 with fractional exhaled nitric oxide, forced expiratory volume in 1 s and wheezing.
Results
Fractional exhaled nitric oxide increased in association with lower promoter methylation of both IL-6 (+29.0%; p = 0.004) and iNOS (+41.0%; p = 0.002). Lower IL-6 methylation was nonsignificantly associated with wheezing during the week of the study (odds ratio = 2.3; p = 0.063).
Conclusion
Our findings support the use of nasal cell DNA for human epigenetic studies of asthma.
doi:10.2217/epi.11.106
PMCID: PMC3297414  PMID: 22332661
airway obstruction; asthma; children; DNA methylation; epigenetics; inflammation
BMJ Open  2012;2(5):e001231.
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.
doi:10.1136/bmjopen-2012-001231
PMCID: PMC3488751  PMID: 23075571
Epidemiology; Genetics
Environmental Health Perspectives  2011;120(2):210-215.
Background: Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses.
Objectives: Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers.
Methods: We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari).
Results: In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (–2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008).
Conclusions: Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial DNA damage and dysfunction.
doi:10.1289/ehp.1103979
PMCID: PMC3279451  PMID: 22005026
benzene; biomarkers; low exposures; methylation; mitochondrial DNA copy number
Epigenetics  2011;6(2):188-194.
Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.
doi:10.4161/epi.6.2.13528
PMCID: PMC3775884  PMID: 20930513
Alu; DNA methyltransferases; LINE-1; SAT-α; chronic lymphocytic leukemia
BMC Medical Genetics  2011;12:150.
Background
Sequence variants in genes functioning in folate-mediated one-carbon metabolism are hypothesized to lead to changes in levels of homocysteine and DNA methylation, which, in turn, are associated with risk of cardiovascular disease.
Methods
330 SNPs in 52 genes were studied in relation to plasma homocysteine and global genomic DNA methylation. SNPs were selected based on functional effects and gene coverage, and assays were completed on the Illumina Goldengate platform. Age-, smoking-, and nutrient-adjusted genotype--phenotype associations were estimated in regression models.
Results
Using a nominal P ≤ 0.005 threshold for statistical significance, 20 SNPs were associated with plasma homocysteine, 8 with Alu methylation, and 1 with LINE-1 methylation. Using a more stringent false discovery rate threshold, SNPs in FTCD, SLC19A1, and SLC19A3 genes remained associated with plasma homocysteine. Gene by vitamin B-6 interactions were identified for both Alu and LINE-1 methylation, and epistatic interactions with the MTHFR rs1801133 SNP were identified for the plasma homocysteine phenotype. Pleiotropy involving the MTHFD1L and SARDH genes for both plasma homocysteine and Alu methylation phenotypes was identified.
Conclusions
No single gene was associated with all three phenotypes, and the set of the most statistically significant SNPs predictive of homocysteine or Alu or LINE-1 methylation was unique to each phenotype. Genetic variation in folate-mediated one-carbon metabolism, other than the well-known effects of the MTHFR c.665C>T (known as c.677 C>T, rs1801133, p.Ala222Val), is predictive of cardiovascular disease biomarkers.
doi:10.1186/1471-2350-12-150
PMCID: PMC3266217  PMID: 22103680
BACKGROUND
Lower blood DNA methylation has been associated with atherosclerosis and high cardiovascular risk. Mechanisms linking DNA hypomethylation to increased cardiovascular risk are still largely unknown.
In a population of community-dwelling elderly individuals, we evaluated whether DNA methylation in LINE-1 repetitive element, heavily methylated sequences dispersed throughout the human genome, was associated with circulating Vascular Cell Adhesion Molecule-1 (VCAM-1), Inter-Cellular Adhesion Molecule-1 (ICAM-1), and C-reactive protein (CRP).
METHODS AND RESULTS
We measured LINE-1 methylation by bisulfite PCR-Pyrosequencing on 742 blood DNA samples from male participants in the Boston area Normative Aging Study (mean age=74.8 years). Mean serum VCAM-1 increased progressively in association with LINE-1 hypomethylation (from 975.2 to 1063.4 ng/ml in the highest vs. lowest methylation quintiles; p-trend=0.004). The association between VCAM-1 and LINE-1 hypomethylation was significant in individuals without ischemic heart disease or stroke (n=480; p=0.001), but not in those with prevalent disease (n=262; p=0.57). Serum ICAM-1 and CRP were not associated with LINE-1 methylation (p-trend=>0.25). All results were confirmed by multivariable analyses adjusting for age, BMI, smoking, pack-years, and ischemic heart disease/stroke.
CONCLUSIONS
LINE-1 element hypomethylation is associated with higher serum VCAM-1. Our data provide new insights into epigenetic events that may accompany the development of cardiovascular disease.
PMCID: PMC3155741  PMID: 20305373
cell adhesion molecules; epidemiology; cardiovascular diseases; risk factors; LINE-1; VCAM-1
Heredity  2010;105(1):105-112.
Purpose of the review
Epigenetics investigates heritable changes in gene expression occurring without changes in DNA sequence. Several epigenetic mechanisms, including DNA methylation and histone modifications, can change genome function under exogenous influence. We review current evidence indicating that epigenetic alterations mediate effects from exposure to environmental toxicants.
Recent findings
Results from animal models indicate that in-utero or early-life environmental exposures produce effects that can be inherited transgenerationally and are accompanied by epigenetic alterations. The search for human equivalents of the epigenetic mechanisms identified in animal models is under way. Recent investigations have identified a number of environmental toxicants that cause altered methylation of human repetitive elements or genes. Some exposures can alter epigenetic states and the same and/or similar epigenetic alterations can be found in patients with the disease of concern. Based on current evidence, we propose possible models for the interplay between environmental exposures and the human epigenome.
Summary
Several investigations have examined the relation between exposure to environmental chemicals and epigenetics, and identified toxicants that modify epigenetic states. Whether environmental exposures have transgenerational epigenetic effects in humans remains to be elucidated. In spite of the current limitations, available evidence supports the concept that epigenetics holds substantial potential for furthering our understanding of the molecular mechanisms of environmental toxicants, as well as for predicting health-related risks due to conditions of environmental exposure and individual susceptibility.
doi:10.1038/hdy.2010.2
PMCID: PMC3133724  PMID: 20179736
Epigenetics; DNA methylation; Histone modifications; Environmental exposures; Transgenerational effects
Environmental Health Perspectives  2011;119(7):964-969.
Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined.
Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM.
Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject.
Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51).
Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic.
doi:10.1289/ehp.1002955
PMCID: PMC3222996  PMID: 21385672
environmental carcinogens; epigenetics; histone modifications; metals; particulate matter
Carcinogenesis  2009;31(2):216-221.
Shorter telomere length (TL) in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter TL has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on TL measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)–DNA adduct], genetic instability (micronuclei, MN) and DNA methylation [p53 promoter and Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements, as surrogate measures of global methylation] in PBLs. TL was measured by real-time polymerase chain reaction. Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index) and exhibited lower TL (P = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations [i.e. anti-BPDE–DNA adduct and MN (P < 0.0001)] and epigenetic changes [i.e. p53 gene-specific promoter and global methylation (P ≤ 0.001)]. TL decreased with longer duration of work as cokeoven worker (P = 0.039) and in all subjects with higher levels of anti-BPDE–DNA adduct (P = 0.042), p53 hypomethylation (P = 0.005) and MN (P = 0.009). In multivariate analysis, years of work in cokery (P = 0.008) and p53 hypomethylation (P = 0.001) were the principal determinants of shorter TL. Our results indicate that shorter TL is associated with chronic PAH exposure. The interrelations with other genetic and epigenetic mechanisms in our data suggest that shorter TL could be a central event in PAH carcinogenesis.
doi:10.1093/carcin/bgp278
PMCID: PMC3491668  PMID: 19892797
Global hypomethylation has been shown to increase genome instability potentially leading to increased cancer risk. We determined whether global methylation in blood leukocyte DNA was associated with gastric cancer in a population-based study on 302 gastric cancer cases and 421 age- and sex-matched controls in Warsaw, Poland, between 1994 and 1996. Using PCR-pyrosequencing, we analyzed methylation levels of Alu and LINE-1, 2 CG-rich repetitive elements, to measure global methylation levels. Gastric cancer risk was highest among those with lowest level of methylation in either Alu (OR = 1.3, 95% CI = 0.9–1.9) or LINE-1 (OR = 1.4, 95% CI = 0.9–2.0) relative to those with the highest levels, although the trends were not statistically significant. For Alu, the association was stronger among those aged 70 or older (OR = 2.6, 95% CI = 1.3–5.5, p for interaction = 0.02). We did not observe meaningful differences in the associations by other risk factors and polymorphisms examined. For LINE-1, the association tended to be stronger among individuals with a family history of cancer (OR = 3.1, 95% CI = 1.4–7.0, p for interaction = 0.01), current alcohol drinkers (OR = 1.9, 95% CI = 1.0–3.6, p for interaction = 0.05), current smokers (OR = 2.3, 95% CI = 1.1–4.6, p for interaction = 0.02), those who rarely or never consumed fruit (OR = 3.1, 95% CI = 1.2–8.1, p for interaction = 0.03), CC carriers for the MTRR Ex5+123C>T polymorphism (OR = 2.3, 95% CI = 1.2–4.4, p for interaction = 0.01) and TT carriers for the MTRR Ex15+572T>C polymorphism (OR = 1.7, 95% CI = 1.0–2.8, p for interaction = 0.06). The association was not different by sex, Helicobacter pylori infection, intake of folate, vitamin B6 and total protein and the remaining polymorphisms examined. Our results indicate that interactions between blood leukocyte DNA hypomethylation and host characteristics may determine gastric cancer risk.
doi:10.1002/ijc.25190
PMCID: PMC3009461  PMID: 20099281
gastric cancer; methylation; global hypomethylation; gastric cancer risk
Loss of genomic DNA methylation has been found in a variety of common human age-related diseases. Whether DNA methylation decreases over time as individuals age is unresolved. We measured DNA methylation in 1,097 blood DNA samples from 718 elderly subjects between 55–92 years of age (1–3 samples/subjects), who have been repeatedly evaluated over an 8-year time span in the Boston area Normative Aging Study. DNA methylation was measured using quantitative PCR-Pyrosequencing analysis in Alu and LINE-1 repetitive elements, heavily methylated sequences with high representation throughout the human genome. Age at the visit was negatively associated with Alu element methylation (β=−.12 %5mC/year, p=0.0005). A weaker association was observed with LINE-1 elements (β=−.06 %5mC/year, p=0.049). We observed a significant decrease in average Alu methylation over time, with a −0.2 %5mc change (p=0.012) compared to blood samples collected up to 8 years earlier. The longitudinal decline in Alu methylation was linear and highly correlated with time since the first measurement (β=−.089 %5mC/year, p<0.0001). In contrast, average LINE-1 methylation did not vary over time [p=0.51]. Our results demonstrate a progressive loss of DNA methylation in repetitive elements dispersed throughout the genome.
doi:10.1016/j.mad.2008.12.003
PMCID: PMC2956267  PMID: 19150625
Environmental Health Perspectives  2010;118(6):763-768.
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 PM1 (< 1 μm in aerodynamic diameter), PM10 (< 10 μm in aerodynamic diameter), coarse PM (PM10 minus PM1), 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.
doi:10.1289/ehp.0901300
PMCID: PMC2898851  PMID: 20061215
epigenetics; etiology; miRNA expression; particulate matter; peripheral blood leukocytes
Environmental Health Perspectives  2010;118(6):790-795.
Background
DNA methylation is an epigenetic mark that regulates gene expression. Changes in DNA methylation within white blood cells may result from cumulative exposure to environmental metals such as lead. Bone lead, a marker of cumulative exposure, may therefore better predict DNA methylation than does blood lead.
Objective
In this study we compared associations between lead biomarkers and DNA methylation.
Methods
We measured global methylation in participants of the Normative Aging Study (all men) who had archived DNA samples. We measured patella and tibia lead levels by K-X-Ray fluorescence and blood lead by atomic absorption spectrophotometry. DNA samples from blood were used to determine global methylation averages within CpG islands of long interspersed nuclear elements-1 (LINE-1) and Alu retrotransposons. A mixed-effects model using repeated measures of Alu or LINE-1 as the dependent variable and blood/bone lead (tibia or patella in separate models) as the primary exposure marker was fit to the data.
Results
Overall mean global methylation (± SD) was 26.3 ± 1.0 as measured by Alu and 76.8 ± 1.9 as measured by LINE-1. In the mixed-effects model, patella lead levels were inversely associated with LINE-1 (β = −0.25; p < 0.01) but not Alu (β = −0.03; p = 0.4). Tibia lead and blood lead did not predict global methylation for either Alu or LINE-1.
Conclusion
Patella lead levels predicted reduced global DNA methylation within LINE-1 elements. The association between lead exposure and LINE-1 DNA methylation may have implications for the mechanisms of action of lead on health outcomes, and also suggests that changes in DNA methylation may represent a biomarker of past lead exposure.
doi:10.1289/ehp.0901429
PMCID: PMC2898855  PMID: 20064768
aging; DNA methylation; epigenetics; lead; metals
Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.
PMCID: PMC2848123  PMID: 19421002
DNA methylation; plasma DNA; biomarker; cancer; repetitive elements; DNA methylation inhibitors; solid tumors; repetitive DNA elements; LINE; MAGE-A1

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