Toxicomethylomics revisited: A state-of-the-science review about DNA methylation modifications in blood cells from workers exposed to toxic agents.

Introduction: Epigenetic marks have been proposed as early changes, at the subcellular level, in disease development. To find more specific biomarkers of effect in occupational exposures to toxicants, DNA methylation studies in peripheral blood cells have been performed. The goal of this review is to summarize and contrast findings about DNA methylation in blood cells from workers exposed to toxicants. Methods: A literature search was performed using PubMed and Web of Science. After first screening, we discarded all studies performed in vitro and in experimental animals, as well as those performed in other cell types other than peripheral blood cells. Results: 116 original research papers met the established criteria, published from 2007 to 2022. The most frequent investigated exposures/labor group were for benzene (18.9%) polycyclic aromatic hydrocarbons (15.5%), particulate matter (10.3%), lead (8.6%), pesticides (7.7%), radiation (4.3%), volatile organic compound mixtures (4.3%), welding fumes (3.4%) chromium (2.5%), toluene (2.5%), firefighters (2.5%), coal (1.7%), hairdressers (1.7%), nanoparticles (1.7%), vinyl chloride (1.7%), and others. Few longitudinal studies have been performed, as well as few of them have explored mitochondrial DNA methylation. Methylation platforms have evolved from analysis in repetitive elements (global methylation), gene-specific promoter methylation, to epigenome-wide studies. The most reported observations were global hypomethylation as well as promoter hypermethylation in exposed groups compared to controls, while methylation at DNA repair/oncogenes genes were the most studied; studies from genome-wide studies detect differentially methylated regions, which could be either hypo or hypermethylated. Discussion: Some evidence from longitudinal studies suggest that modifications observed in cross-sectional designs may be transitory; then, we cannot say that DNA methylation changes are predictive of disease development due to those exposures. Conclusion: Due to the heterogeneity in the genes studied, and scarcity of longitudinal studies, we are far away from considering DNA methylation changes as biomarkers of effect in occupational exposures, and nor can we establish a clear functional or pathological correlate for those epigenetic modifications associated with the studied exposures.

Genomic targets and selective inhibition of DNA methyltransferase isoforms.

BACKGROUND: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. RESULTS: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δß > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (ß: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δß > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. CONCLUSIONS: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome.

Integration of genome-level data to allow identification of subtype-specific vulnerability genes as novel therapeutic targets.

The identification of cancer-specific vulnerability genes is one of the most promising approaches for developing more effective and less toxic cancer treatments. Cancer genomes exhibit thousands of changes in DNA methylation and gene expression, with the vast majority likely to be passenger changes. We hypothesised that, through integration of genome-wide DNA methylation/expression data, we could exploit this inherent variability to identify cancer subtype-specific vulnerability genes that would represent novel therapeutic targets that could allow cancer-specific cell killing. We developed a bioinformatics pipeline integrating genome-wide DNA methylation/gene expression data to identify candidate subtype-specific vulnerability partner genes for the genetic drivers of individual genetic/molecular subtypes. Using acute lymphoblastic leukaemia as an initial model, 21 candidate subtype-specific vulnerability genes were identified across the five common genetic subtypes, with at least one per subtype. To confirm the approach was applicable across cancer types, we also assessed medulloblastoma, identifying 15 candidate subtype-specific vulnerability genes across three of four established subtypes. Almost all identified genes had not previously been implicated in these diseases. Functional analysis of seven candidate subtype-specific vulnerability genes across the two tumour types confirmed that siRNA-mediated knockdown induced significant inhibition of proliferation/induction of apoptosis, which was specific to the cancer subtype in which the gene was predicted to be specifically lethal. Thus, we present a novel approach that integrates genome-wide DNA methylation/expression data to identify cancer subtype-specific vulnerability genes as novel therapeutic targets. We demonstrate this approach is applicable to multiple cancer types and identifies true functional subtype-specific vulnerability genes with high efficiency.

Analysis of retrotransposon subfamily DNA methylation reveals novel early epigenetic changes in chronic lymphocytic leukemia.

Retrotransposons such as LINE-1 and Alu comprise >25% of the human genome. While global hypomethylation of these elements has been widely reported in solid tumours, their epigenetic dysregulation is yet to be characterised in chronic lymphocytic leukaemia, and there has been scant consideration of their evolutionary history that mediates sensitivity to hypomethylation. Here, we developed an approach for locus- and evolutionary subfamily-specific analysis of retrotransposons using the Illumina Infinium Human Methylation 450K microarray platform, which we applied to publicly-available datasets from chronic lymphocytic leukaemia and other haematological malignancies. We identified 9,797 microarray probes mapping to 117 LINE-1 subfamilies and 13,130 mapping to 37 Alu subfamilies. Of these, 10,782 were differentially methylated (PFDR<0.05) in chronic lymphocytic leukaemia patients (n=139) compared with healthy individuals (n=14), with enrichment at enhancers (p=0.002). Differential methylation was associated with evolutionary age of LINE-1 (r2=0.31, p=0.003) and Alu (r2=0.74, p=0.002) elements, with greater hypomethylation of older subfamilies (L1M, AluJ). Locus-specific hypomethylation was associated with differential expression of proximal genes, including DCLK2, HK1, ILRUN, TANK, TBCD, TNFRSF1B and TXNRD2, with higher expression of DCLK2 and TNFRSF1B associated with reduced patient survival. Hypomethylation at nine loci was highly frequent in chronic lymphocytic leukaemia (>90% patients) but not observed in healthy individuals or other leukaemias, and was detectable in blood samples taken prior to chronic lymphocytic leukaemia diagnosis in 9 of 82 individuals from the Melbourne Collaborative Cohort Study. Our results demonstrate differential methylation of retrotransposons in chronic lymphocytic leukaemia by their evolutionary heritage that modulates expression of proximal genes.

Epigenome-wide analysis reveals functional modulators of drug sensitivity and post-treatment survival in chronic lymphocytic leukaemia.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) patients display a highly variable clinical course, with progressive acquisition of drug resistance. We sought to identify aberrant epigenetic traits that are enriched following exposure to treatment that could impact patient response to therapy. METHODS: Epigenome-wide analysis of DNA methylation was performed for 20 patients at two timepoints during treatment. The prognostic significance of differentially methylated regions (DMRs) was assessed in independent cohorts of 139 and 163 patients. Their functional role in drug sensitivity was assessed in vitro. RESULTS: We identified 490 DMRs following exposure to therapy, of which 31 were CLL-specific and independent of changes occurring in normal B-cell development. Seventeen DMR-associated genes were identified as differentially expressed following treatment in an independent cohort. Methylation of the HOXA4, MAFB and SLCO3A1 DMRs was associated with post-treatment patient survival, with HOXA4 displaying the strongest association. Re-expression of HOXA4 in cell lines and primary CLL cells significantly increased apoptosis in response to treatment with fludarabine, ibrutinib and idelalisib. CONCLUSION: Our study demonstrates enrichment for multiple CLL-specific epigenetic traits in response to chemotherapy that predict patient outcomes, and particularly implicate epigenetic silencing of HOXA4 in reducing the sensitivity of CLL cells to therapy.

Environmental epitranscriptomics.

Chemical modifications of RNA molecules have gained increasing attention since evidence emerged for their substantive roles in a range of biological processes, such as the stability and translation of mRNA transcripts. More than 150 modifications have been identified in different organisms to date, collectively known as the 'epitranscriptome', with 6-methyladenosine (m6A), 5-methylcytidine (m5C), pseudouridine and N1-methyladenosine (m1A) the most extensively investigated. Although we are just beginning to elucidate the roles of these modifications in cellular functions, there is already evidence for their dysregulation in diseases such as cancer and neurodevelopmental disorders. There is currently more limited knowledge regarding how environmental exposures affect the epitranscriptome and how this may mediate disease risk, but evidence is beginning to emerge. Here, we review the current evidence for the impact of environmental exposures such as benzo[a]pyrene, bisphenol A, pesticides, metals and nanoparticles upon RNA modifications and the expression of their 'writers' (methyl transferases), 'erasers' (demethylases) and 'readers'. We discuss future directions of the field and identify areas of particular promise and consider the technical challenges that are faced.

Prenatal PM2.5 exposure and the risk of adverse births outcomes: Results from Project ELEFANT.

BACKGROUND: Studies investigating the impact of fine particulate matter (PM2.5) exposure during pregnancy upon adverse birth outcomes have primarily been performed in Western nations with low ambient PM2.5 levels. We examined associations between high levels of PM2.5 exposure during pregnancy and risk of adverse birth outcomes by timing and level of exposure in a Chinese population. METHODS: We analysed data from 10,738 live births within the Project ELEFANT study based in Tianjin, China. Personal mean daily PM2.5 exposures were estimated using data from 25 local monitoring sites across the city, used to compute the days exceeding 50, 100, 150, 200 and 250 µg/m3. Relative risk of pre-term birth (<37 weeks) and low birthweight (<2500 g) were estimated by generalized additive distributed lag models, adjusted for maternal age, sex, region, paternal smoking, parity, maternal occupation, season, temperature and dew point. RESULTS: A dose-response was exhibited for PM2.5 exposure and relative risk (RR) of adverse birth outcomes, with exposure in the second and third trimesters of pregnancy associated with greatest risk of adverse birth outcomes. The RRs of pre-term birth with exposures of >50, >150 and > 250 µg/m3 PM2.5 in the third trimester were 1.09 (95%CI: 1.03-1.16), 1.30 (1.09-1.54) and 2.73 (2.03-3.66) respectively. For low birthweight, exposures of >50, >150 and > 250 µg/m3 PM2.5 in the third trimester were associated with RRs of 0.99 (0.88-1.11), 1.37 (1.04-1.81) and 3.03 (1.75-5.23) respectively. CONCLUSIONS: Exposure to high levels of PM2.5 from the second trimester onwards was most strongly associated with increased risk of pre-term birth and low birthweight, with a dose-response relationship. Our data demonstrates the need to account for both level and timing of exposure in analysis of PM2.5-associated birth outcomes.

DNA methylation patterns of LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes.

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 µg/day) and vitamin B12 (500 µg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD.

Exposure to environmental toxicants reduces global N6-methyladenosine RNA methylation and alters expression of RNA methylation modulator genes.

The epitranscriptome comprises more than 100 forms of RNA modifications. Of these, N6-methyladenosine (m6A) is the most abundantform of RNA methylation, with roles in modulating mRNA transcript processing and regulation. The aims of the study weretoexamine changes inm6A RNA methylation in A549 lung epithelial cells in response to environmental toxicants, anddifferential gene expression of m6A modulator genes ('readers', 'writers' and 'erasers') in human subjects exposed toparticulate matter (PM) and in lung cancer tissueusing publicly-available microarray datasets. Global m6A methylation levelsweremeasured in total RNA after exposuretotwo carcinogens (PM and sodium arsenite) for 24- and 48-h, and totwo endocrine disruptors (bisphenol A and vinclozolin)for 24-h.Global m6A methylation level significantly decreased with exposure to >62 µg/mlPM, >1 µM sodium arsenite, >1  µM bisphenol A (BPA), and0.1  µM vinclozolin. In an analysis of a published dataset derived from a population study, we observed that m6A writers (METTL3 and WTAP), erasers (FTO and ALKBH5) and readers (HNRPC) showed significantly higher expression among participants in the high-PM2.5exposure group compared to those in the low-exposure control group (all p < 0.05). Further, the m6A writer METTL3shows reduced expression in lung tumors in comparison to normal lung epithelia (p < 0.0001). Our findings reveal that m6A RNA methylation can be modified by exposure to environmental toxicants, and exposure to particulate matter is associated with differential expression level of m6A RNA methylation modification machinery.

Psychosocial stress is associated with benign breast disease in young Chinese women: results from Project ELEFANT.

PURPOSE: Psychosocial stress, including bereavement and work-related stress, is associated with the risk of breast cancer. However, it is unknown whether it may also be linked with increased risk of benign breast disease (BBD). METHODS: Our study leveraged 61,907 women aged 17-55 years old from the Project ELEFANT study. BBD was diagnosed by clinician. Self-reported data on psychosocial stress over a 10-year period was retrospectively collected from questionnaires and categorised by cause (work, social and economic) and severity (none, low and high). Odd ratios (ORs) for the development of BBD were estimated using logistic regression. The model was adjusted for age, BMI, TSH levels, smoking, alcohol consumption, family history, age of menarche, oral contraceptive usage, education and occupation. RESULTS: Within our study, 8% (4,914) of participants were diagnosed with BBD. Work-related stress [OR 1.57, 95% confidence interval (CI) 1.46-1.69] and financial stress (OR 1.34, 95% CI 1.24-1.44) were significantly associated with BBD incidence, with a smaller but still significant association with social stress (OR 1.11, 95% CI 1.01-1.21). The associations remained significant after exclusion of participants with first- and second-degree family history of breast disease. The presence of multiple forms of stress did not synergistically increase risk. The neutrophil-lymphocyte ratio (NLR), a marker of systemic inflammation and prognostic marker for breast cancer, was not associated with BBD. CONCLUSIONS: Psychosocial stress, particularly work-related and financial stress, is associated with increased risk of benign breast disease among young Chinese women.

Obesity as an effect modifier of the association between menstrual abnormalities and hypertension in young adult women: Results from Project ELEFANT.

BACKGROUND: The menstrual cycle is regulated by reproductive hormones such as estrogen which has been implicated in the pathogenesis of hypertension and is associated with obesity. However, to date there has scant study of hypertension in relation to menstrual characteristics and abnormalities. We hypothesize that adverse menstrual characteristics are associated with an increase the prevalence of hypertension and that this relationship is exacerbated by obesity. METHODS: Our study leverages 178,205 healthy female participants (mean age = 29) in a population-based cross-sectional study in Tianjin, China. Menstrual characteristics including menstrual cycle length and regularity, menstrual bleeding length, menstrual blood loss and dysmenorrhea were assessed by self-reported questionnaires, and hypertension was diagnosed by physician. Multiple logistic regression models were used to assess the relationships between menstrual characteristics and hypertension. RESULTS: Normal length menstrual cycle (OR = 1.21, 95% CI: 1.03-1.41), oligomenorrhea (OR = 1.54, 95% CI: 1.12-2.07), irregular cycle (OR = 1.54, 95% CI: 1.22-1.93), and light menstrual blood loss (OR = 1.36, 95% CI: 1.06-1.72) were associated with hypertension among women who are overweight or obese, but not among women who are normal weight. Longer menstrual bleeding duration (OR = 1.44, 95% CI: 1.24-1.67) and dysmenorrhea were associated with increased prevalence of hypertension (OR = 1.20, 95% CI: 1.14-1.41) in all young women. CONCLUSIONS: The prevalence of hypertension is higher among women with menstrual abnormalities, and this association is modified by overweight and obesity.

Age at menarche and prevention of hypertension through lifestyle in young Chinese adult women: result from project ELEFANT.

BACKGROUND: Early and late age at menarche are associated with risk of hypertension, but little is known whether modifiable lifestyle can reduce this risk. METHODS: Our study leverages 60,135 healthy young Chinese women from the Environmental and LifEstyle FActors iN metabolic health throughout life-course Trajectories (ELEFANT) study. Menarche age and lifestyle factors were assessed by self-reported questionnaires and hypertension was diagnosed by physicians. We estimated the odds ratios (ORs) of hypertension associated with menarche age using multivariable logistic regression. We further investigated whether modifiable lifestyles (body mass index, BMI; psychological stress; passive smoking; and imbalanced diet) increased risk in joint analyses. RESULTS: The association between age at menarche and hypertension was U-shaped, with age ≤ 12 at menarche giving the highest OR (1.46, 95% confidence interval [CI], 1.27-1.69) and ≥ 16 the second highest (OR = 1.36, 95% CI = 1.15-1.62). Simultaneous analysis of lifestyle risk factors and age of menarche showed that having one or more modifiable risk factors increased the menarche age-hypertension association. The risk of hypertension among participants with menarche age ≤ 12 decreased from OR 13.21 (95% CI = 5.17-29.36) with four high-risk lifestyle factors to 12.36 (95% CI = 9.51-16.05) with three high-risk factors, 5.24 (95% CI = 4.11-6.69) with two, and 2.76 (95% CI = 2.09-3.60) with one, in comparison to individuals with no high-risk lifestyle factors and menarche age 14. CONCLUSIONS: Our results suggest that modification of lifestyle, including maintenance of normal weight and a balanced diet, are associated with substantially reduce the risk of hypertension in high-risk individuals. Early and late age at menarche are risk factors for the development of hypertension in Western populations, and there is limited evidence that this is also true of Chinese populations. Targeted prevention of hypertension in vulnerable populations would be highly beneficial in efforts to reduce the incidence of cardiovascular disease, but it is not currently known whether lifestyle intervention could reduce hypertension risk. In this study, we analysed the risk of hypertension by age at menarche and four modifiable lifestyle factors (BMI, diet, psychological stress, and smoking tobacco) in a cohort of 60,135 young adult Chinese women (mean age 29). We identified that early and late age at menarche are associated with increased risk of hypertension in young Chinese women. There was joint effects between age at menarche and lifestyles on hypertension only participants with age at menarche ≤12 and being overweight or obese. Modification of lifestyle, including maintenance of normal weight and a balanced diet, can substantially reduce the risk of hypertension in high-risk individuals. In conclusion, our study has revealed that early and late menarche age are associated with the development of hypertension in young Chinese women, and that this risk is modified by healthy lifestyle traits.

Dietary Intervention Modifies DNA Methylation Age Assessed by the Epigenetic Clock.

SCOPE: Alterations in DNA methylation patterns are correlated with aging, environmental exposures, and disease pathophysiology; the possibility of reverting or preventing these processes through dietary intervention is gaining momentum. In particular, methyl donors that provide S-adenosyl-methionine for one-carbon metabolism and polyphenols such as flavanols that inhibit the activity of DNA methyltransferases (DNMTs) can be key modifiers of epigenetic patterns. METHODS AND RESULTS: DNA methylation patterns are assessed in publicly available Illumina Infinium 450K methylation datasets from intervention studies with either folic acid + vitamin B12 (GSE74548) or monomeric and oligomeric flavanols (MOF) (GSE54690) in 44 and 13 participants, respectively. Global DNA methylation levels are increased in unmethylated regions such as CpG islands and shores following folic acid + vitamin B12 supplementation and decreased in highly methylated regions, including shelves and open-seas, following intervention with MOF. After supplementation with folic acid + vitamin B12, epigenetic age, estimated by the Horvath "epigenetic clock" model, is reduced in women with the MTHFR 677CC genotype. CONCLUSIONS: The effects of supplementation with folic acid + vitamin B12 and MOF on DNA methylation age are dependent upon gender and MTHFR genotype. Additionally, the findings demonstrate the potential for these dietary factors to modulate global DNA methylation profiles.

Occupational noise exposure is associated with hypertension in China: Results from project ELEFANT.

OBJECTIVES: We investigated the association between occupational noise exposure and the risk of elevated blood pressure and hypertension by stage in young adults. METHODS: We utilized 124,286 young adults (18-40 years) from the Project ELEFANT study. We categorized occupational noise exposure as high (75 dBA noise exposure for more than 4 hours per day) or low, and measured blood pressure (mmHg) and categorized participants by hypertension stage (normal, elevated, Stage 1, Stage 2). We applied adjusted logistic regression models to identify associations with hypertension risk, and we further examined the noise-BMI, noise-gender, and noise-residence interactions on hypertension risk in separate models. RESULTS: High occupational noise exposure was associated with increases in blood pressure among participants with elevated blood pressure (Estimate = 0.23, 95% CI: 1.09, 1.46, p = 0.0009), in Stage 1 hypertension (Estimate = 0.15, 95% CI: 1.06, 1.25, p = 0.0008), and in Stage 2 hypertension (Estimate = 0.41 95% CI: 1.31, 1.73, p<0.0001). Likewise, noise exposure-BMI interaction was consistently positively associated with increases in blood pressure in participants with elevated blood pressure (Estimate = 0.71, 95% CI: 1.55, 2.69, p<0.0001), in Stage 1 hypertension (Estimate = 0.78, 95% CI: 1.82, 2.61, p<0.0001), and in Stage 2 hypertension (Estimate = 2.06, 95% CI: 5.64, 10.81, p<0.0001). The noise exposure-male interaction showed higher risk for hypertension compared to the noise exposure-female interaction in participants with elevated blood pressure (Estimate = 1.24, 95% CI: 2.56, 4.71, p<0.0001), Stage 1 (Estimate = 1.67, 95% CI: 4.34, 6.42, p<0.0001) and Stage 2 hypertension (Estimate = 1.70, 95% CI: 3.86, 7.77, p<0.0001). Finally, we found that noise exposure-urban interaction was consistently associated with an increase in blood pressure in elevated blood pressure (Estimate = 0.32, 95% CI: 1.19, 1.62, p<0.0001) and in Stage 2 hypertension (Estimate = 0.44, 95% CI: 1.31, 1.80, p<0.0001).

The effect of morphine upon DNA methylation in ten regions of the rat brain.

Morphine is one of the most effective analgesics in medicine. However, its use is associated with the development of tolerance and dependence. Recent studies demonstrating epigenetic changes in the brain after exposure to opiates have provided insight into mechanisms possibly underlying addiction. In this study, we sought to identify epigenetic changes in ten regions of the rat brain following acute and chronic morphine exposure. We analyzed DNA methylation of six nuclear-encoded genes implicated in brain function (Bdnf, Comt, Il1b, Il6, Nr3c1, and Tnf) and three mitochondrially-encoded genes (Mtco1, Mtco2, and Mtco3), and measured global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5 hmC) levels. We observed differential methylation of Bdnf and Il6 in the pons, Nr3c1 in the cerebellum, and Il1b in the hippocampus in response to acute morphine exposure (all P value < 0.05). Chronic exposure was associated with differential methylation of Bdnf and Comt in the pons, Nr3c1 in the hippocampus and Il1b in the medulla oblongata (all P value < 0.05). Global 5mC levels significantly decreased in the superior colliculus following both acute and chronic morphine exposure, and increased in the hypothalamus following chronic exposure. Chronic exposure was also associated with significantly increased global 5hmC levels in the cerebral cortex, hippocampus, and hypothalamus, but significantly decreased in the midbrain. Our results demonstrate, for the first time, highly localized epigenetic changes in the rat brain following acute and chronic morphine exposure. Further work is required to elucidate the potential role of these changes in the formation of tolerance and dependence.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study.

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10-5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effects of environmental noise exposure on DNA methylation in the brain and metabolic health.

Environmental noise exposure is associated with adverse effects on human health including hearing loss, heart disease, and changes in stress-related hormone levels. Alteration in DNA methylation in response to environmental exposures is a well-known phenomenon and it is implicated in many human diseases. Understanding how environmental noise exposures affect DNA methylation patterns may help to elucidate the link between noise and adverse effects on health. In this pilot study we examined the effects of environmental noise exposure on DNA methylation of genes related to brain function and investigated whether these changes are related with metabolic health. We exposed four groups of male Wistar rats to moderate intensity noise (70-75dB with 20-4000Hz) at night for three days as short-term exposure, and for three weeks as long-term exposure. Noise exposure was limited to 45dB during the daytime. Control groups were exposed to only 45dB, day and night. We measured DNA methylation in the Bdnf, Comt, Crhr1, Mc2r, and Snca genes in tissue from four brain regions of the rats (hippocampus, frontal lobe, medulla oblongata, and inferior colliculus). Further, we measured blood pressure and body weight after long-term noise exposure. We found that environmental noise exposure is associated with gene-specific DNA methylation changes in specific regions of the brain. Changes in DNA methylation are significantly associated with changes in body weight (between Bdnf DNA methylation and Δ body weight: r=0.59, p=0.018; and between LINE-1 ORF DNA methylation and Δ body weight: =-0.80, p=0.0004). We also observed that noise exposure decreased blood pressure (p=0.038 for SBP, p=0.017 for DBP and p 0. 017 for MAP) and decreased body weight (ß=-26g, p=0.008). In conclusion, environmental noise exposures can induce changes in DNA methylation in the brain, which may be associated with adverse effects upon metabolic health through modulation of response to stress-related hormones.

Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements.

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

Aberrant methylation of imprinted genes is associated with negative hormone receptor status in invasive breast cancer.

Epigenetic regulation of imprinted genes enables monoallelic expression according to parental origin, and its disruption is implicated in many cancers and developmental disorders. The expression of hormone receptors is significant in breast cancer because they are indicators of cancer cell growth rate and determine response to endocrine therapies. We investigated the frequency of aberrant events and variation in DNA methylation at nine imprinted sites in invasive breast cancer and examined the association with estrogen and progesterone receptor status. Breast tissue and blood from patients with invasive breast cancer (n = 38) and benign breast disease (n = 30) were compared with those from healthy individuals (n = 36), matched with the cancer patients by age at diagnosis, ethnicity, body mass index, menopausal status and familial history of cancer. DNA methylation and allele-specific expression were analyzed by pyrosequencing. Tumor-specific methylation changes at IGF2 DMR2 were observed in 59% of cancer patients, IGF2 DMR0 in 38%, DIRAS3 DMR in 36%, GRB10 ICR in 23%, PEG3 DMR in 21%, MEST ICR in 19%, H19 ICR in 18%, KvDMR in 8% and SNRPN/SNURF ICR in 4%. Variation in methylation was significantly greater in breast tissue from cancer patients compared with that in healthy individuals and benign breast disease. Aberrant methylation of three or more sites was significantly associated with negative estrogen-alpha (Fisher's exact test, p = 0.02) and progesterone-A (p = 0.02) receptor status. Aberrant events and increased variation in imprinted gene DNA methylation, therefore, seem to be frequent in invasive breast cancer and are associated with negative estrogen and progesterone receptor status, without loss of monoallelic expression.