Federica Rota

Epigenome-wide association of DNA methylation markers in peripheral blood from Indian Asians and Europeans with incident type 2 diabetes: a nested case-control study.

BACKGROUND Indian Asians, who make up a quarter of the worlds population, are at high risk of developing type 2 diabetes. We investigated whether DNA methylation is associated with future type 2 diabetes incidence in Indian Asians and whether differences in methylation patterns between Indian Asians and Europeans are associated with, and could be used to predict, differences in the magnitude of risk of developing type 2 diabetes. METHODS We did a nested case-control study of DNA methylation in Indian Asians and Europeans with incident type 2 diabetes who were identified from the 8-year follow-up of 25 372 participants in the London Life Sciences Prospective Population (LOLIPOP) study. Patients were recruited between May 1, 2002, and Sept 12, 2008. We did epigenome-wide association analysis using samples from Indian Asians with incident type 2 diabetes and age-matched and sex-matched Indian Asian controls, followed by replication testing of top-ranking signals in Europeans. For both discovery and replication, DNA methylation was measured in the baseline blood sample, which was collected before the onset of type 2 diabetes. Epigenome-wide significance was set at p<1 × 10(-7). We compared methylation levels between Indian Asian and European controls without type 2 diabetes at baseline to estimate the potential contribution of DNA methylation to increased risk of future type 2 diabetes incidence among Indian Asians. FINDINGS 1608 (11·9%) of 13 535 Indian Asians and 306 (4·3%) of 7066 Europeans developed type 2 diabetes over a mean of 8·5 years (SD 1·8) of follow-up. The age-adjusted and sex-adjusted incidence of type 2 diabetes was 3·1 times (95% CI 2·8-3·6; p<0·0001) higher among Indian Asians than among Europeans, and remained 2·5 times (2·1-2·9; p<0·0001) higher after adjustment for adiposity, physical activity, family history of type 2 diabetes, and baseline glycaemic measures. The mean absolute difference in methylation level between type 2 diabetes cases and controls ranged from 0·5% (SD 0·1) to 1·1% (0·2). Methylation markers at five loci were associated with future type 2 diabetes incidence; the relative risk per 1% increase in methylation was 1·09 (95% CI 1·07-1·11; p=1·3 × 10(-17)) for ABCG1, 0·94 (0·92-0·95; p=4·2 × 10(-11)) for PHOSPHO1, 0·94 (0·92-0·96; p=1·4 × 10(-9)) for SOCS3, 1·07 (1·04-1·09; p=2·1 × 10(-10)) for SREBF1, and 0·92 (0·90-0·94; p=1·2 × 10(-17)) for TXNIP. A methylation score combining results for the five loci was associated with future type 2 diabetes incidence (relative risk quartile 4 vs quartile 1 3·51, 95% CI 2·79-4·42; p=1·3 × 10(-26)), and was independent of established risk factors. Methylation score was higher among Indian Asians than Europeans (p=1 × 10(-34)). INTERPRETATION DNA methylation might provide new insights into the pathways underlying type 2 diabetes and offer new opportunities for risk stratification and prevention of type 2 diabetes among Indian Asians. FUNDING The European Union, the UK National Institute for Health Research, the Wellcome Trust, the UK Medical Research Council, Action on Hearing Loss, the UK Biotechnology and Biological Sciences Research Council, the Oak Foundation, the Economic and Social Research Council, Helmholtz Zentrum Munchen, the German Research Center for Environmental Health, the German Federal Ministry of Education and Research, the German Center for Diabetes Research, the Munich Center for Health Sciences, the Ministry of Science and Research of the State of North Rhine-Westphalia, and the German Federal Ministry of Health.

EPIGENETIC EFFECTS OF SHIFTWORK ON BLOOD DNA METHYLATION

In the present study, the authors investigated the effects of shiftwork exposure on DNA methylation using peripheral blood DNA from subjects working in two chemical plants in Northern Italy. The investigation was designed to evaluate (a) DNA methylation changes in Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements as a surrogate of global methylation and (b) promoter methylation of glucocorticoid receptor (GCR), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ). One hundred and fifty white male workers (mean ± SD: 41.0 ± 9 yrs of age) were examined: 100 3 × 8 rotating shiftworkers (40.4 ± 8.7 yrs of age) and 50 day workers (42.2 ± 9.4 yrs of age). The authors used bisulfite-pyrosequencing to estimate repetitive elements and gene-specific methylation. Multiple regression analysis, adjusted for age, body mass index (BMI), and job seniority, did not show any significant association between the five DNA methylation markers and shiftwork. However, job seniority, in all subjects, was significantly associated with Alu (β = −0.019, p = .033) and IFN-γ (β = −0.224, p < .001) methylation, whereas TNF-α methylation was inversely correlated with age (β = −0.093, p < .001). Considering only shiftworkers, multiple regression analysis, adjusted for age, BMI, and job seniority, showed a significant difference between morning and evening types in TNF-α methylation (mean morning type [MT] 11.425 %5mC versus evening type [ET] 12.975 %5mC; β = 1.33, p = .022). No difference was observed between good and poor tolerance to shiftwork. Increasing job seniority (<5, 5–15, >15 yrs) was associated with significantly lower Alu (β = −0.86, p = .006) and IFN-γ methylation (β = −6.50, p = .007) after adjustment for age, BMI, and morningness/eveningness. In addition, GCR significantly increased with length of shiftwork (β = 3.33, p = .05). The data showed alterations in blood DNA methylation in a group of shiftworkers, including changes in Alu repetitive elements methylation and gene-specific methylation of IFN-γ and TNF-α promoters. Further studies are required to determine the role of such alterations in mediating the effects of shiftwork on human health. (Author correspondence: giovanni.costa@unimi.it)

Microvesicle-associated microRNA expression is altered upon particulate matter exposure in healthy workers and in A549 cells

Cardiovascular disease risk has been consistently linked with particulate matter (PM) exposure. Cell‐derived microvesicles (MVs) are released into plasma and transfer microRNAs (miRNAs) between tissues. MVs can be produced by the respiratory system in response to proinflammatory triggers, enter the circulatory system and remotely modify gene expression in cardiovascular tissues. However, whether PM affects MV signaling has never been investigated. In this study, we evaluated expression of microRNAs contained within plasma MVs upon PM exposure both in vivo and in vitro. In the in vivo study, we isolated plasma MVs from healthy steel plant workers before and after workplace PM exposure. We measured the expression of 88 MV‐associated miRNAs by real‐time polymerase chain reaction. To assess a possible source of the MV miRNAs identified in vivo, we measured their miRNA expression in PM‐treated A549 pulmonary cell lines in vitro. MiRNA profiling of plasma MVs showed 5.62‐ and 13.95‐fold increased expression of miR‐128 and miR‐302c, respectively, after 3 days of workplace PM exposure (P < 0.001). According to Ingenuity Pathway Analysis, miR‐128 is part of coronary artery disease pathways, and miR‐302c is part of coronary artery disease, cardiac hypertrophy and heart failure pathways. In vitro experiments confirmed a dose‐dependent expression of miR‐128 in MVs released from A549 cells after 6 h of PM treatment (P = 0.030). MiR‐302c was expressed neither from A549 cells nor in reference lung RNA. These results suggest novel PM‐activated molecular mechanisms that may mediate the effects of air pollution and could lead to the identification of new diagnostic and therapeutic interventions. Copyright

Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells

Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies.

Epigenetic and Transcriptional Modifications in Repetitive Elements in Petrol Station Workers Exposed to Benzene and MTBE

Benzene, a known human carcinogen, and methyl tert-butyl ether (MTBE), not classifiable as to its carcinogenicity, are fuel-related pollutants. This study investigated the effect of these chemicals on epigenetic and transcriptional alterations in DNA repetitive elements. In 89 petrol station workers and 90 non-occupationally exposed subjects the transcriptional activity of retrotransposons (LINE-1, Alu), the methylation on repeated-element DNA, and of H3K9 histone, were investigated in peripheral blood lymphocytes. Median work shift exposure to benzene and MTBE was 59 and 408 µg/m3 in petrol station workers, and 4 and 3.5 µg/m3, in controls. Urinary benzene (BEN-U), S-phenylmercapturic acid, and MTBE were significantly higher in workers than in controls, while trans,trans-muconic acid (tt-MA) was comparable between the two groups. Increased BEN-U was associated with increased Alu-Y and Alu-J expression; moreover, increased tt-MA was associated with increased Alu-Y and Alu-J and LINE-1 (L1)-5′UTR expression. Among repetitive element methylation, only L1-Pa5 was hypomethylated in petrol station workers compared to controls. While L1-Ta and Alu-YD6 methylation was not associated with benzene exposure, a negative association with urinary MTBE was observed. The methylation status of histone H3K9 was not associated with either benzene or MTBE exposure. Overall, these findings only partially support previous observations linking benzene exposure with global DNA hypomethylation.

Abstract 4817: Altered DNA methylation in human lung cell line A549 after treatment with a standard mixture of particulate matter

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BACKGROUND: Numerous health studies have shown acute and chronic particulate air pollution (PM, particulate matter) exposures to be associated with lung cancer, as well as with lung growth impairment, chronic obstructive pulmonary disease (COPD), and asthma. Toxicology studies have shown that PM is only a weak mutagen. Whether PM induces DNA methylation alterations, a primary mechanism in cell transformation, has never been studied in lung epithelial cells. In our study, we found a significative modification of P16 DNA methylation after PM treatement. AIM: To investigate PM on 8 cancer and inflammatory genes (TLR-4, CD14, IFNγ, TNFα, RASSF1A, APC, p53, p16) in A549 human alveolar basal epithelial cells. METHODS: A549 cells were treated with a complex standard mixture of particulate matter (NIST-National Institute of Standards and Technology- Standard Reference Material [SRM] 1648a). SRM 1648a treatments were performed with increasing doses of total mixture (15, 31, 62, 125, and 250 μg/ml) dissolved in colture medium. We used untreated cells as a negative control. Only concentrations that did not affect cell viability were considered. The cells were harvested after 2, 6, 12, 24, and 48 hours of continuous exposure, washed with phosphate buffered saline, and stored as cell pellet at -80°C until DNA extraction. DNA methylation was measured by means of bisulfite-Pyrosequencing. All the experiments were conducted in triplicate to confirm repeatability of our results. RESULTS: Using a t-test assuming that the two distributions have the same variance, we found an increase in p16 methylation for a dose of 15 μg/ml (p16 mean(untreated-treated)=0,83; p-value=0,05); an hypomethylation for a dose of 62 μg/ml (p16 mean(untreated-treated)=−0,77; p-value=0,004) as well as for a dose of 250 μg/ml (p16 mean(untreated-treated)=−0,78; p-value=0,01). We observed also an increase in APC methylation for a dose of 250 μg/ml (APC mean(untreated-treated)=2,90; p-value=0,02). CONCLUSION: This finding indicates that different levels of exposure to PM can induce changes in the DNA methylation levels in vitro. Our hypothesis is that exposure to PM can cause changes in the gene-specific DNA methylation levels thus enhancing cancerogenesis. Additional studies are required to better define the epigenetic mechanisms by which PM and similar environmental agents induce changes in DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2011-4817