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. 2019 May;127(5):57011.
doi: 10.1289/EHP3849. Epub 2019 May 28.

Association of Arsenic Exposure with Whole Blood DNA Methylation: An Epigenome-Wide Study of Bangladeshi Adults

Kathryn Demanelis  1 , Maria Argos  2 , Lin Tong  1 , Justin Shinkle  1 , Mekala Sabarinathan  1 , Muhammad Rakibuz-Zaman  3 , Golam Sarwar  3 , Hasan Shahriar  3 , Tariqul Islam  3 , Mahfuzar Rahman  3   4 , Mohammad Yunus  5 , Joseph H Graziano  6 , Karin Broberg  7 , Karin Engström  8 , Farzana Jasmine  1 , Habibul Ahsan  1   9   10   11 , Brandon L Pierce  1   9   10
Affiliations

Association of Arsenic Exposure with Whole Blood DNA Methylation: An Epigenome-Wide Study of Bangladeshi Adults

Kathryn Demanelis et al. Environ Health Perspect. 2019 May.

Abstract

Background: Arsenic exposure affects [Formula: see text] people worldwide, including [Formula: see text] in Bangladesh. Arsenic exposure increases the risk of cancer and other chronic diseases, and one potential mechanism of arsenic toxicity is epigenetic dysregulation.

Objective: We assessed associations between arsenic exposure and genome-wide DNA methylation measured at baseline among 396 Bangladeshi adults participating in the Health Effects of Arsenic Longitudinal Study (HEALS) who were exposed by drinking naturally contaminated well water.

Methods: Methylation in whole blood DNA was measured at [Formula: see text] using the Illumina InfiniumMethylationEPIC (EPIC) array. To assess associations between arsenic exposure and CpG methylation, we used linear regression models adjusted for covariates and surrogate variables (SVs) (capturing unknown technical and biologic factors). We attempted replication and conducted a meta-analysis using an independent dataset of [Formula: see text] from 400 Bangladeshi individuals with arsenical skin lesions.

Results: We identified 34 CpGs associated with [Formula: see text] creatinine-adjusted urinary arsenic [[Formula: see text]]. Sixteen of these CpGs annotated to the [Formula: see text] array, and 10 associations were replicated ([Formula: see text]). The top two CpGs annotated upstream of the ABR gene (cg01912040, cg10003262 ). All urinary arsenic-associated CpGs were also associated with arsenic concentration measured in drinking water ([Formula: see text]). Meta-analysis ([Formula: see text] samples) identified 221 urinary arsenic-associated CpGs ([Formula: see text]). The arsenic-associated CpGs from the meta-analysis were enriched in non-CpG islands and shores ([Formula: see text]) and depleted in promoter regions ([Formula: see text]). Among the arsenic-associated CpGs ([Formula: see text]), we observed significant enrichment of genes annotating to the reactive oxygen species pathway, inflammatory response, and tumor necrosis factor [Formula: see text] ([Formula: see text]) signaling via nuclear factor kappa-B ([Formula: see text]) hallmarks ([Formula: see text]).

Conclusions: The novel and replicable associations between arsenic exposure and DNA methylation at specific CpGs observed in this work suggest that epigenetic alterations should be further investigated as potential mediators in arsenic toxicity and as biomarkers of exposure and effect in exposed populations. https://doi.org/10.1289/EHP3849.

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Figures

Figure 1A is a Manhattan plot plotting negative log sub 10 (y-axis), ranging between 0 and 12, across chromosome (x-axis), ranging between 1 and 21. Figure 1B is a Volcano plot plotting negative log sub 10 (y-axis), ranging between 0 and 12, across log sub 2 HEALS Urinary Arsenic values, ranging between negative 0.02 and 0.02 micrograms per gram beta value. The relation to island key is as follows: island, non-CpG island, shelf, and shore.
Figure 1.
Genome-wide associations between urinary arsenic concentration and CpG site–specific methylation in the Health Effects of Arsenic Longitudinal Study (HEALS). Using the EPIC (850K) array, the association between log2-transformed urinary arsenic (creatinine adjusted) and methylation was evaluated at 771,192 CpG sites across 396 individuals from HEALS. (A) Manhattan plot of the chromosomal location and p-value for each CpG–arsenic association. (B) Volcano plot presenting association estimate and p-value for each CpG–arsenic association. Colors correspond to CpG relationship to island. In (A) and (B), solid and dashed lines designate the Bonferroni threshold (p=6.5×10−8) and false discovery rate (FDR) 0.05 threshold (p=2.0×10−6), respectively.
Figure 2 is a graph plotting negative log sub 10 values for validation subcohort (BEST) (y-axis), ranging between 0 and 8, across negative log sub 10 values for discovery subcohort (HEALS) (x-axis), ranging between 6 and 12, for direction of association comprising negative, inconsistent, and positive in both cohorts.
Figure 2.
Associations between urinary arsenic and CpG methylation discovered in the Health Effects of Arsenic Longitudinal Study (HEALS) and tested for replication in the Bangladesh Vitamin E and Selenium Trial (BEST). Using results from BEST, we attempted to validate 26 arsenic-associated CpGs that annotated to 450K array among the 67 CpGs identified using the EPIC array in HEALS (p<10−5). The BEST results consisted of associations between log2-transformed urinary arsenic and methylation evaluated for 390,810 CpGs using data on 400 BEST participants with existing 450K array data. Horizontal dashed line represents − log10(p=0.05), and solid vertical line corresponds to − log10[false discovery rate  (FDR)<0.05]. Direction of association summarizes whether results from the cohorts are both positive (triangle), both negative (square), or inconsistent (directions of association differ) (circle). The corresponding results data are presented in Table 2 and Excel Table S1.
Figure 3 is a scatterplot plotting direction of effect plus or minus times negative log sub 10 for HEALS water arsenic group (y-axis), ranging between negative 14 and 10, across direction of effect plus or minus times negative log sub 10 for HEALS urinary Arsenic group (x-axis), ranging between negative 14 and 10.
Figure 3.
Comparison between the associations of urinary arsenic and water arsenic with genome-wide methylation in the Health Effects of Arsenic Longitudinal Study (HEALS). The direction of association and − log10(p) for each CpG–arsenic association are plotted for urinary (x-axis) and water (y-axis) (both log2  transformed). Solid lines and dashed lines designate − log10(p=1) and − log10(p=0.05), respectively. CpGs highlighted in blue diamonds denote log2-transformed urinary arsenic–associated sites [false discovery rate  (FDR)<0.05]. Pearson’s correlation between all urinary and water arsenic CpG associations was 0.67 (p<10−16).
Figure 4A is a Manhattan plot plotting log sub 10 values (minimum FDR values) (y-axis) across chromosomes (x-axis). Figures 4B, 4C, and 4D are coMET plots of Chromosomes 17, 10, and 9, respectively.
Figure 4.
Differentially methylated regions (DMRs) associated with log2-transformed urinary arsenic in the Health Effects of Arsenic Longitudinal Study (HEALS). Using 771,192 CpGs from the EPIC array, DMRs were identified using DMRcate (λ=1,000 base pairs and C=2). (A) Manhattan plot of the minimum false discovery rate (minFDR) for the CpG within all regions identified across EPIC array (no significance threshold set) (n=108,086 regions). Solid and dashed lines designate minFDR of 10−4 and minFDR of 0.05, respectively. (B–D) coMET plots of top three DMRs associated with urinary arsenic. Genomic annotations to gene, CpG islands, chromatin regulation, and DNase clusters are shown below each plot and are from UCSC CpG Island, UCSC DNase Cluster, and the Broad UCSC ChromatinHMM tracks (http://genome.ucsc.edu/, see coMET software documentation for further information and color coding) (Martin et al. 2015).
Figure 5A comprises two graphs, namely, Discovery (EPIC) and Meta-analysis (450 K), plotting proportion of CpG Sites (y-axis), ranging between 0.00 and 1.00, across EPIC Array and FDR less than 0.05 and 450 K array and FDR less than 0.05 (x-axis), respectively, for relation to island comprising shelf, shore, island, and non-CpG island. Figure 5B is a graph plotting gene, promoter, enhancer, TFBS region, and DHS region (y-axis) across fold enrichment or depletion (x-axis), ranging between 0.2 and 2.5, for analysis comprising discovery and meta-analysis.
Figure 5.
Enrichment of arsenic-associated CpGs among genomic features at false discovery rate  (FDR)  of  0.05. (A) Locational (with relation to island) distribution of log2-transformed arsenic-associated CpGs (FDR<0.05) from the Health Effects of Arsenic Longitudinal Study (HEALS) discovery analysis on EPIC array (n=34 CpGs) and meta-analysis of HEALS and the Bangladesh Vitamin E and Selenium Trial (BEST) (n=221 CpGs) compared with distribution of CpGs on entire EPIC (n=771,158) and 450K (n=390,589) array, respectively. Colors correspond to CpG relationship to island. (B) Fold enrichment (FE) is plotted for each genomic feature comparing urinary arsenic–associated CpGs below FDR of 0.05 to the remaining CpGs. CpGs annotating to a promoter region were defined as those annotating to TSS200 or TSS1500. Abbreviations correspond to transcription factor binding site (TFBS) and DNase I hypersensitive site (DHS). P-values were obtained from X2-test comparing distribution of CpGs above and below FDR of 0.05 for each genomic feature [p<0.05 (*), p<10−3 (**), and p<10−6 (***)].
Figure 6a is a QQ plot plotting observed negative log sub 10, ranging between 0 and 16, across expected negative log sub 10 (x-axis), ranging between 0 and 16. Figure 6b is a Manhattan plot plotting negative log sub 10 (y-axis), ranging between 0 and 12, across chromosome (x-axis), ranging between 1 and 21.
Figure 6.
Epigenome-wide meta-analysis of associations between urinary arsenic and DNA methylation in Bangladeshi adults using data from the Health Effects of Arsenic Longitudinal Study (HEALS) and the Bangladesh Vitamin E and Selenium Trial (BEST). Using data from EPIC (HEALS; n=396) and 450K (BEST; n=400) arrays, 390,810 CpG sites were meta-analyzed using METAL [summary statistics from covariate- and surrogate variable (SV)–adjusted model were provided as input]. (A) Q-Q plot of the observed distribution of meta-analysis p-values. The red line represents expected distribution of p-values under the null, and λ corresponds to the genomic inflation factor (see “Methods” section). (B) Manhattan plot of the location of each CpG and p-value for each CpG–arsenic association. Solid and dashed lines designate the Bonferroni threshold (p=1.3×10−7) and false discovery rate (FDR) 0.05 threshold (p=2.8×10−5), respectively.
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