In silico epigenetics of metal exposure and subclinical atherosclerosis in middle aged men: pilot results from the Aragon Workers Health Study

Abstract

We explored the association of metal levels with subclinical atherosclerosis and epigenetic changes in relevant biological pathways. Whole blood DNA Infinium Methylation 450 K data were obtained from 23 of 73 middle age men without clinically evident cardiovascular disease (CVD) who participated in the Aragon Workers Health Study in 2009 (baseline visit) and had available baseline urinary metals and subclinical atherosclerosis measures obtained in 2010-2013 (follow-up visit). The median metal levels were 7.36 µg g^-1, 0.33 µg g^-1, 0.11 µg g^-1 and 0.07 µg g^-1, for arsenic (sum of inorganic and methylated species), cadmium, antimony and tungsten, respectively. Urine cadmium and tungsten were associated with femoral and carotid intima-media thickness, respectively (Pearson's r = 0.27; p = 0.03 in both cases). Among nearest genes to identified differentially methylated regions (DMRs), 46% of metal-DMR genes overlapped with atherosclerosis-DMR genes (p < 0.001). Pathway enrichment analysis of atherosclerosis-DMR genes showed a role in inflammatory, metabolic and transport pathways. In in silico protein-to-protein interaction networks among proteins encoded by 162 and 108 genes attributed to atherosclerosis- and metal-DMRs, respectively, with proteins known to have a role in atherosclerosis pathways, we observed hub proteins in the network associated with both atherosclerosis and metal-DMRs (e.g. SMAD3 and NOP56), and also hub proteins associated with metal-DMRs only but with relevant connections with atherosclerosis effectors (e.g. SSTR5, HDAC4, AP2A2, CXCL12 and SSTR4). Our integrative in silico analysis demonstrates the feasibility of identifying epigenomic regions linked to environmental exposures and potentially involved in relevant pathways for human diseases. While our results support the hypothesis that metal exposures can influence health due to epigenetic changes, larger studies are needed to confirm our pilot results.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Keywords: DNA methylation; cohort study; environmental metals; subclinical atherosclerosis.

Figure 1.

Summary of the in silico analysis and results obtained from DMRs identified in the AWHS-metal pilot study. (a) A subset of 73 AWHS participants had available preliminary metal biomarker and subclinical atherosclerosis data. Among these, 23 participants additionally had whole blood DNA Infinium Methylation 450 K data. We identified 407 and 303 DMRs with respect to subclinical atherosclerosis measures and metals, respectively (p-value area less than 0.05 as estimated by the bumphunter function in the R package ‘minfi’ [18]). Among these regions, 139 were in common for both DMR lists. The exact number of differentially methylated genes for each condition (subclinical atherosclerosis and metals) is shown in the Venn diagrams. (b) After excluding non-protein-coding genomic regions (e.g. non-coding RNAs (ncRNAs)), pathway enrichment analysis showed enriched categories related to inflammation and cellular transport. We subsequently evaluated protein interaction networks among genes encoding proteins with at least one connection with known atherosclerosis-effector proteins, first by ANNs and second by graphically displaying the global protein network. We observed hub proteins, which were directly or indirectly related to metals, pointing to the potential role of epigenetic mechanisms in explaining metal-related cardiovascular risk.

Figure 2.

Protein interaction network between protein attributed to metal and subclinical atherosclerosis-DMRs in the AWHS-pilot study and atherosclerosis-effector protein from public databases included in the BED.