Prenatal Arsenic Exposure and the Epigenome: Identifying Sites of 5-methylcytosine Alterations that Predict Functional Changes in Gene Expression in Newborn Cord Blood and Subsequent Birth Outcomes

Exposure to inorganic arsenic (iAs) at levels that exceed the World Health Organization’s (WHO) recommended limit of 10 µg/l currently impacts the health of individuals in countries around the globe (ATSDR, 2007; WHO, 2006). Chronic iAs exposure is of considerable concern as it is associated with the development of cancers, including the liver, lung, prostate, skin, and urinary bladder as well as other chronic diseases in adults (ATSDR, 2007). In addition to the health consequences from chronic exposure, in utero exposure to iAs is associated with detrimental health consequences in infancy including increased risk for infection and increased risk for both cancer and noncancer endpoints later in life (reviewed in Bailey et al. (2014)). The development of iAs-associated disease likely results from the concerted action of several mechanisms of toxicity including the alteration of protein function via direct binding to sulfhydryl groups as well as the generation of oxidative stress (Jomova et al., 2011). Oxidative stress, in turn, can damage cellular macromolecules such as proteins, lipids, and DNA (Jomova et al., 2011). Exposure to iAs has also been shown to alter the expression of genes involved in key biological pathways such as DNA repair (Andrew et al., 2003). Prenatal iAs exposure has been associated with altered gene expression in human cord blood leukocytes and various target tissues in rodents (Fry et al., 2007; Liu et al., 2004, 2006; Rager et al., 2014a). It is probable that these changes in gene expression are regulated, at least in part, by epigenetic alterations as supported by evidence of changes in genomic 5-methylcytosine patterns associated with prenatal arsenic exposure in human cord blood leukocytes (Kile et al., 2012); however, such a direct linkage has not before been carried out in the context of DNA methylation. During the DNA methylation process in mammals, a methyl group is enzymatically added to the 5′ position of cytosines mainly in the context of CpG dinucleotides (Smith et al., 2012). Alterations in DNA methylation across the genome can lead to changes in genomic integrity and the silencing or expression of genes or entire chromosomes (Smith et al., 2012). DNA methylation patterns are highly dynamic during embryonic development, and changes during this stage may lead to permanent reprogramming and lifelong effects (Tobi et al., 2009). Although it is generally accepted that CpG-based methylation can lead to a repression in gene expression, evidence shows these relationships are far more complex. For example, DNA methylation does not invariably lead to gene silencing, and in multiple cases it has been observed to result in increased expression or to have no relationship with gene expression (Bock et al., 2012; Boellmann et al., 2010; Dosunmu et al., 2012). Previously published research has demonstrated that both chronic exposure in adults as well as prenatal exposure to iAs is associated with DNA methylation changes in humans (Kile et al., 2012, 2014; Smeester et al., 2011). However, the relationship between altered levels of DNA methylation, gene expression, and health outcomes has not been previously examined in newborns exposed to arsenic in utero. To assess these relationships, we utilized samples and data obtained through the Biomarkers of Exposure to ARsenic (BEAR) prospective pregnancy cohort. This cohort includes women from Gomez Palacio, in the state of Durango, Mexico (Rager et al., 2014a). In this area, iAs in drinking water often exceeds 50 µg/l, and adverse health effects associated with iAs exposure, including skin lesions and diabetes mellitus, have been previously identified (as described in Rager et al. (2014a)). We recently assessed the impact of prenatal exposure to arsenic on genome-wide mRNA expression profiles in blood leukocytes of a nested set of newborns within the cohort (Rager et al., 2014a) and identified a role of microRNAs (miRNAs) as mediators of this response. In the present study, we expand upon this research to examine the relationship between DNA methylation levels and mRNA expression in a gene-specific manner. At baseline (ie, independent of iAs), the predicted negative correlation between DNA methylation and gene expression was generally observed. In the context of iAs-associated changes in DNA methylation, we demonstrate that DNA methylation at individual CpG sites and/or methylation averaged across CpG sites for a given gene was not necessarily predictive of gene expression change. A genome position-specific analysis was used to identify the sites that were most predictive of functional change. Furthermore, a subset of genes with altered DNA methylation levels was associated with gene expression as well as birth outcomes.