Abstract
BackgroundEnvironmental factors during perinatal development may influence developmental plasticity and disease susceptibility via alterations to the epigenome. Developmental exposure to the endocrine active compound, bisphenol A (BPA), has previously been associated with altered methylation at candidate gene loci. Here, we undertake the first genome-wide characterization of DNA methylation profiles in the liver of murine offspring exposed perinatally to multiple doses of BPA through the maternal diet.ResultsUsing a tiered focusing approach, our strategy proceeds from unbiased broad DNA methylation analysis using methylation-based next generation sequencing technology to in-depth quantitative site-specific CpG methylation determination using the Sequenom EpiTYPER MassARRAY platform to profile liver DNA methylation patterns in offspring maternally exposed to BPA during gestation and lactation to doses ranging from 0 BPA/kg (Ctr), 50 μg BPA/kg (UG), or 50 mg BPA/kg (MG) diet (N = 4 per group). Genome-wide analyses indicate non-monotonic effects of DNA methylation patterns following perinatal exposure to BPA, corroborating previous studies using multiple doses of BPA with non-monotonic outcomes. We observed enrichment of regions of altered methylation (RAMs) within CpG island (CGI) shores, but little evidence of RAM enrichment in CGIs. An analysis of promoter regions identified several hundred novel BPA-associated methylation events, and methylation alterations in the Myh7b and Slc22a12 gene promoters were validated. Using the Comparative Toxicogenomics Database, a number of candidate genes that have previously been associated with BPA-related gene expression changes were identified, and gene set enrichment testing identified epigenetically dysregulated pathways involved in metabolism and stimulus response.ConclusionsIn this study, non-monotonic dose dependent alterations in DNA methylation among BPA-exposed mouse liver samples and their relevant pathways were identified and validated. The comprehensive methylome map presented here provides candidate loci underlying the role of early BPA exposure and later in life health and disease status.
Highlights
Environmental factors during perinatal development may influence developmental plasticity and disease susceptibility via alterations to the epigenome
Analysis pipeline and quality control for identifying differential methylation We used the MethylPlex- Generation Sequencing (M-NGS) platform to evaluate genome-wide alterations in DNA methylation following perinatal bisphenol A (BPA) exposure in mice, which requires minimal DNA input (~ 50 nanograms) and enriches methylated DNA using a cocktail of methylation-dependent restriction enzymes prior to deep sequencing (Figure 1)
This analysis provides us an estimate of the maximum false discovery rate (FDR) of 15.5% (108/696 * 100%) for our analysis presented below; the actual FDR may be much lower, if true autosomal differences in methylation exist between sexes
Summary
Environmental factors during perinatal development may influence developmental plasticity and disease susceptibility via alterations to the epigenome. An evaluation of circulating blood BPA levels in pregnant women in southeast Michigan indicated exposure levels between 0.5 μg/L and 22.3 μg/ L (mean 5.9 μg/L) [12], and our recent study of human fetal liver samples indicated that there is considerable exposure to BPA during pregnancy and that BPA in fetuses was in a unconjugated form not readily eliminated from the body [13].
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