Perinatal Lead Exposure Promotes Sex-Specific Epigenetic Programming of Disease-Relevant Pathways in Mouse Heart

Abstract

Environmental contaminants such as the metal lead (Pb) are associated with cardiovascular disease, but the underlying molecular mechanisms are poorly understood. In particular, little is known about how exposure to Pb during early development impacts the cardiac epigenome at any point across the life course and potential differences between sexes. In a mouse model of human-relevant perinatal exposures, we utilized RNA-seq and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to investigate the effects of Pb exposure during gestation and lactation on gene expression and DNA methylation, respectively, in the hearts of male and female mice at weaning. For ERRBS, we identified differentially methylated CpGs (DMCs) or differentially methylated 1000 bp regions (DMRs) based on a minimum absolute change in methylation of 10% and an FDR < 0.05. For gene expression data, an FDR < 0.05 was considered significant. No individual genes met the FDR cutoff for gene expression; however, we found that Pb exposure leads to significant changes in the expression of gene pathways relevant to cardiovascular development and disease. We further found that Pb promotes sex-specific changes in DNA methylation at hundreds of gene loci (280 DMCs and 99 DMRs in males, 189 DMCs and 121 DMRs in females), and pathway analysis revealed that these CpGs and regions collectively function in embryonic development. In males, differential methylation also occurred at genes related to immune function and metabolism. We then investigated whether genes exhibiting differential methylation at weaning were also differentially methylated in hearts from a cohort of Pb-exposed mice at adulthood. We found that a single gene, Galnt2, showed differential methylation in both sexes and time points. In a human cohort investigating the influence of prenatal Pb exposure on the epigenome, we also observed an inverse association between first trimester Pb concentrations and adolescent blood leukocyte DNA methylation at a locus in GALNT2, suggesting that this gene may represent a biomarker of Pb exposure across species. Together, these data, across two time points in mice and in a human birth cohort study, collectively demonstrate that Pb exposure promotes sex-specific programming of the cardiac epigenome, and provide potential mechanistic insight into how Pb causes cardiovascular disease.

Keywords: DNA methylation; Developmental Origins of Health and Disease (DOHaD); cardiovascular disease; epigenetic; heart; sex differences; toxicoepigenetics.

Figure 1

Treatment paradigm for developmental exposure to Pb. Dam exposure began 2 weeks prior to mating and continued through gestation and lactation. Animals were sacrificed at weaning on postnatal day 21. Pb exposure occurred via drinking water, which was administered ad libitum. Whole hearts were harvested and snap-frozen in liquid nitrogen prior to extraction of DNA and RNA.

Figure 2

Heart weights as a percentage of body weight (A,B) and total body weights (C,D) for male and female offspring at sacrifice. N = 7 animals per group. *** p < 0.001. Linear mixed effects regression, with litter-specific random effects to account for within-litter correlation, was used to determine statistical significance.

Figure 3

RNA-seq data in weanling mouse hearts. (A,B) Results of KEGG pathway analysis of RNA-seq data, showing the top 15 most significant pathways for males (A) and females (B). Pathways are listed in order of decreasing significance from top to bottom (i.e., smallest to largest FDR). Blue and red arrows indicate reduced and increased expression of the pathway, respectively. (C) Scatter plot of all genes differentially expressed (DE) with Pb exposure with a p-value < 0.01, depicting correlations in log2 fold change with Pb exposure between males and females. Pink, orange and blue dots depict DE genes with a p-value <0.01 in males, females, or both, respectively. The p-value for correlation between males and females was determined using Pearson’s product-moment correlation.

Figure 4

ERRBS in weanling offspring mouse hearts. (A) Numbers of differentially hyper- and hypomethylated cytosines (DMCs) and regions (DMRs) in male and female hearts. (B,C) Volcano plots depicting DMCs in males and females. CpGs with FDR <0.05 and at least 10% absolute change in DNA methylation were considered significant. (D,E) Annotation summary plots depicting the total number of CpGs tested in pink, hypermethylated DMCs in green, and hypomethylated DMCs in blue for each genomic annotation using the R annotatr package for males (D) and females (E).

Figure 5

Sex specificity of Pb-induced changes in DNA methylation. (A,B) Venn diagrams depicting overlap between males and females in DMC-associated genes (A) or DMR-associated genes (B). p-values for overlap between the two groups calculated using a hypergeometric test. Overlap between sexes in DMC-associated genes was significant. (C,D) Polyenrich pathway analysis for DMCs in males (C) and females (D).

Figure 6

Comparison of Pb-induced DNA methylation changes between weaning and 5 months of age. (A,B) Venn diagrams depicting overlap in DMC- and DMR-associated genes between cohorts sacrificed at weaning and 5 months of age in males (A) and females (B). (C,D) bar plots depicting the direction of methylation change in DMRs, comparing the weaning and 5-month cohorts. Galnt2 and Adcy5 had 2 DMRs at 5 months of age. (E) First-trimester blood Pb levels (BLL) vs. GALNT2 methylation in adolescent blood leukocyte DNA. One CpG site annotated to an intron of GALNT2 was statistically significantly associated with Pb when adjusting for gender, batch, and blood cell composition (p = 0.00009). Red points and line: Female subjects and regression line. Blue points and line: Male subjects and regression line. Black line: Regression line for both sexes combined.