Prenatal DEHP exposure predicts neurological disorders via transgenerational epigenetics

Abstract

Recent experimental and observational research has suggested that childhood allergic asthma and other conditions may be the result of prenatal exposure to environmental contaminants, such as di-(2-ethylhexyl) phthalate (DEHP). In a previous epidemiological study, we found that ancestral exposure (F0 generation) to endocrine disruptors or the common plasticizer DEHP promoted allergic airway inflammation via transgenerational transmission in mice from generation F1 to F4. In the current study, we employed a MethylationEPIC Beadchip microarray to examine global DNA methylation in the human placenta as a function of maternal exposure to DEHP during pregnancy. Interestingly, global DNA hypomethylation was observed in placental DNA following exposure to DEHP at high concentrations. Bioinformatic analysis confirmed that DNA methylation affected genes related to neurological disorders, such as autism and dementia. These results suggest that maternal exposure to DEHP may predispose offspring to neurological diseases. Given the small sample size in this study, the potential role of DNA methylation as a biomarker to assess the risk of these diseases deserves further investigation.

Figure 1

Infinium methylation microarray analysis of Methylation changes in DEHP-exposed human placenta samples. (A) The flowchart shows how the pipeline of raw data was processed. DNA methylation was performed using the high-resolution Infinium MethylationEPIC BeadChip Kit interrogating about 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution. By utilizing a cut-off p-value threshold of greater than 0.05 and an FDR of 10%, a total of 1254 probes displaying a minimum of 10% differential methylation were identified. (B) Density of DNA methylation level for 12 human placenta samples exposed to DEHP; blue lines: low DEHP exposure (n = 6), red lines: high DEHP exposure (n = 6). Individual probes with beta (β)-values (range 0–1) are approximate representations of the absolute methylation percentage of specific CpG sites within the sample population. Beta value = 1 indicates complete methylation; beta value = 0 represents no methylation. (C) Density of 10% differentially DNA methylated probes of all samples. Hypomethylation was observed in the DEHP high group (red line), as compared to DEHP low group (blue line). (D) Heatmap showing the 10% differentially DNA methylated probes; left panel: low DEHP exposure; right panel: high DEHP exposure. Heatmap showing differentially methylated cytosine (DMC) sites across the two different exposure dosages of DEHP. Most of the probes observed were hypomethylation in DEHP (red line: methylated, blue line: unmethylated).

Figure 2

Spatial distribution of differentially methylated CpGs (DMCs) and gene-centric annotations. (A) Violin plot of 1261 probes showing beta values of gene body (n = 386), 5’UTR (n = 30), intergenic region (n = 677), and promoters (n = 168). The violin shape shows the quantitative distribution of DNA methylation. Pink denotes the high DEHP group, while blue denotes the low DEHP group. The distribution of (B) hypomethylated sites and (C) hypermethylated sites in the gene body, 5’UTR, intergenic region, and promoters is shown in the pie chart.

Figure 3

Top 30% differential methylated CpG sites between low and high DEHP-exposed groups. (A) Seven hypermethylated sites and (B) five hypomethylated sites with 30% DMC in high (blue) and low (black) DEHP-exposed groups are shown.

Figure 4

Cytoscape and STRING pathway analysis for genes with differential methylated CpG sites of human placenta exposed to low and high levels of DEHP. Pathways that are enriched with (A) hypermethylated genes and (C) hypomethylated genes are shown. Cytoscape analysis of protein–protein interaction (PPI) found that (B) hypermethylated genes are related to the Wnt signaling pathway, while (D) hypomethylated genes are related to neural-related adhesion molecules. The colored box was enlarged in the right panel. These figures on protein–protein interaction (B,D) were generated by Cytoscape version 3.9.1 (https://cytoscape.org/index.html).

Figure 5

Histogram showing beta values of DMC probes located close to imprinting genes in high and low DEHP-exposed groups.