Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations

Abstract

Environmental compounds are known to promote epigenetic transgenerational inheritance of adult onset disease in subsequent generations (F1-F3) following ancestral exposure during fetal gonadal sex determination. The current study was designed to determine if a mixture of plastic derived endocrine disruptor compounds bisphenol-A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) at two different doses promoted epigenetic transgenerational inheritance of adult onset disease and associated DNA methylation epimutations in sperm. Gestating F0 generation females were exposed to either the "plastics" or "lower dose plastics" mixture during embryonic days 8 to 14 of gonadal sex determination and the incidence of adult onset disease was evaluated in F1 and F3 generation rats. There were significant increases in the incidence of total disease/abnormalities in F1 and F3 generation male and female animals from plastics lineages. Pubertal abnormalities, testis disease, obesity, and ovarian disease (primary ovarian insufficiency and polycystic ovaries) were increased in the F3 generation animals. Kidney and prostate disease were only observed in the direct fetally exposed F1 generation plastic lineage animals. Analysis of the plastics lineage F3 generation sperm epigenome previously identified 197 differential DNA methylation regions (DMR) in gene promoters, termed epimutations. A number of these transgenerational DMR form a unique direct connection gene network and have previously been shown to correlate with the pathologies identified. Observations demonstrate that a mixture of plastic derived compounds, BPA and phthalates, can promote epigenetic transgenerational inheritance of adult onset disease. The sperm DMR provide potential epigenetic biomarkers for transgenerational disease and/or ancestral environmental exposures.

Figure 1. Adult-onset testis disease and prostate disease in males from control, plastics and lower dose

(LD) plastics (BPA, DEHP and DBP) lineages. Percentages of males with testis (panel A) or prostate disease (panel B) in F1 and F3 generations are presented. The actual number of diseased rats/total number of rats in each group are shown above the respective bar graphs (* P<0.05; *** P<0.001). Representative micrographs (Scale bar  = 200 μm) showing histopathology images of adult-onset transgenerational testis and prostate disease in plastics (panels D, and G) and lower dose (LD) plastics lineages (panels E and H) compared to F3 control lineage (panels C and F). Testis sections from F3 generation animals in plastics and lower dose (LD) plastics lineages showed histopathology including azoospermic and atretic seminiferous tubules, presence of vacuoles in basal regions of seminiferous tubules, sloughed cells in center of seminiferous tubule and lack of seminiferous tubule lumen (arrows). Prostate sections showed epithelial atrophy and hyperplastic ductular epithelium (arrows).

Figure 2. Adult-onset kidney disease in males or females from control, plastics and lower dose

(LD) plastics (BPA, DEHP and DBP) lineages. Percentages of females (panel A) and males (panel B) with kidney disease in F1 and F3 generations are presented. The actual number of diseased rats/total number of rats in each group are shown above the respective bar graphs (** P<0.01; *** P<0.001). Representative micrographs (Scale bar  = 200 μm) showing histopathology images of adult-onset transgenerational kidney disease in F3 generation plastics (panels D and G) and lower dose (LD) plastics lineages (panels E and H) compared to F3 control lineage (panels C and F). Kidney sections showed Bowman's capsule abnormality and proteinaceous fluid filled cysts (arrows).

Figure 3. Pubertal abnormalities, primordial follicle loss, polycystic ovary disease and tumor development from control, plastics, or lower dose

(LD) plastics (BPA, DEHP and DBP) lineages. Percentages of females (panel A) and males (panel B) with pubertal abnormalities, or those females with primordial follicle loss (panel C) or polycystic ovary disease (panel D), and tumor development in females (panel E) and males (panel F) in F1 and F3 generations are presented. The actual number of diseased rats/total number of rats in each group are shown above the respective bar graphs (* P<0.05; ** P<0.01; *** P<0.001).

Figure 4. Obesity developed in control, plastics, or lower dose

(LD) plastics (BPA, DEHP and DBP) lineages. Percentages of females (panel A) and males (panel B) with obesity in F1 and F3 generations are presented. The actual number of diseased rats/total number of rats in each group are shown above the respective bar graphs (* P<0.05). Abdominal fat deposition in F3 generation 1yr old rats from non-obese (C) and obese (D) animals. Pink colored fat deposition over most organs noted in panel B (arrows).

Figure 5. Adult-onset disease/abnormalities in rats from control, plastics, or lower dose

(LD) plastics (BPA, DEHP and DBP) lineages. Incidences of total female disease (panel A), total male disease (panel B), female multiple disease (panel C) and male multiple disease (panel D) in F1 and F3 generations are presented. The actual number of diseased rats/total number of rats in each group are shown above the respective bar graphs (* P<0.05; ** P<0.01; *** P<0.001).

Figure 6. Gene network analysis for differential DNA methylation regions

(DMR) associated genes in the F3 generation plastics lineage sperm. Chromosomal locations for transgenerational DMR detected with MeDIP-Chip are indicated with arrowheads. The chromosomal size and number are presented. There were 197 DMR in sperm DNA from F3 generation plastics lineage compared to control lineage.

Figure 7. The F3 generation plastics lineage sperm DMR associated gene functional categories.

The number of DMR associated genes correlating to a specific gene functional category is presented including those with unknown function and expressed sequence tags (EST).

Figure 8. Gene network analysis for differential DNA methylation regions

(DMR) associated genes in the F3 generation plastics lineage sperm. Direct connection (functional or binding) genes are shown according to their location in the cell. Genes not shown are not connected. Node shapes code: oval and circle – protein; diamond – ligand; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Arrows with plus sign show positive regulation/activation, arrows with minus sign – negative regulation/inhibition; gray arrows represent regulation, lilac – expression, purple – binding, green – promoter binding, and yellow – protein modification.

Figure 9. Genes with known links with obesity that correlate with F3 generation plastic lineage sperm DMR associated genes.

The correlated DMR associated genes with associations with obesity are presented. The DMR associated genes with indirect connections to the direct connection genes are also presented.