Pesticide methoxychlor promotes the epigenetic transgenerational inheritance of adult-onset disease through the female germline

Abstract

Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

Figure 1. Ancestral exposure to methoxychlor and transgenerational kidney disease.

Percentages of females (panel A, C, E) and males (panel B, D, F) with kidney diseases in the F1 and F3 generation control (open bars) and methoxychlor (black bars) lineages. The number of diseased rats / total number of rats in each lineage are also shown (* P<0.05; ** P<0.01).

Figure 2. Ancestral exposure to methoxychlor and transgenerational pubertal abnormalities, and ovarian and uterine diseases.

Percentages of females F1 and F3 generation (panel A) and males (panel B) with pubertal abnormality or females with ovarian disease (panel C) or uterine infection (panel D). Percentages of the F1 and F3 generation females with primordial follicle loss (panel E) and polycystic ovary disease (panel F) in control and methoxychlor lineages. The number of diseased rats / total number of rats in each lineage are also shown. (** P<0.01; *** P<0.001).

Figure 3. Ancestral exposure to methoxychlor and transgenerational tumor development and transgenerational obesity.

Percentages of F1 and F3 generation females (panel A) or males (panel B) with tumor development and percentages of females (panel C) or males (panel D) with obesity. Representative abdominal adiposity for obese (panel E) animals is presented with arrows indicating the excessive dorsal abdominal and retro-peritoneal adiposity distribution. The number of diseased rats / total number of rats is shown above the respective bar graphs (** P<0.01).

Figure 4. Ancestral exposure to methoxychlor and adult-onset transgenerational disease in rats.

Incidences of F1 and F3 generation total female disease (panel A), total male disease (panel B), female multiple disease (panel C) and male multiple disease (panel D) in control and methoxychlor lineages. The number of diseased rats / total number of rats is shown above the respective bar graphs (* P<0.05; ** P<0.01; *** P<0.001).

Figure 5. Ancestral exposure to methoxychlor promoted adult-onset transgenerational diseases in F4 generation reverse outcross offspring showing female germline transmission.

Incidences of kidney disease in females (panel A), in males (panel B), obesity in females (panel C) and in males (panel D) of the F4 generation outcross (OC) or reverse outcross (ROC) offspring of the control and methoxychlor lineages.

Figure 6. Ancestral exposure to methoxychlor promoted adult-onset transgenerational diseases in F4 generation reverse outcross offspring increasing total female disease, total male disease and multiple male disease incidences.

Incidences of total disease in females (panel A), in males (panel B), multiple female disease (panel C), and multiple male disease (panel D) in the F4 generation outcross (OC) or reverse outcross (ROC) offspring of the control and methoxychlor lineages.

Figure 7. Ancestral exposure to methoxychlor and transgenerational epigenetic changes and induced sperm intersection epimutations.

Chromosomal locations for regions with transgenerational change in DNA methylation (arrowheads). There were 37 differentially methylated regions in sperm DNA from methoxychlor lineage rats compared to control lineage rats for all three experiments.

Figure 8. Ancestral exposure to methoxychlor and transgenerational epigenetic changes and induced sperm average epimutations.

Chromosomal locations for regions with transgenerational change in DNA methylation (arrowheads). There were 311 differentially methylated regions in sperm DNA from methoxychlor lineage rats compared to control lineage rats for all experiments. The box under the line at each chromosome represents a statistically significant over-representation (cluster) of epimutations.

Figure 9. Exposure specific transgenerational sperm epimutations in methoxychlor, vinclozolin, DDT, pesticide (DEET and permethrin) and plastics (BPA and phthalates).

A Venn diagram indicating the total number of intersection epimutations (A) and average epimutations (B) involving overlap between the various epimutation exposure data sets. Total number of epimutations for each exposure are in brackets next to label.