Ancestral dichlorodiphenyltrichloroethane (DDT) exposure promotes epigenetic transgenerational inheritance of obesity

Abstract

Background: Ancestral environmental exposures to a variety of environmental factors and toxicants have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The present work examined the potential transgenerational actions of the insecticide dichlorodiphenyltrichloroethane (DDT) on obesity and associated disease.

Methods: Outbred gestating female rats were transiently exposed to a vehicle control or DDT and the F1 generation offspring bred to generate the F2 generation and F2 generation bred to generate the F3 generation. The F1 and F3 generation control and DDT lineage rats were aged and various pathologies investigated. The F3 generation male sperm were collected to investigate methylation between the control and DDT lineage male sperm.

Results: The F1 generation offspring (directly exposed as a fetus) derived from the F0 generation exposed gestating female rats were not found to develop obesity. The F1 generation DDT lineage animals did develop kidney disease, prostate disease, ovary disease and tumor development as adults. Interestingly, the F3 generation (great grand-offspring) had over 50% of males and females develop obesity. Several transgenerational diseases previously shown to be associated with metabolic syndrome and obesity were observed in the testis, ovary and kidney. The transgenerational transmission of disease was through both female (egg) and male (sperm) germlines. F3 generation sperm epimutations, differential DNA methylation regions (DMR), induced by DDT were identified. A number of the genes associated with the DMR have previously been shown to be associated with obesity.

Conclusions: Observations indicate ancestral exposure to DDT can promote obesity and associated disease transgenerationally. The etiology of disease such as obesity may be in part due to environmentally induced epigenetic transgenerational inheritance.

Figure 1

Transgenerational obesity. Percentages of females (a) or males (b) with obesity in the F1 and F3 generations. The dichlorodiphenyltrichloroethane (DDT) or lower dose (LD) DDT is indicated. The number of diseased rats/total number of rats (n value) is shown above the respective bar graphs. A logistic regression analysis was performed and P values indicate a significant difference from controls (*P <0.05; **P <0.01; ***P <0.001). The representative abdominal adiposity is shown for non-obese (c) and obese (d) rats, with the pink fat tissue deposition indicated (arrows) around the liver, intestines, subcutaneous and epididymis in the obese animal.

Figure 2

Obesity and associated disease. The major diseases previously shown to be associated with obesity are indicated with those identified in the present study shown with a red box.

Figure 3

Transgenerational obesity and associated disease. Percentages of the F1 and F3 generation disease/abnormalities from control (open bars), dichlorodiphenyltrichloroethane (DDT) (black bars) and lower dose (LD) DDT lineages. Testis disease (a), spermatogenic cell apoptosis (b) and sperm counts (c), ovarian disease (d), polycystic ovarian disease (e) and primordial follicle pool loss (f) are presented. Percentages of females (g) and males (h) with kidney diseases and percentages of females (I) and males (J) with incidence of multiple disease. The number of diseased rats/total number of rats (n value) is shown above the respective bar graphs. Those showing numbers above the bars were analyzed with a logistic regression analysis and those with a mean???SEM indicated were analyzed with a t test with the P value indicated (*P <0.05; **P <0.01; ***P <0.001) (Additional file 4: Table S2 and Additional file 5: Table S3).

Figure 4

Transgenerational disease in F4 generation outcross or reverse outcross offspring for both male and female germline transmission. Incidences of obesity in females (a) and in males (b), ovary disease (c), testis disease (d), kidney disease in females (e), in males (f), of the F4 generation outcross (OC) (F3 dichlorodiphenyltrichloroethane (DDT) lineage male cross with wild-type female) or reverse outcross (ROC) (F3 DDT lineage female cross with wild-type male) offspring of the control, DDT, and lower dose (LD) DDT F3 generation lineages. The number of diseased rats/total number of rats (n value) in each lineage are shown above the bar. A logistic regression analysis with P value compared to control is indicated (*P <0.05; **P <0.01; ***P <0.001).

Figure 5

Transgenerational F3 generation sperm epimutations. (a) Chromosomal locations for differential DNA methylated regions (DMR) in sperm DNA from F3 generation dichlorodiphenyltrichloroethane (DDT) lineage rats compared to control lineage (arrowheads). The high stringency intersection epimutations are identified as red arrows, which is a subset of the less stringent ?average? DMR indicated with open arrows. The chromosomal size and number are listed (Additional file 8: Table S6 and Additional file: 9 Table S7). (b) A Venn diagram of DMR from various F3 generation exposure lineages including: vinclozolin, plastics, pesticides, hydrocarbons and DDT. The total number of DMR per exposure lineage in brackets is presented and unique and overlapping DMR identified. (c) The CpG/100 bp is presented and the corresponding number of DMR associated. The density is presented as the number of CpG for 100 bp of the DMR.

Figure 6

Transgenerational F3 generation sperm epimutation-associated gene network. (a) The gene network associated with the differential DNA methylation regions (epimutations) with cellular location indicated. The green labeled genes are those in the high stringency intersection set of 38 differential DNA methylated region (DMR) associated genes. (b) The genes that have a known link to obesity and polycystic ovarian disease that are associated with dichlorodiphenyltrichloroethane (DDT) induced sperm DMRgenes are presented.