Ancestral environmental exposures to a variety of environmental toxicants and other factors have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The current study examined the potential transgenerational actions of the herbicide atrazine. Atrazine is one of the most commonly used herbicides in the agricultural industry, in particular with corn and soy crops. Outbred gestating female rats were transiently exposed to a vehicle control or atrazine. The F1 generation offspring were bred to generate the F2 generation and then the F2 generation bred to generate the F3 generation. The F1, F2 and F3 generation control and atrazine lineage rats were aged and various pathologies investigated. The male sperm were collected to investigate DNA methylation differences between the control and atrazine lineage sperm. The F1 generation offspring (directly exposed as a fetus) did not develop disease, but weighed less compared to controls. The F2 generation (grand-offspring) was found to have increased frequency of testis disease and mammary tumors in males and females, early onset puberty in males, and decreased body weight in females compared to controls. The transgenerational F3 generation rats were found to have increased frequency of testis disease, early onset puberty in females, behavioral alterations (motor hyperactivity) and a lean phenotype in males and females. The frequency of multiple diseases was significantly higher in the transgenerational F3 generation atrazine lineage males and females. The transgenerational transmission of disease requires germline (egg or sperm) epigenetic alterations. The sperm differential DNA methylation regions (DMRs), termed epimutations, induced by atrazine were identified in the F1, F2 and F3 generations. Gene associations with the DMRs were identified. For the transgenerational F3 generation sperm, unique sets of DMRs (epimutations) were found to be associated with the lean phenotype or testis disease. These DMRs provide potential biomarkers for transgenerational disease. The etiology of disease appears to be in part due to environmentally induced epigenetic transgenerational inheritance, and epigenetic biomarkers may facilitate the diagnosis of the ancestral exposure and disease susceptibility. Observations indicate that although atrazine does not promote disease in the directly exposed F1 generation, it does have the capacity to promote the epigenetic transgenerational inheritance of disease.

BackgroundEnvironmental toxicants such as DDT have been shown to induce the epigenetic transgenerational inheritance of disease (e.g., obesity) through the germline. The current study was designed to investigate the DDT-induced concurrent alterations of a number of different epigenetic processes including DNA methylation, non-coding RNA (ncRNA) and histone retention in sperm.MethodsGestating females were exposed transiently to DDT during fetal gonadal development, and then, the directly exposed F1 generation, the directly exposed germline F2 generation and the transgenerational F3 generation sperm were investigated.ResultsDNA methylation and ncRNA were altered in each generation sperm with the direct exposure F1 and F2 generations being predominantly distinct from the F3 generation epimutations. The piRNA and small tRNA were the most predominant classes of ncRNA altered. A highly conserved set of histone retention sites were found in the control lineage generations which was not significantly altered between generations, but a large number of new histone retention sites were found only in the transgenerational generation DDT lineage sperm.ConclusionsTherefore, all three different epigenetic processes were concurrently altered as DDT induced the epigenetic transgenerational inheritance of sperm epimutations. The direct exposure generations sperm epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene associations with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Observations demonstrate all three epigenetic processes are involved in transgenerational inheritance. The different epigenetic processes appear to be integrated in mediating the epigenetic transgenerational inheritance phenomenon.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0178-0) contains supplementary material, which is available to authorized users.

Epigenetic transgenerational inheritance of disease and phenotypic variation can be induced by several toxicants, such as vinclozolin. This phenomenon can involve DNA methylation, non-coding RNA (ncRNA) and histone retention, and/or modification in the germline (e.g. sperm). These different epigenetic marks are called epimutations and can transmit in part the transgenerational phenotypes. This study was designed to investigate the vinclozolin-induced concurrent alterations of a number of different epigenetic factors, including DNA methylation, ncRNA, and histone retention in rat sperm. Gestating females (F0 generation) were exposed transiently to vinclozolin during fetal gonadal development. The directly exposed F1 generation fetus, the directly exposed germline within the fetus that will generate the F2 generation, and the transgenerational F3 generation sperm were studied. DNA methylation and ncRNA were altered in each generation rat sperm with the direct exposure F1 and F2 generations being distinct from the F3 generation epimutations. Interestingly, an increased number of differential histone retention sites were found in the F3 generation vinclozolin sperm, but not in the F1 or F2 generations. All three different epimutation types were affected in the vinclozolin lineage transgenerational sperm (F3 generation). The direct exposure generations (F1 and F2) epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene pathways associated with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Our results show that the three different types of epimutations are involved and integrated in the mediation of the epigenetic transgenerational inheritance phenomenon.

Exposure to vinclozolin has been shown to induce the epigenetic transgenerational inheritance of increased susceptibility to disease, and to induce transgenerational changes to the epigenome. In the current study, gestating F0 generation rats were exposed to vinclozolin, and the subsequent F1, F2 and transgenerational F3 generations were evaluated for diseases and pathologies. F1 and F2 generation rats exhibited few abnormalities. However, F3 generation rats showed transgenerational increases in testis, prostate, and kidney disease, changes in the age of puberty onset in males, and an increased obesity rate in females. Overall there was an increase in the rate of animals with disease, and in the incidence of animals with multiple diseases. The objective of the current study was to analyze the sperm epigenome of F3 generation rats with specific abnormalities and compare them to rats without those abnormalities, in an effort to find epigenetic biomarkers of transgenerational disease. Unique signatures of differential DNA methylation regions (DMRs) in sperm were found that associated with testis disease, prostate disease and kidney disease. Confounding factors identified were the presence of multiple diseases in the analysis and the limited number of animals without disease. These results further our understanding of the mechanisms governing epigenetic transgenerational inheritance, and may lead in the future to the use of epigenetic biomarkers that will help predict an individual’s susceptibility for specific diseases.

Dichlorodiphenyltrichloroethane (DDT) has previously been shown to promote the epigenetic transgenerational inheritance of adult onset disease in rats. The current study investigated the potential that sperm epimutation biomarkers can be used to identify ancestral induced transgenerational obesity and associated pathologies. Gestating F0 generational female rats were transiently exposed to DDT during fetal gonadal sex determination, and the incidence of adult-onset pathologies was assessed in the subsequent F1, F2, and F3 generations. In addition, sperm differential DNA methylation regions (DMRs) that were associated with specific pathologies in the transgenerational F3 generation males were investigated. There was an increase of testis disease and early-onset puberty in the F2 generation DDT lineage males. The F3 generation males and females had significant increases in the incidence of obesity and multiple disease. The F3 generation DDT males also had significant increases in testis disease, prostate disease, and late onset puberty. The F3 generation DDT females had increases in ovarian and kidney disease. Epigenetic alterations of the germline are required for the transgenerational inheritance of pathology. Therefore, the F3 generation sperm was collected to examine DMRs for the ancestrally exposed DDT male population. Unique sets of DMRs were associated with late onset puberty, prostate disease, kidney disease, testis disease, obesity, and multiple disease pathologies. Gene associations with the DMR were also identified. The epigenetic DMR signatures identified for these pathologies provide potential biomarkers for transgenerationally inherited disease susceptibility.