The current study demonstrates that dioxin (TCDD), an environmental toxicant and contaminant present in herbicides such as Agent Orange, can promote epigenetic transgenerational inheritance of diseases in unexposed progeny of the F0 generation females exposed during gestation. These observations extend previous studies in mice 
and rats 
by examining a variety of different disease states in 1-year-old rats. In addition, the epigenetic mechanism of transmission of this adult-onset disease was further examined by characterizing the transgenerational epigenetic changes in the F3 generation sperm. Epigenetic alterations in sperm DNA methylation (termed epimutations) in the F3 generation were observed after dioxin exposure of the F0 generation gestating female ancestors. This transgenerational transmission of adult onset diseases has implications of disease risk for not only the current exposed human and animal populations, but also for future generations 
. For example, Vietnam War Veterans exposed to the Agent Orange descendants may currently be experiencing an increased incidence of disease 
. The toxic effects of direct exposure to dioxin include acute liver damage, weight loss, thymic atrophy, immune suppression and chronic diseases, as well as lymphomas and leukemias in humans 
. The list of diseases seen following exposure of war veterans to Agent Orange (herbicide contaminated with dioxin) during the Vietnam era is growing 
. Similar observations have been made with the Taiwan 
, Seveso Italy 
, China 
and Japan exposures 
Due to the bioaccumulation of dioxin and up to decade long half-life in humans, any woman becoming pregnant even 20 years after dioxin exposure runs the risk of transmitting dioxin effects to her fetus and later generations. A generational study in the Seveso Italy exposed population supports this concept demonstrating health effects in progeny born 25 years following the exposure 
. Few studies have addressed this transgenerational aspect of dioxin exposure. The first animal study demonstrated transgenerational actions of dioxin on mouse fertility 
. Subsequently dioxin effects on F3 generation 120 day old rat disease was demonstrated 
. The current study investigated the adult onset disease in 1 year old F3 generation offspring of F0 generation ancestors exposed to dioxin.
This study did not use toxic doses of dioxin, but used only pharmacological doses based on 0.1% of the oral LD50 dose for dioxin. Therefore, no major toxic effects of dioxin were observed. However, the dose and route of administration used in the current study does not allow risk assessment of dioxin exposure. The objective of the study was to investigate if exposure to TCDD could promote epigenetic transgenerational inheritance of disease/abnormality phenotypes, and not to assess environmental risk of exposure to dioxin. These observations can now be used in future studies with appropriate modes of administration and doses to design more effective risk assessment analysis. However, the current study demonstrates the potential of dioxin to promote epigenetic transgenerational inheritance of disease.
In the current study, the transgenerational diseases/abnormalities observed includes kidney disease, ovary disease/abnormality, and pubertal abnormalities. Kidney disease incidence was higher in the transgenerational F3 generation dioxin lineage males. Chronic kidney disease in humans is correlated with high dioxin levels 
. Prenatal TCDD exposure has been shown to augment renal immune complex deposition, glomerulonephritis, and mesangial proliferation 
. Male rats exposed to TCDD have manifested nephrotoxicity shown by increases in serum creatinine and blood urea nitrogen levels, altered kidney histopathology, and renal oxidative stress 
. Lactational exposure of mice to TCDD caused hydronephrotic kidney 
. The current study is the first to report a transgenerational kidney histopathology in unexposed F3 generation male descendents of F0 generation gestating females exposed to dioxin.
As previously observed 
, the ovarian disease/abnormality identified included primordial follicle loss and polycystic ovarian disease in F3 generation dioxin lineage females. Currently the world’s human female population is facing an increased incidence of primary ovarian insufficiency, characterized by primordial follicle reserve loss, and an increased incidence of polycystic ovarian disease, characterized by the presence of anovulatory cysts 
. Similar to kidney disease, ovary disease phenotypes in the current study also appear to be the outcome of epigenetic transgenerational inheritance mechanisms. In animal studies, effects of dioxin exposure on ovarian function and steroid levels have been demonstrated. Dioxin exposure affects ovarian function 
and results in reduced ovarian weight and reduced numbers of corpora lutea and follicles 
. Further, dioxin causes reduced ovulation rate, failure of follicular rupture, morphologic changes in the ovary, and abnormal cyclicity with disruption of the estrous cycle 
. Dioxin slows follicular maturation 
. Ovarian tumors were induced by chronic TCDD exposures 
. A nonmonotonic dioxin dose-related association was found with risk of earlier menopause (loss of primordial follicle pool reserve) in a population of women residing near Seveso, Italy, in 1976, at the time of a chemical plant explosion 
. In the current study, the diseases of primordial follicle loss and polycystic ovarian disease found in the F3 generation support two previous reports 
of transgenerational ovarian diseases following ancestral exposure to dioxin. It is important to note that the polycystic ovarian disease was observed at an increased frequency in the transgenerational (F3) generation 
. Therefore, ancestral exposure of a gestating female to dioxin promotes an altered fetal gonadal development and epigenetic reprogramming of the male germline that then transmits the altered epigenome to subsequent generations to contribute to the development of these ovarian diseases transgenerationally. All cell types and tissues derived from the altered sperm epigenome have cell specific alterations in transcriptomes and epigenomes. Previous observations showed a transgenerational alteration in both the transcriptome and the epigenome of the ovarian granulosa cells from F3 generation rats of the vinclozolin lineage 
. Epigenetic mechanisms underlie the development of polycystic ovary syndrome in women 
and prenatally androgenized rhesus monkeys 
. These observations suggest an additional epigenetic paradigm be considered for the etiology of primary ovarian insufficiency and polycystic ovarian disease in women.
Pubertal abnormalities were increased only in female F3 generation animals of dioxin lineage. In an earlier study 
it was shown that F3 dioxin lineage females had an alteration of the time of pubertal onset (number of days to pubertal onset reduced). The current study assessed the number of animals with pubertal abnormalities using a time of puberty cutoff of mean of control lineage ±2 standard deviations and reports an increased percent incidence of pubertal abnormalities in F3 generation dioxin lineage females. The current study investigated pubertal abnormalities in part due to the dramatic increase in pubertal abnormalities over the past decades in humans 
. The early and delayed onset of puberty are forerunners to different adult health consequences. For example, early onset of puberty results in accelerated bone mineralization and reduced adult height in girls, as well as susceptibility to breast tumors 
. The delayed onset of puberty leads to reduced bone mineralization, psychological stress and metabolic problems 
. In the current study, equal proportions of F1 generation females of dioxin lineage had early and delayed pubertal onset, while males had increased proportion of delayed pubertal onset, indicating sexually dimorphic and different direct-exposure effects. Previously, perinatal exposure to a low dose of dioxin induced only precocious puberty that included early maturation of the hypothalamic-pituitary axis, the gonads and genitals, in female Long-Evans hooded rats 
. In the current study, the affected F3 generation dioxin lineage females had early onset of puberty, while the affected males showed delayed onset of puberty, indicating sex-specific and different transgenerational effects. In this study pubertal onset in dioxin lineage rats is an example where direct and transgenerational effects are very different. Previously, early onset of puberty in girls has been suggested to be due to environmental exposures to endocrine disruptors 
. Dioxin exposure is suggested to cause early onset of menarche in girls 
. Early onset of puberty in girls disrupts brain development, endocrine organ systems and growth leading to susceptibility to disease. It is interesting to note that puberty onset (an early developmental milestone) in this study is associated with epigenetic transgenerational adult onset ovarian disease in F3 generation females.
The molecular mechanism of epigenetic transgenerational inheritance of phenotypes involves the reprogramming of the germline epigenome during male gonadal sex determination 
. The modified sperm epigenome (DNA methylation) appears to be permanently reprogrammed similar to an imprinted-like site and is protected from DNA demethylation and reprogramming after fertilization and in the following generations. This allows transgenerational transmission of the modified sperm epimutations to then modify all somatic cell and tissue epigenomes and transcriptomes to promote epigenetic transgenerational inheritance of disease phenotypes. The current study further analyzed the altered sperm epigenome and epimutations induced by ancestral dioxin exposure. Transgenerational alterations in an F3 generation sperm epigenome were initially identified following developmental exposure to vinclozolin 
. A recent study demonstrated a variety of different environmental toxicants induce exposure specific differentially DNA methylated regions (DMR), defined as epimutations and epigenetic biomarkers, which included dioxin lineage F3 generation sperm epimutations 
. The list of DMR associated genes from F3 generation sperm dioxin lineage is presented in Table S4
. Therefore, the sperm epimutations correlated with the epigenetic transgenerational inheritance of the disease phenotypes documented.
Transgenerational diseases are promoted by many environmental compounds 
. Vinclozolin exposure resulted in F3 generation testis disease, prostate disease, kidney disease, immune system abnormalities, tumors, uterine hemorrhage during pregnancy and polycystic ovarian disease 
. Further, changes in the methylation patterns of imprinted genes in sperm of F3 generation male mice were found following vinclozolin exposure 
. Exposure of F0 generation gestating rats to Bisphenol-A caused decreased fertility in F3 generation males 
. Environmental factors such as nutrition 
also can promote epigenetic transgenerational inheritance of disease phenotypes. Demonstration of epigenetic transgenerational inheritance in worms 
, flies 
, plants 
and mammals 
suggest this phenomenon will likely be critical in biology and disease etiology 
. Together these observations demonstrate that exposure of gestating females to dioxin during gonadal sex determination promotes epigenetic transgenerational inheritance of adult-onset disease including kidney disease, ovary disease/abnormality and pubertal onset abnormalities. The overall increase in total and multiple diseases/abnormalities in F3 generation are also considerable. Associated with the occurrence of these transgenerational diseases are the epigenetic changes in rat sperm DNA. These epimutations may be useful as early stage biomarkers of compound exposure and adult onset disease. Although not designed for risk assessment, these results have implications for the human populations that are exposed to dioxin and are experiencing declines in fertility and increases in adult onset disease, with a potential to transmit them to later generations.