Session 5 Abstracts

Friday, December 4 (9am-1.00 Pacific / noon-4.00pm Eastern / 6-10.00pm Paris)

HERITABLE IMPACTS OF ENDOCRINE DISRUPTING COMPOUNDS

Sex Differences in Inter- and Transgenerational Effects of EDCs on the Developing Brain
• Andrea Gore, Ross Gillette, Krittika Krishnan, Lindsay M. Thompson, Mandee Bell, and David Crews
Division of Pharmacology & Toxicology, The University of Texas at Austin, Austin, TX 78712

Environmental endocrine-disrupting chemicals (EDCs) are exogenous chemicals that perturb hormones and their actions in exposed individuals and their descendants. Our laboratory has been investigating effects of two EDC classes: a PCB mix (weakly estrogenic) and vinclozolin (anti-androgenic), focusing on neurobiological and behavioral outcomes across generations in a rat model. This talk will present data on how developmental EDC exposures have sex- and lineage- (maternal or paternal) specific effects on the brains of F1, F2, and F3 generations. We will then demonstrate that a second “hit” of an EDC – to the F3 generational dams – shifts the neurobehavioral trajectory in a manner that is EDC-specific, and strongly influenced by sex and lineage, evidenced in the F4 and F6 generations. Last, we will show that DNA methylation profiles of both brain and sperm in F1 and F3 male rats are uniquely altered, as a potential epigenetic mechanism of transfer of information. These studies are highly relevant to humans and wildlife, as all individuals are exposed to environmental chemicals due to their persistence and ubiquity. Furthermore, the finding that prior EDC exposure history influences outcomes to EDC exposures generations later has important implications for the importance of understanding EDC effects, and regulating these chemicals properly.
Supported by NIH ES029464, ES023254.

Intergenerational Epigenetic Effects of Thyroid Hormone
• Arturo Hernandez, PhD, Maine Medical Center Research Institute, MaineHealth

Thyroid hormones (TH) exert pleiotropic effects on mammals, profoundly affecting metabolism and the development of the central nervous system.  Their circulating levels are controlled by the hypothalamic-pituitary-thyroid (HPT) axis in adulthood but are very low in fetal and early neonatal life due to active clearance of maternal TH by the type 3 deiodinase (DIO3). Fifty years ago, rats that experienced neonatal thyrotoxicosis by pharmacological TH administration developed reduced testis size and abnormal maturation and function of the HPT axis, phenotypes that were transmitted to the next generation. Similarly, the offspring of women carrying a mutation in the TH receptor beta experience thyrotoxicosis in utero and show abnormal HPT axis regulation as adults, a trait that is maintained for at least two generations in genetically normal individuals through the paternal line but that is lost in the maternal line. A more robust model of developmental thyrotoxicosis is the DIO3-deficient mouse, which also exhibits abnormal testis size and altered maturation and programming of the HPT axis. Loss of DIO3 reduces DNA methylation in neonatal spermatogonia and changes the adult sperm methylome, impacting neonatal brain gene expression and adult behavior in genetically normal F2 generation descendants. Interestingly, as an imprinted gene, Dio3 is epigenetically regulated and its testicular expression is specific to undifferentiated spermatogonia, suggesting a role in modulating male germline TH action and the associated epigenetic information of the mature sperm. 

Comparative Actions of Environmental Toxicants in Epigenetic Transgenerational Inheritance of Pathology, and Use of Epimutation Biomarkers of Exposure and Disease
• Michael K. Skinner, PhD, Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, Washington, USA

Transgenerational effects of environmental toxicants significantly amplify the biological impacts and health hazards of these exposures. One of the most sensitive periods to exposure is during fetal gonadal sex determination when the germ line is undergoing epigenetic programming and DNA re-methylation occurs. Previous studies have shown that toxicants (eg Glyphostate) can cause an increase in adult onset disease such as infertility, prostate, ovary and kidney disease, cancers and obesity. Interestingly, this effect is transgenerational (F1, F2, F3 and F4 generations) and due to a permanent (imprinted) altered epimutation of the germline. The transgenerational epigenetic mechanism appears to involve the actions of an environmental compound at the time of sex determination to permanently alter the epigenetic (e.g. DNA methylation) programming of the germ line that then alters the transcriptomes of developing organs to induce disease susceptibility and development transgenerationally. In addition to DNA methylation, alterations in sperm ncRNAs and histone alterations have also been observed. A variety of different environmental compounds have been shown to induce this epigenetic transgenerational inheritance of disease including: fungicide vinclozolin, plastics BPA and phthalates, pesticides, DDT, dioxin, hydrocarbons and herbicides like atrazine and glyphosate. Interestingly, exposure specific epigenetic alterations were observed between the specific toxicants. In addition, epigenetic biomarkers for specific disease susceptibilities have recently been observed that can potentially be used to facilitate diagnosis and preventative medicine strategies for clinical management of disease.  The suggestion that environmental toxicants can reprogram the germ line to induce the epigenetic transgenerational inheritance of disease, which is a new paradigm in disease etiology, indicates generational toxicology needs to be assessed in the future. 

Physiologically Relevant Bisphenol A Levels Alter the Developmental Trajectory of an In Vitro Model of Mouse Primordial Germ Cells
• Steen Ooi, PhD, UCLA

Animal-based studies indicate that Bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remain poorly defined. Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to Primordial Germ Cell (PGC) formation alters their differentiation trajectory and unique molecular program. Exposure of EpiLCs to BPA resulted in a dose-dependent increase in cell number, associated with an increase proportion of cells in S-phase as well as a reduced proportion undergoing apoptosis. Exposure also resulted in a greater proportion of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively impact PGC specification and resulted in dose-dependent increase in PGCLC proliferation. However, PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis. Our findings represent the first characterisation of the consequences of early BPA exposure on a model of PGC development, highlighting altered cell behaviour and underlying pathways and molecular processes.

HERITABLE IMPACTS OF DIETHYLSTILBESTROL (DES)

Results and Signals from the Ongoing NCI Multi-Generational DES Follow-up Study 
• Linda Titus, PhD, Dartmouth Geisel School of Medicine, NH, USA

This presentation will provide an overview of the US National Cancer Institute (NCI) DES Combined Cohort Follow-up Study. Strengths of the NCI study include its relatively large size, medical record documentation of DES exposure or lack of exposure, and the extended follow-up of three generations of DES exposed and unexposed individuals. The "first generation" of individuals in the NCI study comprises women who were given DES during pregnancy and those who were not given DES; the "second generation" comprises the sons and daughters of the first generation women; i.e., men and women who were prenatally DES exposed or unexposed; the "third generation," sometimes referred to as the "granddaughters" generation, comprises the daughters of second generation women who were prenatally DES exposed or unexposed. The presentation will focus on the findings from the NCI Third Generation Study -- women whose mothers were or were not prenatally exposed to DES. Findings to date for the third generation DES exposed women, compared to the unexposed, suggest an increased likelihood of menstrual irregularities and a possible excess of ovarian cancer based, however, on only 3 cases. Further work is underway to assess these outcomes, as well as the possibility of adverse reproductive outcomes, which affected their prenatally exposed mothers.

DES Exposure During Pregnancy and Third-Generation Neurodevelopmental Deficits
• Marianthi Kioumourtzoglou, ScD, Columbia University

We investigated the potential consequences of diethylstilbestrol (DES)—a potent endocrine disruptor—use during pregnancy on third-generation neurodevelopment. We used information on 47,540 participants enrolled in the Nurses’ Health Study II. The 3 generations analyzed in this study were the participants (F1 generation), their mothers (F0 generation), and their live-born children (F2 generation). F0 DES use during her pregnancy with F1 was the exposure of interest and physician-diagnosed F2 attention-deficit/hyperactivity disorder (ADHD) was the outcome of interest. Of the 47,540 F0 mothers, 861 (1.8%) used diethylstilbestrol and 46,679 (98.2%) did not while pregnant with the F1 participants. Use of DES by F0 mothers was associated with an increased risk of ADHD among the F2 generation: We estimated an adjusted odds ratio (OR) of 1.36 (95% CI, 1.10-1.67) and an OR of 1.63 (95% CI, 1.18-2.25) if DES was used during the first trimester of pregnancy. We observed no effect modification by the F2 children’s sex. Our study provides evidence that DES use is associated with multigenerational neurodevelopmental deficits. Although the doses and potency level of environmental endocrine disruptors to which humans are exposed today are lower than those of DES, the prevalence of such exposure and the possibility of cumulative action are potentially high. Our findings, thus, have important implications for exposures to other environmental endocrine disruptors during pregnancy and third generation adverse health effects.

To be followed by interactive discussion of DES grandchild effects, including:
• Linda Titus, PhD
• Marianthi Kioumourtzoglou, ScD
• Subhrangsu S. Mandal, PhD, University of Texas at Arlington
• Kari Christianson, MedShadow, and former research director, DES Action USA
• Scott Kerlin, PhD, independent researcher