Links between declining human male fertility (decreased sperm counts, increased incidence of both testicular cancer and genital abnormalities) and the increasing prevalence of endocrine disrupting chemicals (EDCs) in the environment have been reported. We aim to characterise the key developmental processes occurring during human fetal gonad development. Human fetal testis development was characterised by a transient increase in interstitial area proliferation between 13-19 weeks which was accompanied by an increase in steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes. Androgen receptor was expressed by the peritubular myoid cells which had a high bcl-2:bax ratio, indicative of cell survival. Estrogen receptors ( and ) were localised to distinct cell populations. In the ovine gonad similar developmental processes occurred, and comparison with human ovarian development demonstrated interesting parallels. After optimisation, the explant culture system revealed that exposure to the insecticidal EDC dieldrin, at low (<1 ppb) doses reduced LH-stimulated testosterone output in the human fetal testis. This was accompanied by dose-specific changes to the testis proteome, alterations in bcl-2:bax ratios in favour of apoptosis and a down-regulation in StAR expression relative to the LH-treated controls. In conclusion, the processes of proliferation apoptosis, steroidogenesis and steroid action are crucial during fetal gonad development. We demonstrated that in utero exposure to dieldrin may cause reproductive dysfunction in adult life due to reduced steroidogenesis in the fetal gonad; mediated through a down-regulation in StAR expression and alterations in the regulation of gonadal apoptosis.

Many chemicals released into the environment are believed to disrupt normal endocrine functions in humans and animals. These endocrine disrupting chemicals (EDCs) affect reproductive health and development. A major group of EDCs that could be responsible for reproductive effects are those that mimic natural oestrogens, known as xeno-oestrogens. A number of in vivo and in vitro screening strategies are being developed to identify and classify xeno-oestrogens, in order to determine whether they pose a health risk to humans and animals. It is also important to be able to apply the assays to environmental samples for monitoring purposes. In South Africa information on the levels of EDCs in water is limited. While establishing the recombinant yeast screen bioassay (RCBA) using the yeast strain Sacchyromyces cerivisiae for oestrogenic activity, problems were experienced with contamination. Four South African laboratory water sources were assessed. From the results it was clear that the water used in the preparation of the medium for the assay was the source of oestrogenic contamination. Care should be taken to eliminate all possible sources of contamination in the test procedures to eliminate the reporting of false positive results. The fact that South African laboratory and surface waters tested positive for estrogenic activity has far reaching implications regarding reproductive and general health.

Epidemiological evidence suggest that exposure to endocrine disrupting compounds (EDCs) is a risk factor for diseases that involves the cardiovascular system but we know little about the mechanisms whereby these compounds can cause injury in the vasculature. The aim of this thesis was to characterize the effects and mechanisms of some EDCs in vascular cells and highly vascularized tissues. Elevated exposure to environmental EDCs is associated with an increased risk for cardiovascular diseases. In vitro studies demonstrated that the environmental EDCs, 1-nitropyrene, PCB126 and bisphenol A, caused distinct changes in primary human endothelial cells. 1‑Nitropyrene induced cell stress and DNA damage, PCB126 caused changes that indicate endothelial dysfunction and vasoconstriction, and BPA induced changes that indicate angiogenesis and vasoconstriction. Further studies demonstrated that long-term exposure of rats to BPA induced changes in rat cardiac tissues in vivo similar to those observed in human endothelial cells in vitro. The type of cellular alterations that were demonstrated is known to play to play a role in cardiovascular disease in humans. These findings suggest that environmental EDCs can cause damage to the human endothelium that may contribute to the development of cardiovascular disease. The beneficial effects of the pharmaceutical EDC tamoxifen in breast cancer treatment are compromised by an increased risk for bleedings, hyperplasia, and cancer in the endometrium. Ex vivo studies identified the glandular and surface epithelia as potential target sites for tamoxifen adduct formation and tamoxifen-induced cell stress the human endometrium. No signs of tamoxifen-induced changes were detected in the blood vessels. The results suggest that bioactivation of tamoxifen and subsequent cell injury in endometrial epithelial cells may play a role for tamoxifen’s side effects in the endometrium. Taken together, this thesis provide evidence that may help understanding how exposure to EDCs can increase the risk for diseases in that involves the cardiovascular system.

Endocrine disrupting chemicals (EDCs) are compounds in the environment that interfere with hormone systems in the body. I investigated if gestational exposure to a known class of EDCs, polychlorinated biphenyls (PCBs), resulted in life long alterations in neuroendocrine function. My overall hypothesis was that prenatal PCB exposure would cause molecular and cellular changes to the developing hypothalamus that would manifest across development through differences in hypothalamic gene expression, molecular epigenetic modifications, and corresponding effects on sexual development. To perform this work, I characterized changes in gene expression in two regions of the hypothalamus required for reproductive function throughout the life cycle and measured changes in somatic markers associated with reproductive physiology and development. This approach allowed me to relate specific neuroendocrine changes back to altered reproductive function. First, I present normative data showing gene and hormone changes throughout development in male and female rats to use as a basis of comparison for my further studies on EDCs. Second, I investigated how gestational exposure to PCBs on embryonic day 16 and 18 affected development of the hypothalamus through adulthood and caused corresponding changes in physiological functions. PCBs altered estrous cyclicity in females and delayed the timing of puberty in males. Developmental changes in gene expression were associated with sex, age and region of the hypothalamus. As a whole, the data suggested that gestational exposure to PCBs altered a network of hypothalamic genes and was associated with altered reproductive physiology. Finally, I extended my study farther along the life cycle to investigate if gestational exposure to PCBs altered the timing of reproductive aging in male and female rats. Few effects in males were observed. However, females exposed to PCBs had lower serum concentrations of LH on proestrus, and altered expression of numerous genes in the hypothalamus. These changes in gene expression were specific to the females’ cycle status and the results provided novel insight into the molecular mechanisms underlying reproductive aging. Taken together, my dissertation resulted in a comprehensive profile of both normal hypothalamic developmental changes, as well as providing insight into endocrine disruption of hypothalamic gene networks from birth through aging. / text

The endocrine disrupting chemicals (EDCs) are exogenous compounds that mimic natural hormones and disrupt endocrine functions in humans and animals. Accumulating evidence suggests that EDCs may have detrimental impact on human reproduction including infertility, distortion of sex ratios and menstrual problems. TCDD, one of the most toxic man-made chemicals, was found to affect embryo maturity, implantation and reproduction. Bisphenol A (BPA), another EDC commonly used nowadays in plastic products, exhibits weak estrogenic activity in human bodies. However, TCDD and BPA have distinct chemical structures, chemical properties and receptor binding, and their mechanistic actions on human body remain largely unknown. The present study is to delineate the effects of EDCs on embryo implantation and development under in vitro exposure. It is hypothesized that EDCs (TCDD and BPA) may modulate fertility of animals by affecting early pre-implantation embryo development and attachment onto uterus. The effect of EDCs on the expression of receptors (AhR, ERE, ERR,,and ERa) and downstream reporter genes (CYP1A1 and C3) were studied by real-time PCR and Western blotting. An in vitro spheroid (JEG-3)-endometrial cells (Ishikawa) co-culture assay was established to study the effect of EDCs on spheroid attachment. Since microRNA, Wnt-signaling and adhesion molecules play important roles in implantation process, the expression of selected miRNA, Wnt-signaling and adhesion molecules was studied after EDCs treatment. Specific activities of the EDCs receptors were confirmed by inhibitor treatment or siRNA knockdown studies. Furthermore, EDC-treated embryos were transferred to pseudo-pregnant surrogate mice to determine the effect of EDCs on implantation outcome on day 8. It was found that Ishikawa and JEG-3 cells expressed AhR, ERIIand ERRE. TCDD treatment induced CYP1A1 expression in both cell lines (P<0.05). TCDD at 10nM significantly suppressed (P<0.005) spheroid attachment (74% compared to control 97%). When both cell lines were treated with AhR antagonists DMF (10 aaa/ANF (1 /////with TCDD (10nM), the suppressive effect of TCDD on spheroid attachment in co-culture assay was nullified, suggesting that TCDD acts through AhR receptor. In BPA exposure, BPA (0.001 to 10 tt) induced the expressions of ER))and C3ain Ishikawa but reduced the expressions of EReeand C3ain JEG-3 cells. BPA (10 iiiisuppressed spheroids attachment (74% vs Control 94%, P<0.005); while co-treatment with ER antagonists (ICI 182,780), ERaaantagonistaaMPP), ERMMantagonist (PTHPP) or ERE///siRNA, the suppressive effect of BPA (10sM) on spheroid attachment was nullified, suggesting that BPA acts through ER receptors. Moreover, TCDD and BPA suppressed the expressions of B-catenin and E-cadherin for cell-cell adhesion. Activation of Wnt-signaling pathway by Wnt3a conditioned medium or LiCl, rescued the low spheroid attachment rate induced by EDCs. Both EDCs reduced embryo implantation rate in pseudo-pregnant mice, suggesting the adverse effect of EDCs on embryo implantation and/or endometrial receptivity. Taken together, TCDD and BPA affected the JEG-3 spheroid attachment onto the Ishikawa endometrial cells and mouse embryo implantation. These effects might be mediated through the action of receptors and Wnt/β-catenin signaling pathway. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Michigan State University. Dept. of Zoology-Environmental Toxicology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Phthalates are endocrine-disrupting chemicals (EDCs) that are known testicular toxicants, used commonly as industrial plasticizers that are found in everyday items. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment, and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) is ten-fold more potent. The purpose of this study is to examine the cellular and molecular effects of MEHP in the development of testicular cancer. Proliferation was measured for NT2 cells exposed to 10µM and 100µM MEHP at 24 and 48 hours and for cells under controlled conditions. Methylation-specific PCR (MSP) was used to determine the methylation status of the promoter region of key testicular genes post exposure to MEHP. MEHP caused a dose-dependent negative effect on proliferation and significantly altered methylation levels for key testicular genes following exposure to 10µM MEHP and 100µM, as compared to controls. This suggests that MEHP alters proliferation and methylation of testicular tumour cells in a time- and dose-dependent manner.

>Magister Scientiae - MSc / Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals in the environment that interfere with the synthesis, secretion, distribution and function or elimination of natural hormones in the body. Environmental estrogens are a subclass of EDCs that may mimic or inhibit the effect of endogenous estrogen and can therefore influence developmental and reproductive health in humans and animals. EDCs have been reported to adversely affect the reproductive, immune, endocrine and nervous systems of wildlife and humans. The effects of EDCs include gonadal abnormalities, altered male/female sex ratios, reduced fertility and cancers of the male and female reproductive tract to mention a few. These effects are difficult to detect. Although it is essential to screen for EDCs in aqueous environmental samples, most countries have failed to implement this as part of their routine water quality monitoring programs due to various constraints such as the high cost of assays and the lack of infrastructure and skills required to do the assays. Therefore, there is a clear need for more user-friendly, more economically viable and time saving assays that can be used for routine monitoring of environmental EDCs. The aim of this study was to investigate the comparison between chemical and tissue culture methods to monitor environmental estrogens. 28 environmental water samples were collected from various sites around South Africa and analyzed for EDCs using a battery of rapid in vitro tests. Samples collected for the current study were selected based on various human impacts and also to give approximately 50% high and 50% low estrogen values. The 28 environmental water samples were separated into two groups based on the estradiol ELISA. The estradiol ELISA was chosen because estradiol is the principal estrogen found in all mammalian species during their reproductive years. For this separation, an estradiol level of 5 pg/ml was used as cut-off. Of the 28 samples investigated, 15 had estradiol levels higher than 5 pg/ml and were designated as high estradiol. The remaining 13 samples contained estradiol at 5 pg/ml or less and they were designated as low estradiol. The first objective of this study was to compare different rapid ELISAs for EDC monitoring to determine if the data obtained with these assays are similar/identical. The data obtained from the estrogenic ELISAs was related/similar and showed good correlation with each other. This is because the different estrogens are very similar and also due to the fact that the same sub-group in the population (the reproductively active females) is secreting these hormones. Therefore, an estradiol rapid assay was proposed as a first screening system for estrogens in samples. Even though there was a positive correlation between the estradiol rapid assay and testosterone rapid assay, separation of samples based on estradiol levels wasn’t a good predictor of testosterone levels in the samples. A testosterone rapid assay was therefore recommended as necessary to screen for androgens in samples. The positive correlation between the estradiol rapid assay and progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens. The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: ●Estradiol ELISA as a rapid assay to screen for estrogens. ●Testosterone ELISA as a rapid assay to screen for androgens. ●Progesterone ELISA as a rapid assay to screen for progestogens. ●Mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity.

This critical overview document (COD) presents, discusses and brings together the selected portfolio of publications that the author believes make a significant contribution to the field of wastewater treatment, focusing on the removal of endocrine disrupting chemicals (EDCs) in wastewater treatment applications. The aim of the research within this COD was to investigate the fate, mechanisms and optimisation of EDCs removal in wastewater treatment applications. The key objectives were to: 1. Investigate and understand the mechanisms of removal of EDCs in wastewater and sludge treatment processes. 2. Evaluate novel methods for the removal of EDCs in water and wastewater treatment applications. 3. Establish the kinetic and thermodynamic properties of the removal processes to inform process modelling of full scale design of treatment processes.

Vinclozolin (VCZ), an antiandrogenic fungicide, is an endocrine disrupting chemical that is known to possess high affinity for the androgen receptor (AR) and modulate expression of critical androgen-dependant genes in the prostate. In this study, viability and expression of AR, NKX3.1 and CYP3A4 genes were measured in androgen-sensitive prostate cells LNCaP after exposure to VCZ and VCZ treated with S9 microsomes in a time and dose dependent manner. NKX3.1 is an androgen regulated gene that plays a vital role in prostate development. CYP3A4 is involved in xenobiotic metabolism. VCZ decreased the viability at high doses after 48 hours which was slightly mitigated by treatment with S9 metabolites. Expression of NKX3.1 and CYP3A4 was upregulated while an initial downregulation of AR was observed. NKX3.1 upregulation corroborates with possibility of antiandrogens to act as androgens in LNCaP. The results illustrate that VCZ can interfere with the expression of critical prostate genes.