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.

>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.

Endocrine disrupting compounds (EDCs) in the environment have recently emerged as a major issue in Canada and around the globe. The primary objective of this thesis was to investigate the fate of EDCs in two wastewater treatment processes, membrane bioreactors (MBRs) and ultraviolet (UV) disinfection. Two submerged MBR systems using hollow fiber membranes from two membrane manufacturers were tested. The results from a bench-scale and a pilot scale MBR for the treatment of swine wastewater with high concentration of EDCs showed that over 94% of the estrogenic activity (EA) in the influent was reduced through the MBR process. Biological degradation was the dominant removal mechanism for the removal of EDCs in MBRs. Over 85% of the influent EA was reduced by biodegradation through the MBR process. The other MBR system was built to study the removal mechanisms of two estrogens in a hybrid MBR with the addition of powdered activated carbon (PAC). The effects of PAC dosing on MBR overall performance was studied as well. It was found that PAC dosing could increase the removal rates of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) by 3.4% and 15.8%, respectively and result in a slower rate of trans-membrane pressure (TMP) increase during MBR operation, which could significantly reduce the operating cost for membrane cleaning and/or replacement. The operating cost for PAC dosing could be offset by the benefit achieved from reducing the cost for membrane maintenance. The slower rate of TMP increase in the PAC-MBR was associated with the lower concentrations of soluble extracellular polymeric substances and colloidal organic compounds in the PAC-MBR sludge. The degradation kinetics of three estrogens, estrone (E1), E2, and EE2 in de-ionized water by UV irradiation was studied. The experimental results showed both the apparent concentrations and overall EA of all three investigated estrogens in water decreased with direct UV irradiation. To further study the impact of UV on the overall EA of wastewater, the EA of pre-UV and post-UV samples from five wastewater treatment plants were measured in both liquid and solid phase by Yeast Estrogen Screen assay. It was found that the EA of wastewater decreased after UV disinfection in three of the investigated plants whereas it increased in the other two plants. This observation needs to be further studied because it might have significant impacts on the application of UV systems for wastewater disinfection.

Endocrine disrupting compounds (EDCs) in the environment have recently emerged as a major issue in Canada and around the globe. The primary objective of this thesis was to investigate the fate of EDCs in two wastewater treatment processes, membrane bioreactors (MBRs) and ultraviolet (UV) disinfection. Two submerged MBR systems using hollow fiber membranes from two membrane manufacturers were tested. The results from a bench-scale and a pilot scale MBR for the treatment of swine wastewater with high concentration of EDCs showed that over 94% of the estrogenic activity (EA) in the influent was reduced through the MBR process. Biological degradation was the dominant removal mechanism for the removal of EDCs in MBRs. Over 85% of the influent EA was reduced by biodegradation through the MBR process. The other MBR system was built to study the removal mechanisms of two estrogens in a hybrid MBR with the addition of powdered activated carbon (PAC). The effects of PAC dosing on MBR overall performance was studied as well. It was found that PAC dosing could increase the removal rates of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) by 3.4% and 15.8%, respectively and result in a slower rate of trans-membrane pressure (TMP) increase during MBR operation, which could significantly reduce the operating cost for membrane cleaning and/or replacement. The operating cost for PAC dosing could be offset by the benefit achieved from reducing the cost for membrane maintenance. The slower rate of TMP increase in the PAC-MBR was associated with the lower concentrations of soluble extracellular polymeric substances and colloidal organic compounds in the PAC-MBR sludge. The degradation kinetics of three estrogens, estrone (E1), E2, and EE2 in de-ionized water by UV irradiation was studied. The experimental results showed both the apparent concentrations and overall EA of all three investigated estrogens in water decreased with direct UV irradiation. To further study the impact of UV on the overall EA of wastewater, the EA of pre-UV and post-UV samples from five wastewater treatment plants were measured in both liquid and solid phase by Yeast Estrogen Screen assay. It was found that the EA of wastewater decreased after UV disinfection in three of the investigated plants whereas it increased in the other two plants. This observation needs to be further studied because it might have significant impacts on the application of UV systems for wastewater disinfection.

Endocrine disrupting compounds (EDC) are environmental contaminants that disrupt reproduction, development and behaviour in aquatic organisms. A thorough evaluation of the impacts of EDCs on aquatic organisms is currently limited by a lack of robust biomarkers in small model fish, particularly for assessing EDCs with (anti-)androgenic activity. Male sticklebacks build nests using spiggin, an androgen-responsive glycoprotein, which can be used to assess (anti-)androgenic exposure. EDC assessment in the field using threespine stickleback and the spiggin biomarker is limited to coastal and estuarine environments. However, their freshwater relative, brook stickleback (Culaea inconstans), also possess spiggin and their widespread distribution suggests that they may have applications as a bioindicator of EDCs in freshwater systems. Therefore, the overall objective of this thesis was to determine if brook stickleback are a suitable bioindicator species for EDCs by evaluating their response and sensitivity to estrogenic and (anti-)androgenic chemicals. Basal transcript levels of spiggin in kidney and vitellogenin in liver were first measured in wild-caught brook stickleback using qPCR and found to be differentially expressed in males and females. Brook stickleback were then exposed to two model compounds, 17α-ethinylestradiol (EE2) and 17α-methyltestosterone (MT), at 1, 10 and 100 ng/L for 21 days (sampled at 7 and 21 days) via static-renewal to determine the responsiveness of these transcripts to exogenous hormones. The effect of hormone exposure on condition factor, organosomatic indices and histopathology of kidneys was also measured. Exposure to MT and EE2 significantly induced spiggin and vitellogenin transcripts in female kidneys and male livers, respectively. Exposure to EE2 also significantly increased the hepatosomatic index in females after 7 days and in both sexes after 21 days whereas the gonadosomatic index was reduced in females after 21 days. An increase in kidney epithelium cell height was also observed in MT-exposed females and males after 7 days. These results mirror those of threespine stickleback and suggest that brook stickleback are responsive to androgenic and estrogenic chemical exposure and more specifically, possess quantifiable and sensitive biomarkers for exposure to compounds with androgenic activity. In a third experiment, female fish were co-exposed to MT at 500 ng/L and an anti-androgen (flutamide; FL) at 25, 150 and 250 µg/L for 14 days (sampled at 4 and 14 days) to validate this bioassay for the evaluation of anti-androgens using the same endpoints as in the previous two experiments. In females, exposure to MT increased spiggin transcript levels and nephrosomatic index (NSI) but co-exposure to FL did not result in a significant suppression of these endpoints because of high inter-individual variability. In males, exposure to MT increased NSI and co-exposure to FL resulted in a reduction in this endpoint, illustrating anti-androgenic effects. Although the response of brook stickleback to hormone exposure was endpoint-specific and was at times lower than other small model fish species, the ability to simultaneously assess estrogenic and (anti-)androgenic chemical exposure in a single fish using quantitative endpoints is an advantage exclusively held by members of the stickleback family. The results of this thesis suggest that brook stickleback hold promise as an additional small fish model for the evaluation of EDCs, with potential application in EDC biomonitoring in the freshwaters of North America.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Modulation of the male reproductive system occurs as a result of exposure to endocrine disrupting compounds (EDCs) in different life stages. The effects of EDCs on the male reproductive system include infertility, decreased sperm count, function and morphology, abnormal development of secondary sex characteristics, reproductive function and sexual behavior, as well as decreased libido. Phytochemicals are naturally occurring, biologically active chemical compounds in plants. They are divided into different groups. Isoflavonoids and lignans, are the two major groups of phytoestrogens. Phytoestrogens of teas, coffee and buchu have many beneficial effects on body systems such as antimutagenic, antidiabetic, anti-inflammatory, antibacterial and antiviral properties. They also elicit many adverse events, for example, heavy consumption of green and black tea may cause liver damage and added unwanted effects when combined with other herbal beverages. Chronic heavy consumption of coffee is positively correlated to acute myocardial infarction and can elevate serum cholesterol levels. Rooibos tea decreases steroidogenesis by steroid secreting cell lines.This study investigated the effects of these beverages on the male reproductive system, using a minced testes method for determination of cell viability and hormone (testosterone) production. The first objective of this study was to optimize protein supplement for in vitro testosterone production using human serum albumin (HSA) and foetal bovine serum (FBS). Testicular cultures were prepared and exposed overnight to different concentrations of both sera and then incubated for 4 hours with or without luteinizing hormone (LH). The results showed that addition of protein supplements (HSA or FBS) did not have a significant effect on testosterone production. The second objective of this study was to investigate the effects of green tea, black tea, rooibos tea, coffee and buchu on cell viability of testicular cultures. Cells were treated overnight with varying concentrations of the plant extracts followed by incubation with/without LH for 4 hours. The effects of the plant beverages on cellular protein production were determined by the Bradford assay. The results showed that treatment of cells with varying concentrations of the plant extracts (with/without LH-treatment) had no significant effect on total cellular protein. The third objective of this study was to investigate the effects of black, green and rooibos teas, coffee and buchu on testosterone production by testicular cultures. The results obtained from these experiments showed that rooibos tea and buchu did not affect testosterone production in the presence or absence of LH. The results also indicated that green tea, black tea and coffee inhibited testosterone production by mouse testis cultures in the presence of LH, but not in the absence of LH. Black tea was the most potent inhibitor of testosterone synthesis by mouse testis cultures (IC50= 48 μg/ml), followed by coffee (IC50= 64 μg/ml) and green tea (IC50= 173 μg/ml). Green tea, black tea and coffee inhibited LH-stimulated testosterone synthesis, suggesting that these beverages may impair testicular steroidogenesis in mice. Thus, in spite of their acclaimed beneficial effects, consumption of these beverages in high doses raises concerns for their inhibitory effects on male reproductive function. Further in vitro and in vivo studies are warranted to determine their exact mechanisms of action on the male reproductive system in general and testicular function in particular.

No description available.

Background: Evidence from experimental studies suggests that atrazine and its analytes alter the timing of puberty in laboratory animals. Such associations have not been investigated in humans. Objective: To determine the association between in utero exposure to atrazine analytes and earlier menarche attainment in a nested case-control study of the population-based Avon Longitudinal Study of Parents and Children. Methods: Cases were girls who reported menarche before 11.5 years while controls were girls who reported menarche at or after 11.5 years. Seven atrazine analyte concentrations were measured in maternal gestational urine samples (sample gestation week median (IQR): 12 (8–17)) during the period 1991–1992, for 174 cases and 195 controls using high performance liquid chromatography-tandem mass spectrometry. We evaluated the study association using multivariate logistic regression, adjusting for potential confounders. We used multiple imputation to impute missing confounder data for 29% of the study participants. Results: Diaminochlorotriazine (DACT) was the most frequently detected analyte (58%>limit of detection [LOD]) followed by desethyl atrazine (6%), desethyl atrazine mercapturate (3%), atrazine mercapturate (1%), hydroxyl atrazine (1%), atrazine (1%) and desisopropyl atrazine (0.5%). Because of low detection of other analytes, only DACT was included in the exposure–outcome analyses. The adjusted odds of early menarche for girls with DACT exposures≥median was 1.13 (95% Confidence Interval [95% CI]:0.82, 1.55) and exposure Conclusions: This study is the first to examine the association between timing of menarche and atrazine analytes. We found a weak, non-significant association between in-utero exposure to atrazine metabolite DACT and early menarche, though the association was significant in the subset of girls with complete confounder information. Further exploration of the role of these exposures in female reproduction in other cohorts is needed.

In recent years exposure to endocrine-disrupting chemicals (EDCs) in humans and wildlife has become an increasing concern. These compounds have been found ubiquitously in the environment and are suspected to induce adverse effects on the health of aquatic organisms. The results of health effects due to EDCs are clearly presented in many aquatic organisms, such as the feminization of male fish and a near extinction of some species. No clear effects on human health have been documented at this time. The major sources of these contaminants in the environment are discharges from wastewater treatment plants (WWTP) and diffuse pollution. Conventional wastewater treatment processes are not designed to remove such emerging pollutants and removal efficiency depends on many factors, including treatment technology and pollutant species. Passage through WWTPs and changes due to treatment technologies lead to detection of minute concentrations of EDCs in water downstream from discharge points. In New Orleans, Louisiana, discharge from its East Bank WWTP is being considered for potential reuse for wetland restoration. Therefore, effluents must be treated adequately to prevent adverse effects on the natural biota. Since effluents from wastewater treatment plants using conventional technologies may prove potentially unsafe for the environment due to the presence of EDCs, improved and/or new treatment processes for removal of these contaminants are needed. Ferrate (Fe+6) is known as an alternative oxidant for the treatment of wastewater that can be used as an oxidizing, disinfectant, and/or coagulating agent. Because of its redox potential, ferrate has been used as a disinfection agent and has been reported as a tool for enhanced treatment to remove many micropollutants without producing undesirable disinfection byproducts in contrast to other disinfection processes. Recent research has noted the ability of ferrate to deactivate a wide range of EDCs present in wastewater effluents. The negative effect of effluent's soluble organic matter on ferrate has been reported and higher doses of ferrate may be needed to obtain desired effluent quality. This study found that aerobic biological treatment processes reduce more than half of EDCs in wastewater and that free chlorine disinfection increases estrogenic activity in discharged effluent. Higher organic content in wastewater results in increased ferrate demand. The optimum ferrate dosage to deactivate EDCs in lab scale is 6 ppm, and a dosage of 8 ppm is possibly the operational optimum dose. pH neutralization by concentrated sulfuric acid was not found to affect EDCs deactivation efficiency by ferrate when added at the end of designed contact time. Ferrate was observed to have a high oxidation rate in the first10 minutes after application into wastewater and then degraded to other iron states, such as iron III. Higher oxidation rates can be obtained when more organics are present in wastewater as TOC. Higher dosages required longer oxidation reaction times. Ferrate was observed to generate fewer disinfection byproducts as compared to chlorine. Haloacetic acids in ferrate-treated effluent are generated from organics in wastewater and reactions with residual hypochlorite from the incomplete ferrate synthesis process. The reduction of trihalomethanes may be related to EDCs deactivation by ferrate. Because this study was performed on a lab scale, assessment of onsite production and application of ferrate is required to determine the feasibility of the ferrate treatment process at a full-scale treatment plant and to optimize required dosage. / acase@tulane.edu

Previous research indicates that pharmaceutically active compounds (PhACs) and endocrine-disrupting compounds (EDCs) are poorly removed during conventional drinking water treatment processes including coagulation; however, removal efficiency increases in the presence of natural organic matter (NOM). Therefore, this project investigates the link between various NOM types with EDC/PhAC removal. Bench-scale coagulation tests were conducted on three different source waters spiked with environmentally relevant levels (nominally 1000 ng/L) of EDCs/PhACs. Two different coagulants were used: polyaluminum chloride (PACl) and aluminum sulphate (alum). NOM was characterized using size exclusion liquid chromatography-organic carbon detection (LC-OCD). Results for Lake Ontario, Otonabee and Grand River water indicate that certain EDCs/PhACs are significantly removed during coagulation while others increase in concentration. Concurrently, particular NOM fractions (biopolymers and humic substances) are also being removed. Solvents used for EDC/PhAC spiking (acetone and acetonitrile) did not affect coagulation, but contributed to low molecular weight neutral and hydrophobic NOM fractions.