Endocrine-disrupting chemicals (EDCs) are environmental pollutants known to perturb hormone systems and interfere with normal endocrine function. Exposure to EDCs during hormone-sensitive developmental periods can result in profound dysfunction in reproductive physiology and behavior. In this dissertation, effects of gestational exposure to a class of EDCs called polychlorinated biphenyls (PCBs) were examined in the developing hypothalamus, which is known to control reproductive physiology and behavior in vertebrates. The specific hypothesis was that PCBs caused changes in sexually dimorphic hypothalamic nuclei, resulting in perturbation of adult sociosexual behaviors and alteration of neural networks with changes in expression of microRNAs and genes during development and in adulthood. This research focused on two brain areas relevant to understanding the PCB effects on the developing hypothalamus: 1) microRNA and related target gene expression during postnatal development, 2) adult sociosexual behaviors and gene expression. In both sections, molecular changes were examined in two sexually dimorphic hypothalamic nuclei, medial preoptic nucleus (MPN) and ventromedial nucleus (VMN), known for their role in regulation of sociosexual behavior. In the first section of the dissertation, the effects of PCBs were examined on the expression of microRNAs and target genes at four ages during postnatal [P] development (P15, P30, P45, and P90). Age and sex specific effects were observed in both MPN and VMN, with greater effects in the MPN. The second research section of the dissertation explored whether sociosexual behaviors, namely ultrasonic vocalizations and sociosexual preference behaviors, were altered by gestational PCBs. Expression of forty-eight neuroendocrine candidate genes was also examined in the MPN and VMN of the same animals. Several sociosexual behaviors were affected, including number and acoustic properties of ultrasonic vocalizations, and nose-touching with opposite-sex animals. Gene expression was altered in sex and region-specific manner in the brains of behaviorally affected rats. Taken together, these findings suggest that gestational PCBs have lasting effects on molecular mechanisms during postnatal development and in adulthood, and could result in altered sociosexual behavior. These results have implications for human health and disease, as early life exposures to EDCs have been linked to reproductive decline in humans. / text

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Soil-aquifer treatment (SAT) has been proposed as a method for reusing treated municipal wastewater. SAT is characterized by alternating cycles of aerobic and anaerobic conditions in the subsurface, in response to alternating cycles of flooding and drainage of a surface impoundment. It is not yet known how these alternating redox conditions affect the removal of potentially harmful endocrine-disrupting compounds (EDCs) from treated effluent. The overall objective of my doctoral research is to determine the fate of EDCs in alternating aerobic/anoxic/anaerobic conditions under simulated SAT conditions. To assess the fate of EDCs in simulated SAT conditions, I first had to develop appropriate analytical methods. Prior researchers have developed sophisticated analytical methods for measuring low concentrations of EDCs in water. However, it is not inherently clear which of these methods is preferable for analysis of any particular set of environmental samples. Therefore, in order to compare the analytical methods, solid-phase extraction (SPE) and solid-phase micro-extraction (SPME) were compared for the analysis of two EDCs, bisphenol-A (BPA) and 17B-estradiol (E2), in water samples of water. Following extraction by SPE or SPME, the target EDCs were derivatized (silylated) and then analyzed by gas chromatography (GC) with mass spectrometry (MS). Also, the performance of two candidate derivatization agents, N,O-bis-(trimethylsiyl) trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), was compared. SPME is more convenient, is less labor-intensive, and allows for analysis of smaller sample volumes, but it is expensive because fibers need frequent replacement, and the range of linearity was limited. SPE has a lower material cost and allows for the analysis of a broader range of concentrations, but it is more labor-intensive and large sample volumes may be required. Therefore, the selection of which method is "best" depends upon the constraints (time, money, sample volume, acceptable detection limit) associated with any particular set of samples. The two derivatization agents performed equally when used in conjunction with SPE, but MSFTA yielded higher peak areas for headspace (on-fiber) derivatization during SPME. To investigate how alternating redox conditions of SAT may affect the removal of harmful EDCs, a simulated SAT systems were constructed in 4-L reactors with 500 g of sediment (collected from a wetland) and 3 L of treated effluent from a municipal wastewater treatment plant; then BPA and E2 were spiked into reactors, two common EDCs often found in treated wastewater. Redox conditions in the mesocosms were controlled by switching the air between air (to induce aerobic conditions) and nitrogen (to induce anaerobic conditions); the length of the anoxic/anaerobic cycles was varied to determine how this affects biodegradation of the target EDCs. The mesocosm environment was supplemented with either nitrate or sulfate to serve as potential electron acceptors during the anoxic/anaerobic cycles. In addition to monitoring the concentrations of the target EDCs in the mesocosms over time, I also monitored the concentration of dissolved oxygen in the water; the redox potential; the concentrations of nitrate, nitrite, and sulfate; and the concentration of bacteria in the water (estimated via flow cytometry). BPA was biodegraded only during aerobic cycles, but E2 was biodegraded during both aerobic and anoxic/anaerobic cycles. Whenever the redox conditions in the system were switched, there was a temporary drop in the bacterial population, followed by a recovery of the population. When redox conditions were switched from anoxic/anaerobic to aerobic, biodegradation of the target EDCs commenced after a lag period during which no biodegradation was observed. The lag time for biodegradation in the aerobic cycle was longer when the anaerobic cycles were longer in duration. More biodegradation of E2 was observed under anoxic conditions than under anaerobic conditions. SPE and SPME methods that included derivatization agent are useful method for detection and quantification of EDCs in water. I concluded that SAT is a viable technology to produce potable water from treated WWTP effluent, but the optimal length of flooding and drying cycles of SAT required removing the targeted contaminants during infiltration through the vadose zone.

Industrialization and "development" during the last 200 years have led to an increase of pesticides, an intensified use of synthetic chemicals, higher levels of environmental pollution, and more exposure to hazardous working conditions. Environmental toxins, many of which are endocrine disruptors, are stored in fat tissue, increasing reproductive health risks for both women and men. Women’s bodies are particularly vulnerable as sites for creating, growing, feeding, and nurturing the next generation. And yet, women’s lives are consistently devalued, especially in a capitalist economy, so that a woman’s rights to her own reproductive health are no longer guaranteed. In this thesis I first review ecological destruction, environmental policies, and food safety/security issues for women. I then examine neoliberal globalization as an active participant in the destruction of the environment and an attack on global health. I discuss how utilizing feminist theory effectively, and actively, will ensure women the right to their health. I employ postmodern feminist and refigured ecofeminist theory to demonstrate how a feminist perspective is necessary in the development of policies that address the problem of endocrine disruptors in terms of women’s reproductive health and the health of future generations. Finally, I suggest that the precautionary principle must include a feminist perspective to fully succeed.

Biosolids are the nutrient-rich organic residual materials resulting from the treatment of domestic sewage at a wastewater treatment facility, and are increasingly land-applied for agricultural and land-reclamation purposes as part of the wastewater management process. While the presence and fate of estrogenic endocrine-disruptors (eEDCs) in wastewater has been extensively studied, much less focus has been given to examining the presence and fate of eEDCs during biosolids treatment. In particular, little work has been done to measure the net estrogenic potency of biosolids using in vitro bioassays, such as the Yeast Estrogen Screen (YES) assay. This is despite the fact that widespread land-application of biosolids provides for the direct introduction of eEDCs into terrestrial and aquatic environments. The relative scarcity of bioassay-based net estrogenicity data for sludges and biosolids is in large part due to the analytical challenges involved in working with such a complex sample matrix. Comprehensive sampling at wastewater treatment plants in Guelph and London, ON, demonstrated that the estrogenicity of anaerobically-treated biosolids is considerably lower (12.0-19.7 ng/g estradiol-equivalents) than that reported in earlier published studies. The results of the present study were made possible due to the development of a sample preparation methodology that overcame the toxic effects that sludge and biosolid samples typically exert on yeast cells in the YES assay. An anti-estrogenicity assay was also applied for the first time to sludges/biosolids to measure the extent to which antagonistic compounds ‘block’ the response of the YES assay. The results of these tests suggest that although the net estrogenicity of anaerobically treated solids is indeed low, up to twice the amount of estrogenicity measured by the YES assay may be masked in biosolids by the presence of antagonistic compounds. While aerobic treatment conditions reduced net estrogenicity to at-or-below detectable levels, net estrogenicity remained relatively constant throughout the unit processes of the anaerobic treatment train. Biosolid ageing during storage led to an overall decrease in net estrogenicity of both conventionally-treated “restricted use” and advanced-treated “unrestricted use” anaerobic biosolids. However, levels of net estrogenicity were observed to spike during the early stages of storage, particularly under freeze/thaw conditions. / Natural Science and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship, Water Environment Association of Ontario, Canadian Water Network

A LLE-GC-MS method was developed to detect PPCPs in surface water samples from Big Cypress National Park, Everglades National Park and Biscayne National Park in South Florida. The most frequently found PPCPs were caffeine, DEET and triclosan with detected maximum concentration of 169 ng/L, 27.9 ng/L and 10.9 ng/L, respectively. The detection frequencies of hormones were less than PPCPs. Detected maximal concentrations of estrone, 17β-estradiol, coprostan-3-ol, coprostane and coprostan-3-one were 5.98 ng/L, 3.34 ng/L, 16.5 ng/L, 13.5 ng/L and 6.79 ng/L, respectively. An ASE-SPE-GC-MS method was developed and applied to the analysis of the sediment and soil area where reclaimed water was used for irrigation. Most analytes were below detection limits, even though some of analytes were detected in the reclaimed water at relatively high concentrations corroborating the fact that PPCPs do not significantly partition to mineral phases. An online SPE-HPLC-APPI-MS/MS method and an online SPE-HPLC-HESI-MS/MS method were developed to analyze reclaimed water and drinking water samples. In the reclaimed water study, reclaimed water samples were collected from the sprinkler for a year-long period at Florida International University Biscayne Bay Campus, where reclaimed water was reused for irrigation. Analysis results showed that several analytes were continuously detected in all reclaimed water samples. Coprostanol, bisphenol A and DEET’s maximum concentration exceeded 10 µg/L (ppb). The four most frequently detected compounds were diphenhydramine (100%), DEET (98%), atenolol (98%) and carbamazepine (96%). In the study of drinking water, 54 tap water samples were collected from the Miami-Dade area. The maximum concentrations of salicylic acid, ibuprofen and DEET were 521 ng/L, 301 ng/L and 290 ng/L, respectively. The three most frequently detected compounds were DEET (93%), carbamazepine (43%) and salicylic acid (37%), respectively. Because the source of drinking water in Miami-Dade County is the relatively pristine Biscayne aquifer, these findings suggest the presence of wastewater intrusions into the delivery system or the onset of direct influence of surface waters into the shallow aquifer.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and have been detected in drinking water. Although various water treatment processes can remove EDCs, chemicals can migrate from pipes that transport water and contaminate drinking water. Globally bottled water consumption is steadily rising as an alternative to tap water, but EDCs have also been detected in bottled water. Sources of EDCs in bottled water include contamination of the water source, contamination through the production process or the migration of EDCs from the packaging material. There is limited information on EDCs in drinking water and bottled water from South Africa. The aim of this study was to determine the estrogenic activity, levels of selected EDCs and the potential health risks associated with the consumption of water from selected distribution points in Pretoria (City of Tshwane) and Cape Town as well as bottled water. The study consisted of 3 phases. Phase 1 included the analysis of drinking water samples from ten water distribution points in Pretoria and Cape Town collected over four sampling periods. In phase 2, ten brands of bottled water were analysed after exposure to different storage conditions (20°C, 40°C, light and dark) for 10 days. Samples were extracted using a C18 solid phase extraction method. Estrogenic activity was assessed using the recombinant yeast estrogen screen (YES) bioassay and the T47D-KBluc reporter gene bioassay. The extracts were analysed for di(2-ethylhexyl) adipate (DEHA), di(2-ethylhexyl) phthalate (DEHP), diisononylphthalate (DINP), dibutyl phthalate (DBP), bisphenol A (BPA), nonylphenol (NP), 17β-estradiol (E2), estrone (E1) and ethynylestradiol (EE2) using UPLC-MS. Phase 3 consisted of a scenario based health risk assessment to assess the carcinogenic and toxic human health risks associated with the consumption of distribution point and bottled water. All the samples were below the detection limit (dl) in the YES bioassay, but estrogenic activity was detected in bottled and distribution point water using the T47D-KBluc bioassay. All samples were below the 0.7 ng/L trigger value for estrogenic activity in drinking water. NP was below the dl for all the samples, E2 was detected in five distribution point samples and E1, EE2, DEHA, DEHP, DINP, DBP and BPA were detected in distribution point and bottled water samples. The estrogenic activity and levels of target chemicals were comparable to the levels found in other countries. Hazard quotients for BPA, DEHA and DINP were higher in bottled water compared to distribution point water. The greatest non-carcinogenic health risk was posed by E1 in distribution point water from Pretoria and the highest cancer risk by levels of DEHP in distribution point water from Cape Town. However, overall, health risk assessment revealed acceptable health and carcinogenic risks associated with the consumption of distribution point and bottled water. Although the potential health risks posed by the EDCs found in the water samples in this study were low, the fact that potential EDCs were found in the water samples are still of concern. A monitoring strategy that also includes water from other municipalities and other brands of bottled water are therefore recommended. / Thesis (PhD)--University of Pretoria, 2016. / School of Health Systems and Public Health (SHSPH) / PhD / Unrestricted

Selective serotonin reuptake inhibitors (SSRIs), like fluoxetine, are widely used to treat depressive disorders during pregnancy. These antidepressants reach water reservoirs through sewage treatment facilities and expose the aquatic vertebrates, including fish. It has been shown that early-life exposure to fluoxetine could disrupt the normal function of the stress axis by decreasing the level of circulating glucocorticoids in humans, rodents, and teleosts. Our lab recently showed that early life exposure to fluoxetine resulted in transgenerational hyporcortisolism and altered exploratory behaviour in adult male zebrafish and their descendant male adults for at least three generations. In the current study, we used a stress-responsive transgenic zebrafish line (SR4G) that expresses green fluorescence protein (eGFP) under the control of six consecutive glucocorticoid response elements. The effects of developmental exposure to fluoxetine on the transcriptional profiles of genes in the larval head and male adult telencephalon and hypothalamus were analysed using high throughput RNA sequencing. We also assessed the potential of eGFP mRNA to evaluate blunted stress response as an alternative to cortisol immunoassay measurements. The effects of bisphenol A, vinclozolin and fluoxetine were ytested in the SR4G line. Developmental exposure to fluoxetine resulted in a life-long dysregulation of pathways involved in nervous system development, stress response, and lipid metabolism in both larvae and adult zebrafish. Numerous differentially expressed genes in zebrafish are orthologous to genes in Homo sapiens linked the development of the major depressive disorder and epigenetics regulation and include bdnf, trkb, npas4, per1, per2, dnmt3a, adarb1, adaeb2, hdac4, hdac5, hdac8, and atf2. It is suggested that the dysregulation of the primary transcription regulators of circadian rhythm (clocka) and stress response (nr3c1), amongst others, were the potential drivers of the observed life-long effects. Furthermore, we report on a significant positive linear correlation between cortisol levels and eGFP mRNA levels in SR4G transgenic zebrafish larvae (R2> 0.9). Random forest and logistic regression models trained by eGFP mRNA levels both correctly predicted the blunted stress response. The negative predictive value (NPV) for both models was 100%. Models based on the mRNA levels of 11 genes associated with neurogenesis, stress response and depression resulted in a similar 100% NPV. These findings provide evidence for a life-long effect of developmental exposure to fluoxetine. This study also provides a proof-of-concept for an in vivo biomonitoring assay to screen chemicals for their stress-disrupting potential.

Prenatal exposure to mixture N1, a chemical mixture consisting of four phthalate diesters,three pesticides and Bisphenol A, has been associated with behavioural changes as well as changes in gene expression in mice. In this study it was investigated whether the changes in gene expression could be explained by changes in DNA methylation. Mixture N1 was found to significantly change DNA methylation in three different genes (Nr3c1, Nr3c2 and Crhr1) on totally eight different Cytosine Guanine dinucleotides (CpG) positions. To further investigate whether these changes could be induced already during differentiation of cells, a hippocampal cell model HT22 was exposed to mixture N1 during differentiation. In this mode, mixture N1 induced a statistically significant change in the promoter region of Nr3c1. Unfortunately, this change could however not be validated, and the experiments would need to be repeated. In conclusion, this study showed that exposure to mixture N1 can result in changes in DNA methylation.

Cryptorchidism is a male congenital disorder with an unspecified, multifactorial etiology. This review evaluated the strength of select factors in the development of cryptorchidism to better understand its etiology. The strength of relationship between factors and their respective functions during testicular descent was evaluated. Factors evaluated in the causal pathway include the signaling mechanisms Desert Hedgehog (DHH), Insulin-like Hormone 3 (INSL3) and Platelet-Derived Growth Factor (PDGF), as well as sex hormone regulation (androgen: estrogen ratio, aromatase expression). Articles supporting a factor in testicular descent were evaluated and scored. These scores were summed to create the “Step Score” for each step in the causal pathway. An arrow system was developed which ranked the strength of each pathway step as either “weak”, “moderate” or “strong”. Thus, step scores and the strength of factors in the pathological pathway were determined: DHH (15-moderate), PDGF (10-weak), INSL3 (24-strong) and Androgen: Estrogen ratio, Aromatase (23-strong). The pathological pathway produced by this review represents a literature based perspective of the research regarding cryptorchidism etiology. Literature indicates that prenatal exposure to endocrine disrupting chemicals in animals and humans may lead to abnormal genital development. Recently, prenatal maternal cigarette smoke was demonstrated to be a risk factor for cryptorchidism. This controversial finding was explored in the context of endocrine disrupting chemicals. However, literature has provided very little evidence in support of this hypothesis and more research is needed to better evaluate prenatal maternal smoking as a risk factor for undescended testis.