Human health depends on a well-functioning endocrine system to regulate hormone release for normal bodily functions. Endocrine disrupting chemicals (EDCs) constitutes a group of chemicals, included in many commonly used products, (e.g., PVC flooring), with properties proven or suspected to interact with the natural hormone system in humans and animals. EDCs are manmade compounds (there are also natural compounds with such properties) that can bind to cell receptors and mimic or block the functions of natural hormones potentially resulting in adverse human health effects. One type of widely concerning EDC is phthalates. Since phthalates create weak chemical bonds when they are added into different products, they readily leach into the surrounding environment. Phthalate metabolites can therefore be frequently measured in human biological samples. Major public health concerns regarding EDCs over the past three decades have focused on phthalates. According to the World Health Organization, EDCs are suspected to be associated with altered reproductive function in males and females, increased incidence of breast cancer, abnormal growth patterns and neurodevelopmental delays in children, as well as changes in immune function. The thesis shows that PVC flooring in the home is a source for human uptake of phthalates, that replacement of phthalates in soft PVC products have an impact on human uptake of these chemicals, and that exposure for phthalates in early life increase the risk for airway disorders in children. As we have shown, ongoing regulations on phthalates is of importance for human uptake. Ultimately, individuals can make small changes in their consumer product choices that can lead to changes in uptake of chemicals with endocrine disrupting properties. Philosophically, we all have a responsibility to protect future generations from dangerous chemicals. / Human health depends on a well-functioning endocrine system to regulate hormone release for normal bodily functions. Endocrine disrupting chemicals (EDCs) constitutes a group of chemicals, included in many commonly used products, (e.g., PVC flooring), with properties proven or suspected to interact with the natural hormone system in humans and animals. One type of widely concerning EDC is phthalates. Since phthalates create weak chemical bonds when they are added into different products, they readily leach into the surrounding environment. Phthalate metabolites can therefore be frequently measured in human biological samples. Major public health concerns regarding EDCs over the past three decades have focused on phthalates resulting in implementation of regulations. The thesis shows that PVC flooring in the home is a source for human uptake of phthalates, that replacement of phthalates in soft PVC products have an impact on human uptake of these chemicals, and that exposure for phthalates in early life increase the risk for airway disorders in children. This means that regulation and consumers’ product choices can lead to changes in uptake of EDCs of importance for human health. Philosophically, we all have a responsibility to protect future generations from dangerous chemicals.

Endocrine Disrupting Chemicals (EDC) are exogenous agents that mimic endogenous hormone activity in the body. EDC exposure during the critical period of neonatal development can potentially cause life-long neurological, behavioural and physiological disease. This thesis focuses on the EDC Bisphenol A (BPA), a synthetic xenoestrogen widely prevalent in everyday materials that has significant environmental relevance given its ubiquitous presence in humans around the world. The central research question of my thesis is: Does perinatal exposure to BPA affect rat pup development? A rodent model was selected to study the effects of BPA on the adrenal component of the hypothalamic-pituitary-adrenal axis (HPA axis) stress pathway, which has not been extensively studied. Rat dams were divided into five groups (vehicle control (VEH), positive control diethylstilbestrol (DES), BPA 5, BPA 50 and BPA 500 μg/kg bw/day) and dosed daily throughout gestation and for four days of lactation. Rat pups were sacrificed at two time-points at the beginning and the end of the stress hyporesponsive period (SHRP), at postnatal day (PND) 5 and PND 15. Changes in three morphometric parameters (bodyweight, crown-rump (CR) length and anogenital distance (AGD) were assessed based on the factors of Treatment and Sex. Adrenal gland glucocorticoid receptor (GR) and 18SrRNA expression was determined by qPCR in male pups at PND 5 and PND 15. At PND 5, compared to the VEH group, the BPA 50 pups were significantly heavier (ANOVA, Dunnett’s post-hoc) and the DES and BPA 50 pups had significantly longer CR lengths (ANOVA, Dunnetts’ post-hoc). At PND 15, xenoestrogen treatment significantly influenced CR length (ANOVA). At both time-points, males had significantly longer AGD than females, as physiologically expected (ANOVA). Adrenal gland GR expression in male pups was not significantly affected by treatment, but there was an effect of treatment in18SrRNA gene expression at PND 5 (Kruskal-Wallis). Using the Ct method to determine GR and 18SrRNA fold changes, we cautiously suggest that our experimental doses resulted in a non-monotonic dose response to BPA in the PND 5 animals and a monotonic dose response to BPA exposure in the PND 15 animals. This study highly values the importance of investigating the effects of environmentally relevant, low dose BPA exposure during the critical window of development, given the little that is known about potentially permanent alterations to the stress pathway due to exposure during this delicate period of development.

D.Phil. / The endocrine system regulates important physiological processes in an organism. Numerous natural and synthetic chemicals are released into the environment and can disrupt the normal functioning of the endocrine system, influencing physiological processes even at relatively low concentrations. These chemicals are known as endocrine disrupting chemicals (EDCs). These compounds are highly persistent in nature due to a resistance to biodegradation, and because of their hydrophobicity. EDCs bio-accumulate especially in the fatty tissue of aquatic and terrestrial organisms. Developmental and reproductive disorders are potentially harmful effects of exposure. EDC sources include agricultural and urban runoff, and industrial effluent discharges that eventually pass through sewage- and wastewater treatment plants. These chemicals degrade at various rates and to varying extents during treatment processes, forming many new chemicals. Methods to predict the occurrence of endocrine disruption include the occurrence of intersex, and changes in spermatogenesis and secondary sexual characteristics (SSCs) of supposedly exposed organisms. The urogenital papilla (UGP) is a SSC whose development and morphology is hormonally controlled and may be influenced when exposed to EDCs. The sharptooth catfish (Ciarias gariepinus) is often used in environmental toxicity studies. Sex is determined by the external dimorphic UGP found only in males. Intersex has been observed in this species in South African freshwater systems. A field study was carried out in the Rietvlei (RVD) and Marais (MD) dams in the Rietvlei Nature Reserve (RNR). MD acts as a sedimentation dam before water enters RVD, and is theoretically more polluted. The RNR system receives effluent from various domestic and industrial sources including agriculture, industries, informal settlements and municipal sewage treatment plants. Four sampling surveys, two low and two high flow, were carried out over a period of two years. Supposedly male catfish (n=97) were collected and identified as male or possible intersex based on the UGP. Water and sediment was collected for target EDC and endocrine disrupting metal (EDM) analyses. Fish length and mass, and UGP length and width were measured. Laparotomy was performed and gonads were inspected macroscopically for alterations. A section of the gonad and the entire UGP was removed and processed according to standard techniques for histological analysis. The gonadosomatic- (GSI), hepatosomatic- (HSI) and UGP length (UGPLI) indices were calculated. Fish fat was removed and analysed for target EDCs.

L’objectif général de cette thèse concerne la mise en place et l’évaluation d’une approche de type effect directed analysis (EDA), couplant bioessais in vitro et analyses physico-chimiques, pour la caractérisation de la contamination des milieux aquatiques par les perturbateurs endocriniens (PE) et leur identification. De nouveaux outils in vitro ont été développés et intégrés à une approche multi-bioessais pour la détection de polluants émergents afin de permettre un large diagnostic de la contamination par les PE. Parmi eux, le bioessai basé sur le pregnane X receptor humain s’est avéré complémentaire des cibles classiquement étudiées (e.g. récepteurs des œstrogènes (ER), des androgènes (AR) ou des hydrocarbures aromatiques (AhR)). Combinés, ces bioessais ont permis de déterminer des profils intégrés de la contamination inédits dans le sédiment et les eaux de surface, mettant en lumière la nécessité de considérer d’autres cibles biologiques que ER et AhR et de s’intéresser à l’ensemble des compartiments du milieu aquatique pour une meilleure évaluation de la contamination. En particulier, l’utilisation combinée de ces nouveaux outils à des capteurs passifs de type POCIS (polar organic compound integrative sampler) s’est révélée pertinente pour évaluer la contamination de la colonne d’eau par des composés hydrosolubles. Enfin, une démarche EDA, intégrant ces bioessais et des méthodes physico-chimiques d’extraction et de fractionnement adaptées, a été validée pour l’identification des composés détectés. Son application sur deux sites soumis à des contaminations chimiques différentes (mixte urbaine/agricole et pharmaceutique) a permis d’évaluer la contribution des polluants cibles, dont les prioritaires, dans les réponses des bioessais et d’identifier de nouveaux PE. / The aim of this thesis was to develop and to evaluate an effect directed analysis (EDA) approach, combining in vitro bioassays and chemical analysis for the characterization of aquatic systems contamination by endocrine disrupting chemicals (EDCs) and their identification. For integrative contamination assessment, new in vitro tools have been developed and evaluated for the detection of emerging pollutants, and then integrated in a multi-bioassays approach. In particular, the human pregnane X receptor (PXR) based bioassay provided further information on bioactive contaminants and thereby enhanced the environmental diagnostic as regard to classical biological target (e.g. estrogen (ER), androgen (AR) or aryl hydrocarbon receptors (AhR)). Such multi-receptors approach allowed determining novel integrative toxicity profiles in sediment and surface water. Overall, our results demonstrated the need to integrate other biological target than ER and AhR, and to assess contamination partitioning for a better hazard/risk assessment. For such purpose, combined use of polar organic compound integrative sampler and in vitro bioassays provided suitable tools to assess active hydrosoluble chemicals in surface water. Finally, in order to identify the compounds responsible for the detected activities, the multi-receptors approach has been integrated in EDA strategy applied to two river sites under different chemical pressures (urban/agricultural and pharmaceutical). To tackle this challenge, extraction and fractionation methods were developed and evaluated in order to encompass a diversity of active chemicals that are detected by in vitro bioassays. The EDA approach allowed determining partial contribution of priority pollutants in biological responses as well as the identification of new EDCs.

Endocrine disrupting chemicals (EDCs) are emerging contaminants that can interfere with the hormone system and may cause cancers, birth defects and reproductive system disorders. Prevalence of endocrine-related dysfunction and disease has increased steadily over the past decades. Although accumulating data suggest that these diseases have fetal origins, associations of EDC exposure during pregnancy and adverse health effects on both mothers and fetuses have not been thoroughly evaluated, particularly at multiple points in time. We firstly developed an analytical method for quantification of 28 EDCs (9 phthalates, 8 bisphenols, 5 parabens, 5 benzophenones and triclosan) in urine samples using ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometer. The method was applied to measure targeted compounds in a total of 5220 urine samples collected from 951 pregnant women at three trimesters and 1501 pregnant women at one or two trimesters in Wuhan, China between 2014 and 2015. Based on the quantification results, exposure patterns and health risks of 28 EDCs on participants were evaluated and discussed in detail below. Among these samples, bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), 4-hydroxybenzophenone (4-OH-BP), 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), triclosan, mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHHP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MnBP), monoisobutyl phthalate (MiBP) were determined with detection rates exceeding 50%, in which BPA, BP-3, MeP and MnBP were the predominant compounds. We found the U-shaped trends of urinary concentrations of phthalate metabolites over trimesters. Parabens, benzophenones and triclosan displayed a downward trend over three visits. We also found the levels of targeted compounds varied by exposure-related factors, such as sampling seasons, physical activities, computer using time and decoration information. In addition, multiple EDCs were mostly determined at low doses over trimesters, indicating that real-world exposure of pollutants were dominated by low-dose mixtures. We then evaluated the combined health hazards induced by EDC exposure via calculating the estimated daily intakes on the basis of average urinary concentrations at three trimesters. It was found that 24.9% of participants had potential health risks caused by exposure to phthalate mixtures. The most frequency of cumulative risks occurred in women who were exposed to a high dose of one specific phthalate, di-n-butyl phthalate (DnBP) or di(2-ethylhexyl) phthalate (DEHP). We also evaluated the cumulative health risks of BPA and its alternatives and found that about 1.6% of participants were at risks induced by bisphenol exposure. Combined health hazards were mainly driven by one specific bisphenol (BPS or BPA). Our findings suggested that regional interventions of DnBP, DEHP, BPA and BPS in application and production should be tighten and/or taken. Considering the low-dose effects of BPA, we further investigated the associations of BPA and three major natural estrogens, including estrone (E1), estradiol (E2) and estriol (E3), at three trimesters of pregnancy. We observed non-monotonic dose-response relationships of BPA to E1, E2 and E3 over trimesters even when BPA concentrations were below the current safety thresholds. In the gender-stratified models, we found significant negative relationships (β < 0, p < 0.05) between BPA and E2 among mothers with male fetuses in the first trimester. However, we found that no significant relationship between BPA and E2 among mothers with female fetuses over three trimesters. Significant non-monotonic associations (from significant negative to positive associations) between BPA and E3 were observed among mothers with female fetuses in the second trimester. The above mentioned findings suggested the gender-specific and trimester-specific effects of BPA on estrogens. Our findings also indicated that the current tolerance daily intake value maybe not safe enough to evaluate the potential health risks induced by BPA exposure. We next investigated the effects of maternal exposure to phthalates on both mothers and fetuses. Associations of phthalate exposure with the risks of gestational diabetes mellitus (GDM) and plasma glucose levels were evaluated based on a nested case-control study design. It was found that the levels of phthalate metabolites in women with GDM were significantly higher than those without GDM. Meanwhile, positive associations between urinary concentrations of phthalate metabolites and the risks of GDM were obvious, indicating that phthalate exposure may be a risk factor for GDM. In addition, phthalate levels were related to the increased plasma glucose levels after 75 g oral glucose tolerance test. Our findings suggested that phthalates might disturb the glucose homeostasis and increase GDM risks. Furthermore, we assessed the trimester-specific and gender-specific effects of DEHP exposure on fetal growth, birth size and postnatal growth at 6, 12 and 24 months. We found that among male offspring, 1st-trimester DEHP was negatively related to fetal growth (β < 0, p < 0.05), but positively related to 24-month body mass index (BMI). 2nd-trimester DEHP was negatively related to fetal growth, birth weight and birth length, but positively related to the weight gain rates from birth to 12 months old. 3rd-trimester DEHP was positively (β > 0, p < 0.05) associated with birth weight, BMI at 6 and 12 months. However, among females, 1st-trimester DEHP was associated with increased birth length, while 2nd-trimester DEHP was negatively associated with BMI at 6 and 12 months. A negative association between DEHP and weight gain rates at 6 months was noted among females. Our findings indicated the second trimester maybe the sensitive window of DEHP exposure for offspring growth since 2nd-trimester DEHP levels were related to the decreased fetal growth, decreased birth size, but increased weight gain rates in early childhood age among male offspring. To investigate the mechanism underlying the associations of DEHP exposure with glucose and lipid metabolism, we investigated the biotransformation of DEHP and the disturbed metabolisms induced by MEHP, the putative toxic metabolite of DEHP, in human normal liver cell L02 using metabolomics and lipidomics. We found that MEHP was the major metabolite of DEHP. Decreased uptake of glucose and accumulation of glucose in liver cells were obvious after MEHP exposure. Phospholipid remodeling, incomplete fatty acid β-oxidation, inhibition of purine metabolism and glycolysis, and increased oxidative stress were noted in MEHP-exposed L02 cells, which were related to insulin resistance. In this work, we measured 28 EDCs in a total of 5220 urine samples provided by 951 pregnant women (three trimesters) and 1501 pregnant women (one or two trimesters) and then evaluated the exposure levels, exposure patterns (variations, variability and correlations), health risks and health effects of these compounds on pregnant women and fetuses. Our data suggested that participants had potential health risks induced by exposure to phthalates or bisphenols. Phthalate exposure was related with the increased plasma glucose levels and risks of GDM. Prenatal DEHP exposure may induce the intrauterine growth restriction and catch-up growth among males, which supported the evidence of fetal origin. To explore the underlying mechanisms of MEHP on glucose and lipid metabolic disorders, we exposed the human normal hepatic L02 cells with MEHP, and applied metabolomic and lipidomic approaches for finding potential biomarkers and disturbed pathways. We found that MEHP exposure inhibited glucose uptake, caused phospholipid remodeling and increased oxidative stress in L02. These findings suggest that the usage of products containing EDCs, particularly phthalates, in pregnant women should be limited in China, intervention of BPS should be considered, and threshold values of BPA are called for reevaluation.

Estrogen compounds are a subgroup of the environmental pollutants named Endocrine Disrupting Chemicals. It is a large group of chemicals which are capable of causing hormonal imbalance of an organism and affecting its reproductive, developmental or behavioral functions. In the environment, they are found in very low concentrations (below ng/l). This makes their detection and elimination highly problematic. The Research Centre for Toxic Compounds in the Environment (RECETOX) has been developing an experimental device which employes reverse osmosis (RO) to concentrate the trace amounts of micropollutants (e.g. estrogens). The aim is to lower the detection limit of subsequent analytical methods. RO seems to be a promising alternative to commonly used methods which are more time consuming and costly. The aim of this thesis is to create a mathematical model of RO, which would describe the process of estrogen concentration and therefore could contribute to the optimization of experimental device. The mathematical model was created in MATLAB-simulink and verified by comparison to thirteen experimental results. The solutions used to test the model were NaCl at 0.002–2 g/l and drinking water with 17-ethinylestradiol of 25 ng/l. In order to concentrate the retentate on a flat-sheet RO membrane the model achieves a mean relative error in the range of 0.9–4.9 % for NaCl and drinking water. For the concentration of estrogens the deflection is 3.6 %. Comparing the model and experimental concentration of NaCl on a spiral wound RO-membrane, the deviation is in the range of 5.9–8.7 %. So far, ratio of retentate volume from the beginning to the end of the process has been used to determine the process recovery. However, due to the results obtained in the thesis, this approach appears inaccurate. A more accurate recovery would likely be achieved by using the theoretical concentration value obtained by the proposed mathematical model. However, to increase reliability, the further measurements with estrogens are needed, and also to determine the causes of deflection of model and experimental retentate volume values at the end of the concentration

In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...

Magister Scientiae (Medical Bioscience) - MSc(MBS) / 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: v 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.

Thesis (DTech (Environmental Health))--Cape Peninsula University of Technology, 2012. / Environmental pollution with persistent organic chemicals and inorganic trace metals is an increasingly important issue. Recently, a variety of chemicals are introduced in a very large scale on the surface water network. The main pathway of these pollutants into the environment was identified as wastewater treatment plants (WWTPs). The extended use of chemicals in many product formulations and insufficient WWTPs has lead to an increase in the levels of the detected micro-pollutants wastewater effluents. The majority of these compounds are characterized by a rather poor biodegradability. A large spectrum of pollutants present in waste as traces has been reported to exert adverse effects on human and wildlife. Even though compounds are found in wastewater in a very small amount, they may have the undesirable capability of initiating health effect on various high forms of life. This survey constitutes the first study in the City of Cape Town to report data for a variety of priority substances (phenols and phthalate esters) in WWTP effluents and receiving rivers. These results are of critical importance since the data generated are used to generate potential health risk associated with both the organic and inorganic compounds analyzed.

Spiral artery (SA) remodeling is a fundamental process during early pregnancy that involves the action of vascular smooth muscle cells (VSMCs), maternal immune cells, but also fetal extravillous trophoblast cells (EVTs) and the extracellular matrix (ECM). Mast cells (MCs) and their mediator chymase, have been identified as prominent players for a sufficient SA remodeling process at the fetal-maternal interface in vivo. In contrast, endocrine disrupting chemicals (EDC), especially Bisphenol A (BPA) and 17-α-ethinylestradiol (EE2), have been shown to have a negative impact on SA remodeling in animal models in vivo. However, neither the mechanisms underlying the positive effects of MCs and chymases for the remodeling process, nor the interference of EDCs on SA remodeling, have been elucidated. The purpose of the present work was to evaluate the effect of MCs and recombinant human chymase CMA1 (rhuCMA1) on the phenotype and/or behavior of VSMCs, EVTs and ECM in vitro. Moreover, the influence of BPA and EE2 on the functionality of EVTs in vitro was observed. Using an immortalized human trophoblast cell line (HTR8/SV-neo), a mouse trophoblast cell line (SM9-2), human primary uterine vascular smooth muscle cells (HUtSMCs) and mouse primary uterine smooth muscle cells (SMCs), we assessed the effects of the human MC cell line HMC-1, the mouse mast cell line MC/9 and rhuCMA1 on VSMCs, EVTs and ECM. Additionally, the HTR- 8/SV-neo cells functionality was evaluated after treatment with BPA or EE2. We found that mouse MC/9 cells induce fibronectin expression and migration in SMCs. Furthermore MC/9 cells increase the proliferation and migration of SM9-2 cells. Both human HMC-1 cells and rhuCMA1 stimulate the migration, proliferation, and change of synthetic/contractile marker expression in HUtSMCs. In addition, HMC-1 cells increase the proliferation and migration of HTR8/SVneo trophoblast cells while having an impact on the expression of tissue remodeling genes. HTR8/SVneo cells presented increased migration rates along with decreased expression of the matrix-metalloproteinase regulator genes (TIMPs) upon treatment with rhuCMA1. Moreover, BPA interfered with HTR-8/SV-neo cell proliferation and reduced MMP2 expression in HTR-8/SV-neo. Interestingly, EE2 had no impact on proliferation or migration but suppressed the MMP2 expression in HTR-8/SV-neo cells. The obtained results indicate that MCs, and partly their mediator chymase CMA1, shape the phenotype and modulate the functionality of VSMCs and EVTs. Collectively, possible mechanisms by which MCs and specifically rhuCMA1 promote SA remodeling were identified. The findings are relevant for the understanding of this crucial step in pregnancy and thus, for the comprehension of dysregulated pathways that can lead to pregnancy complications like fetal growth restriction and preeclampsia. Moreover, this work contributes to the knowledge about how EDCs impact on early pregnancy and highlights the high risk of EDCs exposure disturbing the fundamental reproductive process of SA remodeling.