Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Malgré l’efficacité thérapeutique avérée des agents anti-angiogéniques et des agents anti-vasculaires (VDA), le mécanisme d’action précis des stratégies ciblant les vaisseaux sanguins tumoraux, les raisons de leur efficacité ainsi que les mécanismes de résistance à ces drogues sont encore mal compris. Il est rapidement apparu essentiel d’identifier des biomarqueurs capables de refléter l’angiogénèse tumorale ou les effets sur la vascularisation tumorale de ces traitements. Compte tenu de leur importance dans des pathologies vasculaires, les cellules endothéliales matures circulantes (CEC) et les progéniteurs endothéliaux circulants (CEP) ont d’emblée été pressenties comme des candidats intéressants pour être des biomarqueurs de réponse aux stratégies ciblant la vascularisation tumorale. Nous avons exploré l’intérêt de ces cellules en tant que biomarqueurs de l’angiogénèse dans des tumeurs pédiatriques, et leur rôle en tant que biomarqueurs de traitement par des agents anti-angiogéniques chez des sujets adultes atteints de cancer. Ces travaux ont mis en lumière l’intérêt des CEP et ont été à la source d’un travail plus « mécanistique » où nous avons étudié dans différents modèles murins le rôle des CEC et CEP dans le mécanisme d’action des agents anti-vasculaires et plus particulièrement le rôle fonctionnel des CEP dans la résistance à ces molécules. Par des stratégies d’association d’agents anti-angiogéniques aux VDA destinées à inhiber les CEP, nous montrons l’augmentation de l’activité anti-tumorale des VDA et offrons un rationnel mécanistique pour optimiser les schémas thérapeutiques actuels des traitements anti-vasculaires. Nos données apportent des arguments en faveur du rôle potentiel de ces cellules en tant que biomarqueurs de l’angiogénèse, des traitements anti-angiogéniques et de la résistance aux traitements anti-vasculaires. / Despite their therapeutic impact and clinical benefit, the mecanisms of action of anti-angiogenic agents and vascular disrupting agents (VDA), the reasons for their efficacy as well as the mechanisms underlying resistance to these drugs are not fully understood. Thus, identifying surrogate biomarkers of tumor angiogenesis and of the effects of these new therapeutic agents targeting tumor blood vessels has become a crucial objective. Because of their importance in vascular diseases, mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (CEP) were suggested to be potential candidate biomarkers of disease response and relapse to vascular targeting strategies. We investigated the role of these cells as biomarkers of tumor angiogenesis in pediatric solid tumors, as well as biomarkers of response to anti-angiogenic therapies in adult cancer patients. By revealing the particularly important role of CEP, these initial studies led to a more “mechanistic” study in which the cellular and molecular effects of a VDA were evaluated with regard to CEC and CEP in different mouse models; in particular, the “catalytic” role of CEP was explored as a mechanism of resistance to VDA. By combining anti-angiogenic agents aimed to inhibit CEP mobilized by the VDA, we demonstrate an increase in the anti-tumor activity of the VDA and offer a mechanistic rational to optimize VDA-based therapeutic strategies. Our data support the role of CEC and CEP as biomarkers of angiogenesis, of anti-angiogenic strategies and of resistance to vascular-disrupting therapies.

Endocrine-disrupting chemicals (EDCs) are known to cause endocrine disruption (ED), developmental neurotoxicity (DNT), infertility and impaired embryo development. EDCs do therefore impose a threat to humans, wildlife and the environment. The present study investigated the effects of the reference compounds dihydrotestosterone, estradiol, benzo(a)pyrene, rosiglitazone, as well as the EDCs bisphenol F and perfluorooctanesulfonic acid at various concentrations on zebrafish embryos. The scientific questions of the study were to investigate how these environmental pollutants impact the development of zebrafish, what their molecular mechanisms are and what the link between ED and DNT is. Zebrafish embryos were exposed for 5 days to the compounds and various parameters on development were collected at different time points. The expression of 41 genes (qPCR) related to ED and DNT, and the levels of 23 steroids (LC-MS/MS) were determined. Gene correlations were determined with Pearson’s correlation test and paired t-tests were used to determine significantly altered gene activities. The significant gene expression changes were further related to the pathways of steroids in order to connect how gene activity impacted steroid levels. Exposure to estradiol, dihydrotestosterone and bisphenol F induced cyp19a1b expression which can affect personality traits. Perfluorooctanesulfonic acid interferes with thyroid hormone transport by binding to TTR causing profound effects on neurodevelopmental processes and cognitive functions. The compounds influenced genes that can disrupt endocrine systems which can cause neurodevelopmental impairments.

No description available.

Humans experience ubiquitous exposures to estrogenic environmental chemicals from food, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. In our study, pregnant CD-1 mice were orally exposed to BPS or ethinyl estradiol (EE2, a positive control for estrogenicity) from gestational day 9 through postnatal day (PND) 2, the period when many estrogen-sensitive organs are developing. After weaning, the offspring were administered either oil (vehicle) or an estrogen challenge (1 μg EE2/kg/day) for ten days starting at PND21 (prior to puberty), PND80 (early adulthood), or PND260 (later adulthood). Timing of puberty was evaluated in females by noting the date on which vaginal opening occurred. After the 10 day estrogen challenge, we evaluated the response of endocrine sensitive organs through measurements of organ weight, tissue morphology, and gene expression in both males and females. We observed dose- and sex-specific effects of BPS and EE2 treatment, as well as alterations in the responses of males and females to the estrogen challenge. This study sheds light on the effects of low dose xenoestrogen exposures on estrogen-sensitive organs including the reproductive tract and mammary gland. Furthermore, it improves our understanding of the influence of environmental chemicals on secular trends of earlier age of puberty in girls reported over the past few decades.

Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted, followed by a qPCR and bisulfite-pyrosequencing to investigate the changes in gene expression and DNA methylation of NR3C1 and GRIN2B. This study established the differentiation protocol but revealed no significant results regarding the chemical exposure. However, some chemical exposures showed a clear tendency towards an impact of the chemicals on the gene expression and the DNA methylation. Furthermore, a negative correlation between DNA methylation at 2 CpG sites and gene expression in NR3C1 could be observed. In conclusion, the DNA methylation at promoter region in NR3C1 is important for the gene expression.

This study was carried out to evaluate the performance of Soil Aquifer Treatment (SAT) under different loading regimes, using wastewater of much higher strength than usually encountered in SAT systems, and also to investigate the removal of the endocrine disruptors triclocarban (TCC), estrone (E1), 17¿-estradiol (E2) and 17¿- ethinylestradiol (EE2). SAT was simulated in the laboratory using a series of soil columns under saturated and unsaturated conditions. Investigation of the removal of Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD), Dissolved Organic Carbon (DOC), nitrogen and phosphate in a 2 meter long saturated soil column under a combination of constant hydraulic loading rates (HLRs) and variable COD concentrations as well as variable HLR under constant COD showed that at fixed HLR, a decrease in the influent concentrations of DOC, BOD, total nitrogen and phosphate improved their removal efficiencies. It was found that COD mass loading applied as low COD wastewater infiltrated over short residence times would provide better effluent quality than the same mass applied as a COD with higher concentration at long residence times. On the other hand relatively high concentrations coupled with long residence time gave better removal efficiency for organic nitrogen. Phosphate removal though poor under all experimental conditions, was better at low HLRs. In 1 meter saturated and unsaturated soil columns, E2 was the most easily removed estrogen, while EE2 was the least removed. Reducing the thickness of the unsaturated zone had a negative impact on removal efficiencies of the estrogens whereas increased DOC improved the removal in the saturated columns. Better removal efficiencies were also obtained at lower HLRs and in the presence of silt and clay. Sorption and biodegradation were found to be responsible for TCC removal in a 300 mm long saturated soil column, the latter mechanism however being unsustainable. TCC removal efficiency was dependent on the applied concentration and decreased over time and increased with column depth. Within the duration of the experimental run, TCC negatively impacted on treatment performance, possibly due to its antibacterial property, as evidenced by a reduction in COD removals in the column. COD in the 2 meter column under saturated conditions was modelled successfully with the advection dispersion equation with coupled Monod kinetics. Empirical models were also developed for the removal of TCC and EE2 under saturated and unsaturated conditions respectively. The empirical models predicted the TCC and EE2 removal profiles well. There is however the need for validation of the models developed / Netherlands Organisation for International Cooperation in Higher Education (Nuffic) / The Appendix files for this thesis are unavailable online via Bradford Scholars.

<p> Per- and polyfluoroalkyl substances (PFAS) are a class of persistent environmental contaminants that have become ubiquitous, resulting in widespread exposure among humans and wildlife. Amphibians are regularly exposed in the field, making them susceptible to sublethal effects of PFAS exposure. In amphibians exposed to PFAS, deleterious effects have been observed, including reduction in body condition measured using the scaled mass index (SMI) and degraded hepatic condition, among others. PFAS may dysregulate lipid metabolism by altering signaling cascades regulated by peroxisome proliferator activated receptors (PPAR), but whether changes in energy stores can explain changes in amphibian SMI and/or hepatic condition remain underexplored. Since lipids are a critical energy reserve for anurans, understanding whether lipid metabolism is being perturbed is critical. The central objective of this thesis was to investigate the effect of PFAS on lipid homeostasis in <em>Xenopus laevis </em>tadpoles within the context of a PPAR mechanism of action (MOA), considering apical, molecular, and lipidomic endpoints. I conducted three studies: (a) a study to characterize SMI and the relative expression of the hepatic xPPARα/β/γ during metamorphosis, (b) a pharmaceutical exposure to assess the <em>in vivo</em> effects of xPPARα/β/γ agonism on hepatic gene expression for select downstream targets (<em>apoa5, fabp1, acox1,​ pck1</em>), and (c) a chronic PFAS exposure to investigate the effects of environmentally relevant concentrations (PFOS, PFHxS, PFOA, PFHxA at 0.5 ppb; binary mixture of PFOS:PFHxS at 1 ppb) on lipid homeostasis through apical endpoints (mass, snout vent length, SMI, hepatic condition), relative hepatic gene expression, and Multiple Reaction Monitoring (MRM) profiling of the hepatic lipidome for changes in relative class abundance. In study (a), I identified SMI and hepatic expression of <em>xPPARα/β/γ</em> is dynamic during late metamorphosis, indicating the potential for heightened susceptibility. However, in study (b), pharmaceutical agonists had no effect on <em>X. laevis</em> at high doses. For study (c), I did not observe effects on a majority of apical endpoints, including SMI, but detected a significant sex-specific reduction in hepatic condition for male<em> X. laevis</em> tadpoles exposed to single-chemical perfluorosulfonic acid (PFSA) treatments. For gene expression, I observed a transient downregulation for apolipoprotein-V (<em>apoa5</em>) at Nieuwkoop and Faber (NF) stage 62 for <em>X. laevis</em> tadpoles exposed to single-chemical perfluorocarboxylic acid (PFCA) treatments. Lipid profiling detected transient dysregulation of predominantly membrane lipids in-response to short-chain PFAS treatments at NF 58. Overall, our findings indicate PFAS may exert toxicity during anuran metamorphosis through multiple mechanisms of action (MOA) with sex-specific and developmental-stage specific outcomes.</p>

Yes / Despite significant preclinical promise as anticancer agents, vascular-disrupting agents have yet to fulfil their clinical potential due to systemic toxicities. ICT2588 is a tumour-selective MT1-MMP-targeted prodrug of azademethylcolchicine, ICT2552. We investigate activation of ICT2588 and subsequent release of ICT2552 in tumour cells, and examine its ability to induce G2/M cell cycle arrest. We also explore synergism between ICT2588 and ATR inhibition, since colchicine, in addition to its vascular-disrupting properties, is known to induce G2/M arrest, DNA damage, and trigger apoptosis. Several ATR inhibitors are currently undergoing clinical evaluation. The cellular activation of ICT2588 was observed to correlate with MT1-MMP expression, with selective release of ICT2552 not compromised by cellular uptake and prodrug activation mechanisms. ICT2588 induced G2/M arrest, and triggered apoptosis in MT1-MMP-expressing cells, but not in cells lacking MT1-MMP expression, while ICT2552 itself induced G2/M arrest and triggered apoptosis in both cell lines. Interestingly, we uncovered that the intracellular release and accumulation dynamics of ICT2552 subsequent to prodrug activation provided synergism with an ATR inhibitor in a way not observed with direct administration of ICT2552. These findings have important potential implications for clinical combinations of ICT2588 and DNA repair inhibitors.

Food is one of the main routes by which xenobiotic (synthetic) chemicals enter the body of man and wildlife. The routes could be from wrappers in which the foods are presented with possible transfer of the compounds to consumers, hence need for regular screening. The research work is aimed at investigating possible prevalence of phthalate esters in selected foods (cheese, polony and vienna) and their plastic wrappers from commercial stores in Tshwane metropolis. Food samples were purchased from selected stores, taken to the laboratory and stored at 4oC until analysed. Analysis was done by soxhlet extraction while determination and quantification of phthalates was carried out using Gas Chromatography-Flame Ionization Detection (GC-FID). Quality assurance of the process was by standard addition of the phthalate ester standards. Results obtained revealed good chromatographic separation of the analysed esters which ranged from 5.55 min for Dimethyl phthalate (DMP) to 8.96 min for Benzylbutyl phthalate (BBP). Instrumental detection limit of the esters varied from 0.03 - 0.05 μg/kg. The percentage recovery of the phthalate esters ranged from 75 – 90% from spiked cheese samples; 33 – 66% from spiked polony samples and 69 – 99% from spiked vienna samples. These recoveries are quite acceptable and applicable to the analysis and quantification of the compounds in the samples with the exception of Dibutyl phthalate (DBP) (33%); DMP (34%) and BBP (46 %) in polony samples. Results from chromatographic quantification revealed the absence of or non-detection of most of the analysed phthalate esters in the selected food samples. However, level of 0.031 μg/kg of BBP - 0.816 μg/kg of DMP were obtained in some of the analysed samples. / Environmental Sciences / M.Sc. (Environmental Science)