The endocrine system is vulnerable to a range of chemicals in the environment. Endocrine disrupting chemicals (EDCs) are exogenous agents that can induce responses on the endocrine system because of their hormone-like activity and toxicity. Specific to this study are thyroid disrupting chemicals (TDCs), these are EDCs that specifically disrupt the thyroid hormone signalling pathway, and this may result in adverse health effects. Thyroid hormones play a crucial part in metabolism, growth, maintenance of brain function and fertility; hence disruption of the thyroid signalling axis implicates human health. We are exposed to TDCs regularly, and studies have shown an association between TDC exposure and neurobehavioural disorders, reproductive abnormalities and obesity. There is a lack of data associated to thyroid hormone receptor activity in surface and drinking water. Hence, the potential human health risks posed by thyroid disruption may therefore be underestimated. The aim of the study was to optimise and validate the GH3.TRE.Luc reporter gene bioassay that can measure thyroid hormone receptor mediated activity and cytotoxicity in drinking and source water, with relevance to water monitoring. The GH3.TRE.Luc reporter gene bioassay was established, optimized and validated to detect thyroid hormone receptor activity. The luciferace assay was used to test for metabolic activity and the resazurine cell proliferation assay was used to assess cell viability. The assay was applied to compounds with agonistic and antagonistic properties; triidothyronine (T3), thyroxine (T4), triac, tetrac, amiodarone, sodium arsenite, pentachlorophenol (PCP), ethylene thiourea, 2,2,4,4-tetrahydroxybenzophenone (THBP) and methimazole. It was also applied to environmental and drinking water samples from the Global Water Research Coalition (GWRC). Finally, the assay was applied to 48 water samples from a water treatment plant in South Africa, collected over a period of 12 months. Every month, four samples were collected. Two samples were source water samples, with one going into the treatment plant and coming out as 2 distribution pipelines (drinking water). For optimisation and validation, the dose response curves obtained for T3, T4, tetrac and triac (agonists) were comparable to literature. Antagonistic behaviour was seen in sodium arsenite, amiodarone, PCP and methimazole. Spiked water samples from the GWRC showed thyroid hormone receptor activity. Sixteen of the 48 water samples collected from the water treatment plant were positive for thyroid hormone disruptor activity. Highest activity was seen in the winter season, accounting for seasonal variations. High TDCs activity reported in the source water may be due to activities occurring near the dam. The water treatment plant seemed effective for only one of the distribution pipelines, and not the other. This study confirms that GH3.TRE.Luc Reporter Gene Bioassay is a sensitive and effective tool to identify and quantify TDC activity in pure chemicals and in complex environmental mixtures present in water. Further monitoring of water sources for TDCs is recommended to ensure water quality and safety. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

No description available.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) and the active ingredient in Prozac®, is found in the environment and disrupts feeding and metabolism in exposed fish. The objective of this research was to investigate the mechanisms involved in the feeding and metabolism disruption in the model goldfish (Carassius auratus). Two short-term waterborne fluoxetine exposures (7- and 14-days) were performed using two environmentally relevant doses of fluoxetine (0.5 and 1 μg/L) and metabolic effects at the level of the brain, liver, serum and bile in goldfish were investigated. Abundances of mRNA transcripts coding for six feeding neuropeptides were examined to determine which may be involved in the initial neural changes associated with decreased appetite in goldfish. The 7-day fluoxetine exposure at 1 μg/L caused corticotropin-releasing factor (CRF) mRNA levels to increase by 2-fold in female hypothalamus and telencephalon, indicating that CRF may be one of the first of the feeding neuropeptides to be altered. Six hepatic miRNAs were also evaluated in the goldfish liver that were previously associated with fluoxetine exposure in zebrafish (Danio rerio). Following the 7-day exposure at 1 μg/L, miR-22b, miR-140, miR-210, miR-301a and miR-457b levels increased in the female goldfish liver by 4-6 fold. The 14-day fluoxetine exposure at 1 μg/L caused 2-fold increases in miR-210, miR-301a, miR-457b and let-7d in male goldfish liver. These miRNAs were associated with the down-regulation of anabolic metabolic pathways in zebrafish, indicating a conservation of miRNA and fluoxetine effect between fish species. Serum and bile metabolite profiles of fluoxetine exposed goldfish were evaluated using ultra performance liquid chromatography coupled to quadrupole time of flight mass spectrometry. Following the 14-day exposure at 1 μg/L, the bile metabolite profiles of male goldfish were significantly different from controls as detected by cluster analysis and fluoxetine was tentatively identified in the serum. No other discriminant metabolites were identified as of yet. The data presented suggest that fluoxetine causes metabolic disruption in goldfish at multiple organ levels. Because of the widespread detection of fluoxetine and other emerging SSRIs in the aquatic environment, future research is required to firmly establish this pharmaceutical class as a metabolic and endocrine disrupting chemical.

Developing a sensitive and effective in vitro bioassay to detect endocrine disrupting chemicals (EDCs) would reduce the cost, eliminate the possibility of low dose effects, detect the non-monotonic dose responses, and identify mechanisms of actions. The “chemical nose” sensing method using supramolecular complexes composed of cationic monolayer functionalized gold nanoparticles (AuNPs) and fluorescent proteins (FPs) can successfully distinguish serum proteins, mammalian cells, tissue lysates, and chemotherapeutic drug mechanisms. EDCs regulate target cells via genomic or non-genomic pathways in terms of proliferative effect and response time. In this thesis, green fluorescent protein-gold nanoparticle (GFP-AuNP) sensors were used to detect the proliferative effect of 17b-estradiol (E2) and bisphenol A (BPA) on MCF7 and T47D cell lines at fM or pM dose range. Non-monotonic dose responses were also observed at different exposure times. The dose-response relationships using GFP-AuNP sensors could be correlated to the cell cycle analysis. Interestingly, tamoxifen, an estrogen antagonist, showed distinct patterns at low doses on HepG2 cells using triple channel FP-AuNP sensors, which might indicate different mechanisms of actions in this dose range.

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

Orientador: Luis Fernando Barbisan / Coorientador: Wellerson Rodrigo Scarano / Banca: Daniel Araki Ribeiro / Banca: Luis Antonio Justulin Junior / Resumo: O crescimento, a maturação e a manutenção estrutural e funcional da próstata são eventos hormônio-dependentes, principalmente de andrógenos, e alterações endócrinas no período crítico de desenvolvimento prostático poderão causar efeitos adversos sobre a próstata, inclusive câncer. Bisfenol A (BPA) é um desregulador endócrino, presente em embalagens de alimentos e detectado em praticamente todos os fluidos humanos, capaz de alterar o desenvolvimento do trato reprodutivo de ratos machos por meio de exposição gestacional/pré-natal. A genisteína (GEN), um fitoestrógeno da soja, mostrou propriedades quimiopreventivas contra carcinogênese em roedores. Este estudo objetivou avaliar se a exposição gestacional ao BPA causa efeitos deletérios precoces e tardios sobre a morfogênese prostática da prole de machos Sprague-Dawley, e se a exposição concomitante à GEN modificaria estes efeitos deletérios. Fêmeas prenhes foram divididas em grupos que receberam BPA (25 ou 250 μg/kg p.c.) ou dimetilsulfóxido (DMSO - grupo controle)/óleo de canola, por gavage e dieta basal ou contendo GEN (250 μg/kg). A prole recebeu apenas ração basal e foi eutanasiada nos dias pós-natal (DPN) 21 ou 180. A exposição gestacional à menor dose de BPA induziu alterações prostáticas precoces e tardias. A ingestão gestacional de GEN reverteu os efeitos deletérios do BPA sobre os níveis de proliferação celular no epitélio prostático, a arquitetura prostática e a expressão de receptores androgênicos na prole no DPN 21. Um aumento na incidência de inflamação multifocal e de hiperplasia prostática atípica foi observado na prole, no DPN 180, das mães tratadas com a menor dose de BPA, enquanto a ingestão gestacional de GEN atenuou estes efeitos. Desta forma, os resultados do presente estudo indicam que a ingestão de GEN durante a gestação tem ação preventiva contra efeitos adversos do BPA sobre a próstata da prole / Abstract: Not available / Mestre

Endocrine disrupting chemicals (EDCs) are compounds that can interfere with hormone signaling pathways and are now recognized as pervasive in estuarine and marine waters. One prevalent EDC in California’s coastal waters is the xenoestrogen 4-nonylphenol (4-NP), which has been shown to impair reproduction, development, growth, and in some cases immune function of marine invertebrates. To further investigate effects of 4-NP on marine invertebrate immune function we measured total hemocyte counts (THC), relative transcript abundance of immune-relevant genes, and lysozyme activity in Pacific oysters (Crassostrea gigas) following bacterial infection. To quantify these effects we exposed oysters to dissolved phase 4-NP at high (100 μg l-1), low (2 μg l-1), or control (100 μl ethanol) concentrations for 7 days, and then experimentally infected (via injection into the adductor muscle) the oysters with the marine bacterium Vibrio campbellii. 4-NP significantly altered the effects of bacterial infection had on THC. Oysters exposed to both high and low 4-NP did not experience a bacteria-induced increase in THC, as seen in control oysters. We also determined that V. campbellii infection induced differential expression of a subset of immune-related genes tested (Cg-bigdef2, Cg-bpi1, Cg-lys1, Cg-timp) in some, but not all, tissues; 4-NP exposure altered expression patterns in two of these genes (Cg-bpi1 and Cg-tgase). Exposure to 4-NP alone also caused differential expression in some genes (Cg-bpi1, Cg-galectin1, Cg-clec2). Lastly, low levels of 4-NP significantly increased lysozyme activity 24 h post-infection. These results suggest that exposure to 4-NP can alter both cellular and humoral immune responses to bacterial infection in C. gigas.

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.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24222号 / 工博第5050号 / 新制||工||1788(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 松井 康人, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)