Orientador: Arielle Cristina Arena / Banca: Daniela Cristina Ceccatto Gerardin / Banca: Carla Dal Bianco Fernandez / Banca: Luis Antonio Justulin Junior / Banca: Luis Gustavo de Almeida Chuffa / Banca: Cândida Aparecida Leite Kassuya / Banca: Patrícia Fernanda Felipe Pinheiro / Resumo: Fipronil é um inseticida amplamente utilizado na agricultura, medicina veterinária e saúde pública, e recentemente tem sido listado como um provável desregulador endócrino. Estudos demonstram que este composto pode afetar a função da tireoide, parâmetros reprodutivos e o sistema nervoso central. No entanto, a maioria dos estudos realizados com o fipronil avaliou sua toxicidade aguda, e poucos se referem aos seus possíveis efeitos reprodutivos, especialmente se a exposição ocorrer durante a gestação e lactação. Este estudo objetivou avaliar os efeitos resultantes da exposição perinatal ao fipronil e suas possíveis repercussões tardias sobre parâmetros reprodutivos masculinos e femininos, em ratos. Ratas prenhes foram expostas a três doses de fipronil (0,03; 0,3 ou 3 mg/kg) do dia gestacional 15 até o dia pós-natal 7, por gavage. Durante o tratamento, foram monitorados o consumo de água e ração e o peso corporal das ratas expostas. Após o desmame, foram coletados o sangue das mães para análises bioquímicas e órgãos para registro do peso. A prole masculina foi avaliada através dos seguintes parâmetros: peso corporal, distância anogenital e idades da separação prepucial e da descida testicular. Na vida adulta, peso de órgãos reprodutores, contagens, morfologia e motilidade espermática, histo-morfometria do testículo e epidídimo, contagem de células de Sertoli, comportamento e preferência sexual, fertilidade e expressão de receptores de andrógenos no testículo foram investigados. Nos filhotes do sexo feminino foram avaliados a distância anogenital, idade de instalação de puberdade, ciclo estral, dosagem hormonal, fertilidade, comportamento sexual e histologia de útero e ovário. O tratamento não alterou os parâmetros bioquímicos e os pesos da maioria dos órgãos analisados das mães expostas ao fipronil, com exceção do peso da hipófise, que apresentou uma redução no grupo exposto a menor dose... / Abstract: Fipronil is an insecticide widely used in agriculture, veterinary medicine and public health, and recently has been listed as a possible endocrine disruptor. Studies demonstrate that this compound can affect the thyroid function, reproductive parameters and central nervous system. However, most of studies performed with fipronil evaluated the acute toxicity, and a few of them refer to its reproductive effects, especially if the exposure occurs during gestation and lactation. This study aimed to evaluate the effects resulting from perinatal exposure to fipronil and its possible late repercussion on male and female reproductive parameters, in rats. Pregnant rats received three doses of fipronil (0.03; 0.3 or 3 mg/kg) from gestational day 15 to postnatal day 7, by gavage. During the treatment, water and food consumptions as well as the body weight of females exposed were investigated. After weaning, we collected the blood of mothers for biochemical analysis and the organs to verify the weight. The male offspring was evaluated for body weight, anogenital distance and ages of preputial separation and testicular descent. At adult life, weight of reproductive organs, sperm counts, morphology and motility, testicular and epididymal histomorphometry, sertoli cells count, sexual behavior and preference, fertility and patterns of expression of androgen receptor in the testis were evaluated. On female offspring was investigated anogenital distance, puberty onset, estrous cycle, hormonal levels, fertility, sexual behavior and histology of uterus and ovaries. The treatment did not alter the biochemical parameters and the weight of most of analyzed organs of mothers exposed to fipronil, however the weight of pituitary gland presented a reduction in the group exposed to a lower dose. On males, the perinatal exposure to fipronil did not affect the body weight, anogenital distance and puberty onset. Similarly, at adult life, fipronil did not alter the organ... / Doutor

Organic and inorganic compounds are present as contaminants in varying concentrations throughout our water cycle. Examples of these contaminants include the endocrine disrupting compounds (EDCs) bisphenol-A (BPA) and 17β-estradiol (E2) from plastics and pharmaceutical use. It can be necessary to obtain the concentration of these compounds within the water cycle for analysis by interested parties such as research groups, regulatory agencies, and private organizations. These concentrations, however, can be too dilute within the initial sample for analysis. Therefore it is necessary to concentrate the compound of interest (analyte) prior to analysis. One such way to do this is by way of Solid Phase Extraction (SPE). SPE uses a small cartridge which contains chromatographic packing material to chemically extract analytes from a water sample onto a solid phase. To increase concentration, these analytes are then transferred (eluted) to a substantially smaller volume of organic solvent for eventual analyses. These commercially available cartridges are relatively inexpensive, approximately $5 each. However, these cartridges are labeled as single use. In large-scale analyses, this can quickly add up to a sizable percentage of the analysis budget. Additionally, sizable waste volumes can be generated from these analyses in the form of non-degradable polypropylene plastic. If these cartridges can be re-used, material costs as well as waste volumes can be substantially reduced. However, little is known regarding how the quality of analysis degrades with cartridge re-use. The objective of this project is to evaluate the number of times SPE cartridges can be reused without compromising the results of the subsequent analyses. Based on a review of prior literature, I identified and developed protocols for extracting analytes (BPA and E2) from water via SPE, then analyzing them with gas chromatography and mass spectrometry (GC-MS). These protocols have been developed to mimic those employed by research labs, industry, and other entities for which the results of this study would be most applicable. The only deviation is the re-use of the cartridge rather than disposal and replacement. One type of commercially available SPE cartridge (Oasis HLB, Waters Inc., Milford, MA) was used and two water types were tested. The water was spiked with fixed concentrations of BPA and E2, and then analyzed by way of SPE/GC-MS. For both water types, I performed multiple SPE runs on 10 cartridges each. I tracked the history of GC-MS peak areas, which indicate apparent analyte concentration. Peak area data were analyzed as a function of the number of analyses performed (run number), and evaluated for statistically significant changes as well as overall trends. Statistically significant change and/or trends would indicate that the cartridge had exceeded the maximum allowable number of re-uses and would thereby identify the number of times the “single-use” cartridge can reliably be re-used. Peak area history for 20 SPE runs per cartridge for pure water samples and 10 SPE runs for wastewater effluent showed no statistically significant changes or trends on peak area. This indicates that cartridges can be re-used at least 10 times without compromising the integrity of water sample analysis for the EDCs considered in this study.

In this thesis, we have analysed the effects of EE2 on non-reproductive behaviours and fertility. We have showed that two doses of EE2 in male adult short-term exposures evokes opposite behaviours in the novel tank test. A lower dose induced increased bottom-dwelling, a sign of increased anxiety and a higher dose increased surface-dwelling, which would likely expose themselves to predation in a natural environment. Increased shoaling was observed in both exposures, possibly affecting feeding and reproduction opportunities. Fertility analysis of these fish demonstrated a complete inhibition of spawning in the highest dose group. To investigate mechanisms behind the spawning failure, we examined expression levels of genes involved in zebrafish sex differentiation and maintenance of gonadal function. We found downregulated transcription levels of male-predominant genes, suggesting a demasculinization of the testes contributing to functional sterility in these fish. We have demonstrated that non-reproductive behaviour in zebrafish is highly sensitive to EE2 exposure during development. After exposing male and female zebrafish to low doses of EE2 followed by remediation in clean water until adulthood, the fish displayed increased anxiety and shoaling behaviour, demonstrating persistent effects of EE2. Furthermore, behavioural effects were transferred to their progeny. Decreased fertilisation success of the developmentally exposed fish was observed in both sexes when mated to untreated animals of the opposite sex. These fertility effects persisted although the fish had a long remediation period, implying likely reduced fitness of fish populations in aquatic environments. Based on our findings on non-reproductive behaviours and fertility, we performed RNAsequencing analysis of the brain and testes in order to investigate possible biological mechanisms behind the persistent effects. There is a need for biomarkers allowing detection of both reversible and irreversible effects in animals exposed to estrogenic substances, hopefully contributing to better risk assessments for EDCs. Results from RNA-sequencing would serve as a basis for continued studies in pursuit of potential biomarkers.

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

Notre étude a porté sur le diagnostic de la présence des perturbateurs endocriniens dans le golfe de Gabès (Tunisie), leurs effets sur la reproduction d’une espèce bioindicatrice de pollution, la palourde R. decussatus ainsi que leurs mécanismes d’action. Pour atteindre ces objectifs, nous avons fait appel à plusieurs outils méthodologiques, des expositions in vivo (effluents urbains, effluents industriels, 17β-œstradiol) et des prélèvements in situ afin d’évaluer le risque encouru par l’écosystème du golfe de Gabès tout entier en utilisant une approche multiparamétrique basée sur différents niveaux de l’organisation biologique (allant du niveau biochimique jusqu’au niveau populationnel). Nous avons mis en évidence la présence des composés reprotoxiques tout au long de la côte Sud-Est de la Tunisie. Les résultats obtenus ont révélé une corrélation entre la contamination chimique (cadmium, activités œstrogéniques et androgéniques au niveau du sédiment) et certains paramètres biologiques liés à la reproduction (diamètre ovocytaire, vitellogénine, glycogène) et un site très divergent de tous les autres sites (El hofra). Cette station d’El hofra semble être touchée par une perturbation endocrinienne liée probablement à une synergie entre la contamination par le Cd et la présence des composés à activité œstrogénique et androgénique dans le milieu. En parallèle à cette étude in situ, trois expérimentations au laboratoire ont été réalisées. Les résultats ont montré que les effluents de la station d’épuration de Sfax (cocktail de contaminants) sont capables d’affecter la reproduction de la palourde en induisant la synthèse de la vitellogénine et stimulant la reproduction chez les deux sexes. L’exposition des palourdes à un contaminant pur, le 17β-œstradiol, a révélé qu’il peut contribuer à expliquer les perturbations du cycle reproducteur observées dans l’expérience précédente. Cette hormone naturelle a un effet et sans doute un rôle dans la physiologie de la palourde. Dans le but d’étudier son mécanisme d’action via le récepteur aux œstrogènes (RE), nous avons cloné une partie de l’ADNc codant pour les RE. Cependant, l’analyse de la réponse transcriptionnelle n’a pas montré de changement significatif dans l'expression du gène aussi bien chez les mâles que chez les femelles suite à une exposition aux œstrogènes (17β-œstradiol, effluents urbains et industriels). / Our study aimed to assess the presence of endocrine disrupting compounds in the Gulf of Gabes area (Tunisia), their effects on the reproductive potential of the marine bivalve, Ruditapes decussatus as well as elucidating their mechanisms of action. To achieve these goals, several methodologies have been deployed, such as laboratory in vivo exposures (to urban or industrial effluents, estradiol), and in situ sampling to assess the risk of such substances on this ecosystem by using a multiparametric approach based on different levels of biological organization in clams (from biochemical level to the population level). We have demonstrated the presence of reprotoxic compounds along the south-eastern coast of Tunisia. The results revealed, a strong correlation between chemical contamination (cadmium, estrogenic and androgenic activities in sediment) and some reproductive parameters (oocyte diameter, vitellogénine and glycogen levels) and, a particular site (El hofra) that differs from all the other studied sites in this polluted context. Clams from this station seem to suffer from endocrine disruption probably resulting from a synergistic effect between both cadmium contamination and the presence of chemicals acting as estrogens and androgens in the environment. In parallel to this in situ study, three laboratory experiments were performed. The results showed that exposure to a mixture of chemical compounds released by the Sfax wastewater treatment plant had an adverse impact on the reproductive potential of R. decussatus, by inducing vitellogenin synthesis and stimulating reproduction in both sexes. Results of the exposure of organisms to 17β-estradiol alone, revealed that the estrogenic compound could contribute to the observed effects on the reproductive cycle of the test animals in the previous experiment. This natural hormone has induced deleterious effects and may play a role in the physiology of the clam. To study its mechanism of action via estrogen receptors, a partial cDNA of the gene was isolated and characterized from the clam R. decussatus. However, analysis of the transcriptional response did not show a significant change in the gene expression in males as well as in females following exposure to estrogens (17β-estradiol, urban and industrial effluents).