Endocrine disruptors represent a group of chemical compounds that are able to negatively influence the hormonal system of vertebrates. Environmental Protection Agency (U.S.EPA) defines these compounds as exogenous substance or mixture that interferes with the synthesis, secretion, transport, binding, activity, or degradation of natural hormones. This can be observed at the level of the individual organism, its progeny, populations and subpopulations. All these changes have negative effects on homeostasis, reproduction, development or change the behavior of the affected animals. This work focuses on 7 endocrine disruptors - natural estron, 17β estradiol, estriol, and synthetic 17α-ethynylestradiol, irgasan (triclosan), 4-nonylfenol, bisphenol A in the influent and effluent of wastewater plants in the Czech Republic. The thesis contains an optimization of endocrine disruptors determination in wastewater, a preliminary screening to determinate concentration levels, 24 hours composite samples and monitoring of one selected wastewater plant during a day. The analytical procedure is based on filtration, solid-phase extraction (SPE), gel permeation chromatography (GPC), derivatization and gas chromatography coupled with mass spectrometry (GC/MS). Keywords: endocrine disruptors, wastewaters, Czech...

Les perturbateurs endocriniens (PE) sont une préoccupation majeure de par leurs effets sur les grandes fonctions physiologiques des organismes etparticulièrement sur la fonction de reproduction. Chez les vertébrés, la reproduction est sous le contrôle d’un axe neuroendocrinien nommé axegonadotrope. Celui-ci comprend différents acteurs cérébraux (gonadolibérines et kisspeptines), hypophysaires (gonadotropines) et gonadiques(stéroïdes sexuels) dont la mise en place et le fonctionnement sont régulés finement. Parmi les mécanismes de régulation, les stéroïdes vont jouer unrôle important en effectuant des boucles de rétrocontrôle. Les PE mimant ces stéroïdes sont donc potentiellement capables d’impacter ces régulations.L’objectif de ce travail était d’évaluer les effets d’un xénooestrogène sur les mises en place des acteurs cérébraux impliqués dans l’axe gonadotrope chezle bar européen (Dicentrarchus labrax). Nous avons d’abord décrit l’ontogénèse des systèmes à kisspeptine, à gonadolibérine et à gonadotropine aucours du développement larvaire. Nous avons mis en évidence une régulation différentielle de ces acteurs suggérant un rôle durant ce processusdéveloppemental. De plus, nous montrons que le 17α-éthinylestradiol (EE2) est capable de perturber l'ontogenèse des systèmes à GnRH2 et à GnRH3au cours des stades précoces du développement larvaire.Ces données constituent une base permettant l’évaluation des effets des PE sur l’ontogénèse des systèmes neuroendocriniens. / Endocrine disruptors (EDs) are a major concern because of their effects on the main physiological functions, particularly on the reproductive function.In vertebrates, reproduction is under the control of a neuroendocrine axis called hypothalamic-pituitary-gonadal (HPG) axis. It includes various actors atthe brain (gonadotropin releasing hormones (GnRH) and kisspeptins), pituitary (gonadotropic hormones) and gonadal (sex steroids) levels. Theirdévelopment and functioning are finely regulated. Among the regulators, steroids play an important role by performing feedback loops. EDs mimickingsteroids are therefore potentially able to impact these regulations. The objective of this work was to evaluate the effects of xenoestrogens on theimplementation of cerebral actors involved in the HPG axis in the European sea bass (Dicentrarchus labrax). We first described the ontogeny of thekisspeptin, GnRH and gonadotropin systems during larval development. We have highlighted a differential regulation of these actors suggesting a roleduring this developmental process. In addition, we show that 17α-ethinylestradiol (EE2) is able to disrupt the ontogeny of GnRH2 and GnRH3 systemsduring the early stages of larval development.These data provide a basis for evaluation of the EDs effects on the ontogeny of neuroendocrine systems.

Les réseaux neuroendocrines qui contrôlent la reproduction sont mis en place pendant le développement sous l’action des stéroïdes sexuels endogènes. Toute perturbation de l’équilibre hormonal pendant ces phases critiques pourrait être à l’origine de troubles de la fonction de reproduction chez l’adulte. Ce travail vise à identifier l’impact d’une exposition développementale à un oestrogène de synthèse, l’éthinyloestradiol (EE2), sur les réseaux neuroendocrines et les conséquences physiologiques et comportementales chez l’adulte et sa descendance. Nos résultats ont montré que l’EE2 induit des perturbations des comportements sexuels chez les mâles et chez les femelles et une modification des réseaux hypothalamiques à GnRH et des neurones à calbindine qui contrôlent la physiologie et les comportements reproducteurs. Nous avons également montré que certains effets de l’EE2 sont transmis jusqu’à la quatrième génération, mettant ainsi en évidence le caractère transgénérationnel de ces perturbations. Ces résultats mettent en évidence la sensibilité des réseaux neuroendocrines aux perturbateurs endocriniens et la nécessité de prendre en compte ces paramètres dans l’évaluation de leurs effets sur la santé et la reproduction. / Neuroendocrine networks controlling reproductive function are established during development by the action of endogenous sex steroids. Any disturbance in the hormone balance during these critical phases may cause several disorders in reproductive function in adulthood. This work aims at identifing the consequences of a developmental exposure to the synthetic pharmaceutical estrogen ethinylestradiol (EE2) on the neuroendocrine and behavioral outcomes of the reproductive function in adult individuals and their offspring. Our findings showed that EE2 induced disturbances of sexual behaviors in males and females and modified the GnRH and the calbindin hypothalamic networks of exposed animals. We also showed that some effects of EE2 were transmitted up to the fourth generation, pointing out the transgenerational character of certain effects. All these results highlight the sensitivity of neuroendocrine networks to endocrine disruptors and the need to consider these parameters in assessing their effects on health and reproduction.

Orientador: José Teixeira Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agrícola / Made available in DSpace on 2018-08-25T00:46:54Z (GMT). No. of bitstreams: 1 Campos_JulyenneMeneghetti_M.pdf: 7123166 bytes, checksum: da20e59802401636508451e97ba79194 (MD5) Previous issue date: 2014 / Resumo: Leitos cultivados tem sido citados como um método alternativo de redução da concentração de alguns agrotóxicos e hormônios levemente hidrofóbicos, como o 17-?-etinilestradiol, que é um dos principais responsáveis pela causa da alteração no sistema endócrino de humanos e animais. Neste contexto, o presente projeto visou avaliar a eficiência da redução de pH, 17-?-etinilestradiol, oxigênio dissolvido (OD), cor aparente, turbidez, nitrogênio total Kjeldahl (NTK), nitrogênio amoniacal (NH3), nitrito (NO2-), nitrato (NO3-), demanda química de oxigênio (DQO) e fósforo total (PT), utilizando-se três leitos tendo brita como meio suporte ¿ dois deles cultivados com a macrófita Eichhornia crassipes (aguapé), com 43 dias de diferença de cultivo entre eles, e outro leito sem cultivo apenas com a brita para controle e comparação dos resultados. A vazão de entrada nos leitos cultivados variou entre 312 e 1.059 L.dia-1, o TDH médio permaneceu entre 2 e 3 dias, as médias máximas de retenção da concentração de DQO, cor, turbidez, NTK, NH3 e PT pelos leitos foram 94,2%, 76,7%, 90,0%, 42,7%, 39,6% e 51,0%, respectivamente. A biomassa dos dois leitos cultivados com aguapé absorveram juntos, 163 g.kg-1 de nitrogênio e 47,2 g.kg-1 de fósforo, durante o período monitorado. Dentre os parâmetros de eficiência analisados o leito cultivado com Eichhornia crassipes com maior grau de desenvolvimento foi o mais eficiente na retenção da maioria dos parâmetros avaliados com nível 5% de significância. A metodologia adaptada para análise do hormônio 17-?-etinilestradiol por Cromatografia Líquida de Alta Eficiência acoplada à Detector de Arranjo de Diodos obteve limite de detecção de 1,26 µg.L-1, e limite de quantificação de 2,52 µg.L-1, porém as amostras analisadas ficaram abaixo do nível de detecção, não sendo possível a determinação das concentrações das amostras. Conclui-se que a macrófita Eichhornia crassipes fixada em meio suporte obteve retenções de nutrientes consideradas satisfatórias em tratamento de esgoto doméstico, e as informações obtidas por este trabalho poderão contribuir com a melhoria e desenvolvimento de novas tecnologias para retenção e métodos analíticos para quantificação de 17-?-etinilestradiol em águas residuárias, diminuindo o lançamento destes interferentes endócrinos nos corpos hídricos / Abstract: Constructed wetlands have been quoted as an alternative method for the removal of slightly hydrophobic pesticides and hormones, such as 17-?-ethinylestradiol, which is one of the main hormones responsible for humans and animals endocrine systems changes. In this context, this project tried to evaluate the reduction efficiency with regards to pH, 17-?-ethinylestradiol, dissolved oxygen (DO), apparent color, total Kjeldahl nitrogen (NTK), ammonia nitrogen (NH3), nitrite (NO2-), nitrate (NO3-), chemical oxygen demand (COD) total phosphorus (PT), using three constructed wetlands having gravel as support media ¿ two of them cultivated with Eichhornia crassipes (water hyacinth), with 43 growth days gap between, and another with gravel only for control and results comparison. The entrance flow rate in the constructed wetlands ranged between 312 to 1,059 L.dia-1, the average HRT stayed between 2 and 3 days, the maximum chemical oxygen demand retention averages, color, turbidity, NTK, ammonia nitrogen and total phosphorus by the constructed wetlands were 94,2%, 76,7%, 90,0%, 42,7%, 39,6% and 51,0%, respectively. The biomass of both water hyacinth constructed wetlands absorbed together 163 g.kg-1 of nitrogen and 47,2 g.kg-1 of phosphorous during the monitored period. Among the analyzed efficiency parameters, the constructed wetland cultivated with Eichhornia crassipes, also 43 days older, was the most efficient in the majority of the parameters evaluated at a 5%significance level. The suitable methodology for analyzing 17-?-ethinylestradiol by high efficiency liquid chromatography attached to a Diode Arrange Detector, had a detection limit of 1.26µg.L-1, and a quantification limit of 2.53 µg.L-1, but the analyzed samples stayed below the detection level, becoming impossible determine the samples¿ concentrations. The conclusion is that the macrophyte Eichhornia crassipes fixed in a support media had satisfactory nutrients retention from domestic sewage, and the obtained information by this work can contribute for an improvement and development of new technologies for retention and analytical methods for 17-?-ethinylestradiol wastewater quantification, decreasing the discharge of these endocrine disruptors in water bodies / Mestrado / Agua e Solo / Mestra em Engenharia

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.

Submitted master's thesis is dealing with the problem of occurrence of residues of specific anthropogenic pollution of drinking water sources, with accent on possibilities of elimination of these compounds during drinking water treatment. There was pay attention to two estrogenic hormones – estradiol and ethinylestradiol. In laboratory scale there was tested efficiency of coagulation, activated carbon adsorption and ozonation in elimination of studied compounds from artificially contaminated water.

17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...

Although wastewater treatment plants are a line of defense in minimizing indiscriminate output of microconstituents to natural waters, we do not possess a fundamental understanding of the mechanisms involved in microconstituent removal during wastewater treatment. With this in mind, experiments were designed to investigate the factors that can influence the fate of four microconstituents, carbamazepine (CBZ), 17alpha-ethinylestradiol (EE2), iopromide (IOP), and trimethoprim (TMP), during biological suspended culture treatment. Specifically, the role that various ecological members of biological treatment systems play in biotransforming these compounds was evaluated. Sorption assays were performed with inactivated biomass samples (ammonia oxidizing bacteria (AOB), laboratory enriched heterotrophic cultures free of active nitrifiers with low (Ox⁻) or high (Ox⁺) oxygenase activity, and a nitrifying activated sludge (NAS) from a full-scale wastewater treatment plant) to determine whether partitioning dictates removal of individual microconstituents. No microconstituents sorbed to the AOB culture. Neither CBZ nor IOP sorbed to Ox⁻, Ox⁺ and NAS cultures; however, EE2 and TMP sorbed to the Ox⁻, Ox⁺ and NAS biomass. Sorption was positively influenced by the presence of exopolymeric substances (EPS) associated with the cultures. The protein content of EPS affected EE2 and TMP sorption more appreciably than the polysaccharide content of EPS. Further experiments were performed to investigate microconstituent biodegradation by AOBs, Ox⁻ and Ox⁺ cultures. The influence of growth state and oxygenase activity on biotransformation by each culture was also evaluated. Results indicate that EE2 was the only microconstituent that was amenable to biotransformation by batch cultured AOB and heterotrophic cultures. EE2 was biotransformed but not mineralized by AOB chemostat and batch cultures. TMP was not transformed by AOB batch or chemostat cultures; however both EE2 and TMP were transformed by Ox⁻ and Ox⁺ chemostat cultures. Radiolabeled studies showed that EE2 was mineralized by this culture. Kinetically, AOBs dominated EE2 transformation to monohydroxylated metabolites; however, both Ox⁻ and Ox⁺ cultures further degraded and mineralized EE2 and metabolites generated by AOBs. These results indicate that biotransformation of EE2 by NAS may be limited by heterotrophic activity whereas TMP fate may be a function of heterotrophic activity only. Oxygenase activity did not limit EE2 or TMP biotransformation in chemostat cultures. Subsequent experiments that were performed to identify the factors that influence heterotrophic degradation of EE2 and TMP indicated that the presence of readily biodegradable substrates slows EE2 and TMP biotransformation. The impact of slowly biodegradable substrates like EPS on EE2 and TMP degradation was unclear. These results suggest that EE2 and TMP are most amenable to biodegradation in bioreactors where endogenous conditions dominate. / Ph. D.

O controle da fertilidade é obtido principalmente pela inibição da ovulação por meio da atividade combinada de dois componentes principais: estrogênio e progestina. Desta forma, novas formulações vêm sendo desenvolvidas com baixa dose de etinilestradiol associado a um novo agente progestógeno, a drospirenona, com o objetivo de garantir equilíbrio entre eficácia, segurança e controle de ciclo menstrual. Este trabalho tem como objetivo desenvolver, validar e comparar métodos analíticos para a identificação e quantificação de etinilestardiol e drospirenona presentes em uma mesma formulação por cromatografia em fase líquida de alta eficiência (CLAE) e a eletroforese capilar (EC). O método por CLAE, foi realizado empregando-se uma coluna LiChoCART® 100RP - C18, (125 x 4 mm) 5 µm, fase móvel constituída por MeCN/H2O 50:50 (v/v) e vazão de 1.0 mL min. Utilizou-se um detector UV 200 nm para drospirenona e um de fluorescência λex = 280 nm e λem = 310 nm, para o etinilestradiol. O método por EC foi validado utilizando-se como eletrólito, tampão de tetraborato de sódio 30 mM, dodecil sulfato de sódio 20 mM e acetonitrila 30% a pH 9,2, capilar de sílica fundida de 31,2 cm sendo 21 cm efetivos, com 75 µm de diâmetro interno. O método por CLAE apresentou um coeficiente de correlação (r2) de 0,9989 para o etinilestradiol e 0,999 para a drospirenona. No método por EC, o coeficiente de correlação (r2) encontrado foi de 0,9988 para o etinilestradiol e 0,998 para a drospirenona. Os métodos podem ser considerados eficientes e confiáveis para serem empregados em análise de rotina para controle de qualidade destes produtos farmacêuticos. / The fertility control is achieved mainly by inhibiting ovulation through the combined activity of two main components: estrogen and progestin. Thus, new formulations have been developed associating appropriately low dose of ethinylestradiol to a new drug, the drospirenone, in order to ensure the balance between efficacy, safety and cycle control. The objective of this research was to develop, to validate and to compare two analytical method for separation and quantification of ethinylestardiol and drospirenone using high performance liquid chromatography and capillary electrophoresis (CE). The HPLC method, was performed using a LiChoCART® 100RP - C18, (125 x 4 mm) 5 µm column, a mobile phase constituted of acetonitrile:water (50:50 v/v) and flow rate of 1.0 mL min. UV detection was made for drospirenone at 200 nm coupled with a fluorescence detector at λex= 280 nm and λem = 310 nm for ethinylestradiol. The CE method was validated using a solution of sodium tetraborate buffer 30 mM, sodium dodecil sulphate 20 mM and acetonitrile 30%, pH 9.2 and a fused silica capillary of 31,2 cm with 21 cm effective and 75 µm of diameter. The HPLC method showed a correlation coefficient (r2) of 0.9989 for ethinylestradiol and 0,999 for drospirenone. For CE method, the correlation coefficient was (r2) 0,9988 for ethinylestradiol and 0,9989 for drospirenone. The methods showed to be efficient and reliable, and can be used in routine analysis for quality control of these pharmaceutical preparations.

A poluição da água doce no estado de São Paulo ocorre por diversas causas. Entre elas está a ineficiência do serviço de tratamento de esgoto sanitário. Alguns componentes do esgoto sanitário são desreguladores endócrinos, entre eles estão os hormônios naturais e sintéticos excretados pelos seres humanos. Estas substâncias causam modificações no sistema reprodutivo como, por exemplo, câncer, feminização, alterações na transcrição genética, alteração nas gônadas, indução à síntese de vitelogenina, entre outros. O objetivo desse trabalho foi testar a atividade endócrina das águas do rio Paraíba do Sul, em Pindamonhangaba, SP. Para tanto, amostras de água do rio foram utilizadas para a quantificação dos hormônios, 17-etinilestradiol, 17-estradiol e levonorgestrel por meio da técnica de cromatografia líquida de alta eficiência (HPLC). Além disso, foram realizados testes crônicos com peixes (Danio rerio) no interior do rio e em laboratório, e uma posterior quantificação de vitelogenina. Os resultados não indicaram concentrações detectáveis dos hormônios pelo método empregado. Entretanto, foi detectada a indução de vitelogenina nos machos de Danio rerio. Esse fato indica a atividade estrogênica da água utilizada. São necessários outros estudos para a avaliação de quais substâncias presentes na água, estão causando as alterações endócrinas nos peixes, entre eles a utilização de índices para priorizar o local inicial dessas pesquisas. Esses estudos são importantes para a conservação da biodiversidade aquática do rio Paraíba do Sul / Freshwater pollution in São Paulo state, Brazil, occurs due a several reasons. Among them there is the inefficiency of the sewage treatment service. Some sewage components are endocrine disrupters, including natural and synthetic hormones excreted by humans. These substances cause modification on reproductive system such as cancer, feminization, gene transcription and gonad alterations, vitellogenin synthesis induction, among others. The aim of this work was to evaluate the endocrine activity of the river Paraíba do Sul, at Pindamonhangaba city, Brazil. For this, river water samples were collected for the quantification of the hormones, 17- etinylestradiol, 17-estradiol and levonorgestrel by high performance liquid chromatography (HPLC). Furthermore, chronic assays were performed with fish (Danio rerio) into the river and in the laboratory, following quantification of biomarker vitellogenin. The studied hormones were not detected by employed methods. However, induction of vitellogenin in male of Danio rerio was detected indicating estrogenic activity of water. Further studies are necessary for the assessment of which substances into the water are causing the endocrine disruption in fish. These studies are important for the preservation of aquatic biodiversity of the Paraiba do Sul River