17β-nandrolone (17βN) is illegally used in sport and in livestock as a growth promoter.17βN and some of their metabolites have been described as endogenous compounds in some animals, such as adult boars and entire male horses. The aim of this thesis was to identify markers of exogenous administration of 17βN in boars and horses. A method to quantify and identify metabolites of 17βN in the different metabolic fractions has been optimized and validated in boar and horse urine. The method has been applied to urine samples collected after administration of 17βN preparations and to urine samples from non-treated animals to identify endogenous compounds. The following metabolites were identified after the administration of 17βN: 17βN, NorA, NorE, NorEpia, BAB and ABB in boars and 17βN, 17αN, NorEpiA, ABA and ABB in horses. The endogenous compounds detected were: 17βN in boars and horses, and ABA and 17αN only in entire male horses. A chromatographic method by LC/MS/MS has been evaluated to quantify and 17βN sulfate in horse urine and it has been applied to analyze samples from non-treated and treated horses. Concentrations above 30 ng/ml of 17βN sulfate would be evidences of the exogenous administration of 17βN in entire male horses. Preliminary results indicate the capability of GC/C/IRMS analysis to differentiate the endogenous or exogenous origin of 17βN and their metabolites in boars and horses. / La 17β-nandrolona (17βN) s’usa il•legalment en l’esport i com a promotor del creixement en el bestiar. Tant la 17βN com alguns dels seus metabòlits s’han descrit com a compostos endògens en alguns animals, com els porcs adults i els cavalls mascles. L’objectiu de la tesi va ser identificar els marcadors de l’administració exògena de 17βN a porcs i cavalls. Es va optimitzar i validar un mètode basat en la CG/EM per quantificar i identificar els metabòlits de la 17βN en orina de porcs i cavalls en les diferents fraccions metabòliques i es va aplicar a l’anàlisis de mostres procedents d’estudis d’excreció de 17βN i de mostres procedents d’animals no tractats per identificar els compostos endògens. Els metabolits identificats després l’aministració de 17βN van ser: 17βN, NorA, NorE, NorEpiA, BAB i ABB en porcs i 17βN, 17αN, NorEpiA, ABA i ABB en cavalls. Els compostos endògens detectats van ser: 17βN per porcs i cavalls mascles i ABA i 17αN únicament pels cavalls. S’ha avaluat un mètode per CL/EM/EM per quantificar 17βN sulfat en orina de cavall que ha estat aplicat a l’anàlisi de mostres procedents de cavalls no tractats i tractats amb 17βN. Concentracions superiors a 30 ng/ml de 17βN sulfat en orina, seria indicació d’una administració exògena de 17βN. Resultats preliminars obtinguts pel mètode de GC/C/IRMS permeten indicar que mitjançant aquesta tècnica és possible diferenciar l’origen endogen o exogen de 17βN i els seus metabòlits en porcs i en cavalls.

Thesis (Ph.D. in Experimental Pathology, Program in Cancer Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 144-158). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

La pression toxique induite par les perturbateurs endocriniens sur l’environnement ainsi que sur la santé de l’homme a augmenté de manière significative durant les dernières décennies. Il est devenu urgent de mettre en place des outils pour détecter et surveiller les perturbateurs endocriniens et pour déterminer la possibilité de cause de perturbations endocriniennes pour les produits chimiques nouvellement introduits. Le développement de nouveaux tests biologiques in vivo en utilisant les larves d'organismes aquatiques tels que medaka (Oryzias latipes) ou Xenopus laevis semble être une stratégie adéquate pour identifier les perturbateurs endocriniens. Durant cette thèse, trois nouveaux modèles transgéniques à base de l’embryon de medaka ont été développés. Le modèle transgénique de medaka ChgH -gfp permet la détection rapide des œstrogènes et des inhibiteurs de l'aromatase en seulement 24 heures avec une sensibilité de 15 ng / L d’éthinylestradiol. Le modèle transgénique de medaka 42sp50-gfp_CgH-gfp montre une fluorescence dans le foie en réponse aux œstrogènes, en plus de la fluorescence émise dans les œufs en développement. C’est donc un système approprié pour étudier le lien entre la signalisation de l'axe ostrogénique et des aberrations de la détermination du sexe chez les poissons. Un embryon transgénique de medaka spiggin - GFP a aussi été développé pour détecter les androgènes et anti-androgènes dans un test de 96 heures avec une sensibilité de 1,5 ug / L de 17α–méthyl testostérone et 276 ug / L de flutamide. Les nouveaux modèles de medaka transgéniques rapporteurs développés dans cette thèse permettent une détection rapide, simple et fiable des perturbateurs d'axe ostrogénique, androgénique et de l’aberration de détermination du sexe chez le medaka. Ils ont été appliqués pour détecter les perturbateurs endocriniens dans les eaux de surface de l'environnement et pour évaluer le rôle potentiel des produits chimiques dans les perturbations endocriniennes. Cela a permis de démontrer l’applicabilité de ces embryons fluorescents comme outils biologiques dans la procédure de détection et la caractérisation des perturbateurs endocriniens. / The toxic pressure of endocrine disruptors on biodiversity and human health has increased significantly over recent decades. As a consequence tools are needed to detect and monitor endocrine disruptors in surface water and to determine the endocrine disrupting potential of newly introduced chemicals. Fish and amphibian larvae, notably the medaka (Oryzias latipes) and Xenopus laevis, offer multiple advantages in this context. In the research carried out in the context of this thesis, different novel medaka-based transgenic models were developed. First, transgenic ChgH-gfp medaka model was designed and optimized for the rapid detection of estrogens and aromatase inhibitors. The model shows significant response within 24 hours with a sensitivity of 15 ng/L ethinylestradiol. Second, a double transgenic 42sp50-gfp_ChgH-gfp medaka which exhibits fluorescence both in the liver in response to estrogens and in developing oocytes as a function of phenotypic sex. It is therefore a suitable model for studying the link between estrogen axis signalling and aberrations of sex determination in fish. Third, a novel spiggin-gfp medaka model was developed to detect androgens and anti-androgens. This model can be exploited in a 96-hour assay with a sensitivity of 1.5 μg/L 17α- methyltestosterone and 276 μg/L flutamide. The novel transgenic medaka models developed in this thesis allow rapid, simple and reliable detection of estrogen and androgen axis disruption and aberrations in medaka sex determination. They have been successfully applied to detect endocrine disruptors in environmental surface water and to assess chemicals with unknown endocrine disrupting potential. Taken together these results demonstrate the applicability of medaka reporter larvae as biological tools in the procedure of detection and characterization of endocrine disruptors.

Made available in DSpace on 2015-05-14T13:21:39Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5619556 bytes, checksum: f89abc7c11080124052c031ad8d11131 (MD5) Previous issue date: 2014-08-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / With the increasing use of manufactured products such as pharmaceuticals, cosmetics and toiletries, a new category of water bodies pollutants, called emerging contaminants, is gaining prominence in the environmental issues and preservation scenario of the planet's fresh water. Among these are endocrine disruptors, such as estrogens that has concerned the scientific community that has been warning for need to establish maximum concentration levels of these types of substances in the drinking water. Parallel to the legislation s rises to control these pollutants is necessary the development of rapid analytical methodologies, robust and with low waste generation. Almost all the analytical methods for quantification of estrogens in water samples make use of liquid or gas chromatography coupled to mass spectra associated to long chromatographic runs. In this work a fast method is presented for simultaneous quantification of four estrogens (E1, E2, E3 and EE2) in surface water samples using HPLC-DAD and multiway calibration with the purpose to exploit the potentialities of the second order advantage to circumvent the problems of sample complexities. Second order calibration models based on PARAFAC, MCR-ALS, N and U-PLS/RBL methods were constructed using individual pattern of analytes (pure standard) solutions. These models were then validated using a set of synthetic mixtures of these analytes with two potential interferences (daizein and biochanin A) to simulate the organic load and to demonstrate second order advantage. Satisfying statistical validation parameters were obtained, especially for U and N-PLS/RBL models. Finally river and sewage samples fortified with four estrogens were subjected to a pre-concentration and sample clean up using SPE-C18 cartridge followed by chromatographic analysis. The application of the calibration model to the fortified waters' samples led to, most cases, a recovery between 70 and 120%, reflecting the good accuracy of the proposed method / Com o uso cada vez maior de produtos industrializados, como os farmacêuticos, os cosméticos e os de higiene pessoal, uma nova categoria de poluentes dos corpos aquáticos, chamados de contaminantes emergentes, vem ganhando destaque no cenário das questões ambientais e preservação da água potável do planeta. Entre estes estão os alteradores endócrinos, como é o caso dos estrógenos que têm preocupado a comunidade científica que já vem alertando para necessidade de se estabelecer limite máximo de concentração dessas substâncias nas águas de abastecimento. Paralelamente ao surgimento de legislação para controle desses poluentes é necessário que ocorra o desenvolvimento de metodologias analíticas rápidas, robustas e com baixa geração de resíduos. Em quase sua totalidade os métodos analíticos para quantificação de estrógenos em amostras de águas fazem de uso da cromatografia líquida ou gasosas acopladas a espectros de massa, associada a longas corridas cromatográficas. Neste trabalho é proposta uma metodologia rápida para quantificação simultânea de quatro estrógenos (E1, E2, EE2 e E3) em amostras de águas superficiais empregando HPLC-DAD e calibração multivias com propósito de explorar as potencialidades da vantagem de segunda ordem para contornar os problemas da complexidade da matriz. Modelos de calibração de segunda ordem baseados nos métodos PARAFAC, MCR-ALS, N e U-PLS/RBL foram construídos usando soluções padrão individual dos analitos (padrões puros). Estes modelos foram depois validados empregando-se um conjunto de misturas sintéticas destes analitos com mais dois potenciais interferentes (a daizeina e biochanina A) para simular a carga orgânica e demonstrar a vantagem de segunda ordem. Estas misturas sintéticas foram construídas usando planejamento Taguchi. Parâmetros estatísticos de validação satisfatórios foram obtidos, em especial para os modelos U e N-PLS/RBL. Por fim amostras de rio e esgoto fortificadas com os quatro estrógenos foram submetidas a uma pré-concentração e limpeza da amostra usando cartuchos SPE-C18, seguido da análise cromatográfica. A aplicação dos modelos de calibração as amostras de águas fortificadas levou para a maioria dos casos a uma recuperação entre 70 e 120%, refletindo a boa exatidão do método proposto

Orientador: Sebastião Roberto Taboga / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T17:10:13Z (GMT). No. of bitstreams: 1 Rochel-Maia_SabrinaSantos_D.pdf: 11931909 bytes, checksum: 6487540b50b593f34ec82d39617d9a12 (MD5) Previous issue date: 2010 / Resumo: O presente trabalho teve por objetivos: 1) Avaliar a atividade das MMPs na próstata de gerbilos machos e fêmeas adultos intactos, procurando comparar a glândula prostática de machos e fêmeas nas diferentes fases do ciclo estral (artigo I); 2) Avaliar o efeito da testosterona sobre o lobo ventral masculino e sobre a próstata feminina quanto à localização de MMP-2 e TIMP-2, enzimas envolvidas na remodelação estromal e na progressão do câncer (artigo II) e 3) Caracterizar a distribuição dos receptores de estrógeno, ER? e ER?, nos tecidos prostáticos de gerbilos machos e fêmeas controles e submetidos à manipulação hormonal (artigo III). Foram realizados experimentos de castração (7 ou 21 dias) nos gerbilos machos adultos, além de experimentos de aplicação hormonal (7 ou 21 dias) em fêmeas adultas na fase de proestro (cipionato de T, 1 mg/kg ou DHEA, 1 mg/kg). Os tecidos foram avaliados por análises bioquímicas, histológicas e imuno-histoquímicas. A atividade gelatinolítica das MMPs obtida por zimografia difere entre os lobos prostáticos masculinos e a glândula feminina, que apresenta os níveis mais altos para MMP-2 e -9, provavelmente devido à constante remodelação estromal promovida pelo ciclo estral. Com relação ao ciclo estral, a maior atividade de MMP-2 e -9 foi encontrada na fase estro, indicando influência do estrógeno. A castração elevou a expressão de MMP-2 na próstata ventral masculina, e o mesmo efeito foi observado após 21 dias de aplicação de T nas fêmeas, indicando que MMPs e TIMPs são influenciados por andrógenos, mas de forma distinta entre machos e fêmeas uma vez que tanto a falta como o excesso de T levaram ao mesmo efeito, provavelmente, devido a machos e fêmeas apresentarem diferentes padrões de expressão para os receptores nucleares, responsáveis por regular a resposta hormonal pelos tecidos. Segundo o estudo há notável diferença na imunolocalização de ER? entre machos e fêmeas, cujas células estromais prostáticas apresentaram duas vezes mais ER? do que as células estromais da próstata ventral masculina. Esse número foi reduzido drasticamente após aplicação de T por 21 dias nas fêmeas, mas não após a castração dos machos. A reação ao ER? foi similar entre machos e fêmeas intactos, mas a castração dos machos ou a aplicação hormonal nas fêmeas promoveu resposta específica, como total ausência após 21 dias de T nas fêmeas ou intensa imunoreação em algumas regiões da próstata após 21 dias de castração nos machos. Adicionalmente, lesões epiteliais presentes nos grupos experimentais tiveram reação ER?-positivas e ER?-negativas, corroborando os estudos que relacionam o ER? com o desenvolvimento e progressão de doenças prostáticas e o ER? como um fator de prevenção e tratamento do câncer. Os resultados obtidos reforçam o conceito de que o equilíbrio estrógeno/testosterona é essencial para a homeostase prostática, e que tanto andrógenos quanto estrógenos atuam sobre as células epiteliais, através dos receptores nucleares, mas também sobre o compartimento estromal, regulando componentes como as MMPs. No caso da próstata feminina, essa interação é ainda mais complexa pois envolve as alterações cíclicas do ciclo estral / Abstract: The present study aimed to: 1) Acess the activity of MMPs in the prostate gland of intact adult male and female gerbils, trying to compare the male prostate lobes with the female gland in different phases of the estrous cycle (Article I); 2) Evaluate the effect of testosterone on ventral prostate of males and on female prostate in the proestrus phase over the location of MMP-2 and TIMP-2, enzymes involved in stromal remodeling and cancer progression (Article II); and 3) Characterize the distribution of estrogen receptors, ER? and ER? in prostatatic tissue of male and female controls and under hormonal manipulation (Article III). Castration experiments (7 and 21 days) were conducted in adult male gerbils as well as hormone injection experiments (7 and 21 days) in adult females in the proestrus phase (T cypionate, 1 mg/kg or DHEA, 1 mg/kg). Tissues were evaluated by biochemical, histological and immunohistochemical methods. The gelatinolytic activity of MMPs, measured by zymography gel, differs from male prostatic lobes and the female gland, which presents the highest levels of MMP-2 and -9, probably due to its constant stromal remodeling promoted by the estrous cycle. Regarding the estrous cycle, the highest activity of MMP-2 and -9 was found in the estrus phase, indicating the influence of estrogen. Castration increased the expression of MMP-2 in the male ventral prostate, and this same effect was observed after 21 days of T injection in females, indicating that MMP and TIMP regulatory mechanism is also influenced by androgens, although differently between males and females since both the failure as the excess of T lead to the same effect, probably due to males and females presenting different patterns of expression for nuclear receptors, which are responsible for regulating the hormonal response by the tissues. According to our results, there is a remarkable difference in the immunolocalization of ER? between males and females, whose stromal cells presented two times more ER? than stromal cells of the male ventral prostate. This number was drastically reduced after 21 days of T injection in females, but not after castration of males. The immunoreaction to ER? was similar between intact males and females, but the castration of males or the hormonal injection in females promoted specific response, such as total absence of reaction after 21 days of T treatment in females or intense immunoreaction in some regions of ventral prostate after 21 days of castration. Additionally, epithelial lesions found in the experimental groups, were ER?-positives and ER?-negative, corroborating studies that relate ER? with the development and progression of prostatic diseases and ER? as a factor in preventing and treating cancer. The results obtained in this study therefore reinforce the concept that the estrogen/testosterone balance is essential for prostate homeostasis, and that either androgens or estrogens have an effect on the prostatic epithelial cells, via nuclear receptors, but also on the stromal compartment by regulating components such as MMPs. In the case of the female prostate, this interaction is even more complex since it involves the cyclical changes of the estrous cycle / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-11T18:59:23Z No. of bitstreams: 2 Dissertação - Thiago Sardinha de Oliveira - 2014.pdf: 1412743 bytes, checksum: d9f93cb6e80442a38393a204842830c9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-12T09:26:51Z (GMT) No. of bitstreams: 2 Dissertação - Thiago Sardinha de Oliveira - 2014.pdf: 1412743 bytes, checksum: d9f93cb6e80442a38393a204842830c9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-12T09:26:51Z (GMT). No. of bitstreams: 2 Dissertação - Thiago Sardinha de Oliveira - 2014.pdf: 1412743 bytes, checksum: d9f93cb6e80442a38393a204842830c9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-19 / Endogenous estrogens have been associated with greater vascular protection in premenopausal women, and the increased risk of cardiovascular diseases in postmenopausal women can be associated to the decrease in plasma estrogen levels. Furthermore recently studies showed that the use of estrogens, like estrone, exhibits remarkable vascular effect when used on isolated arteries, however any investigation was made to elucidate the mechanism of action of this compound. So, the present study was designed to investigate the ability of estrone to induce vascular relaxation and modulate NO-dependent signaling pathway and analyzed the role of estrogens receptor on estrone-mediated vascular relaxation, compared with the effects promoted by 17β-estradiol. 12 week-old male Wistar rats were used to the vascular reactivity, which was performed in an organ bath study for an isometric tension recording. To the experimental protocols, concentration-response curves (0.1 - 100μM) to estrone or 17β - estradiol were performed. The mechanism contributing to estrone-induced effects were determined comparing with the vascular effects induced by 17β - estradiol that have its effect vascular well characterized. It was observed that the vascular relaxation promoted by estrone is dependent on the endothelium and the estrogen receptor. The vasorelaxant effect promoted by estrone was significantly altered in the presence of the inhibitor of PI3K signaling pathway (wortmannin) and the Ca2+-CaM complex inhibitor (calmidazolium), showing the involvement of PI3K/Ca2+-CaM signaling pathways. This study demonstrate that estrone promoted vasorelaxant effect on rat thoracic aortic on endotheliumdependent manner and its effect depends on the estrogen receptors that activate the PI3K pathway and the Ca2+-calmodulin complex which subsequently activates the NO/cGMP pathway. These results contribute to the better understanding of the role of estrone in the conjugated equine estrogen (CEE) which could be associated to the benefits effects of estrogens in the CEE therapy. / Os estrogênios endógenos têm sido associados com uma maior proteção do sistema vascular em mulheres na pré-menopausa, uma vez que os riscos de doenças cardiovasculares em mulheres na pós-menopausa são maiores, alterações estas que se devem à diminuição nos níveis plasmáticos de estrogênios. Além disso, estudos recentes mostraram que o uso de estrogênios, como a estrona, apresenta notável efeito vasorelaxante quando avaliado seu efeito em artérias isoladas, no entanto, nenhuma investigação foi realizada para elucidar o mecanismo de acção deste composto. Assim, o presente estudo procurou investigar o efeito da estrona em aorta de ratos, verificando seu efeito em induzir o relaxamento vascular e modular a via de sinalização dependente do óxido nítrico (NO), e ainda o papel dos receptores de estrogênios, comparando com os efeitos promovidos pelo 17β- estradiol. Os animais utilizados neste estudo foram ratos Wistar com 12 semanas de idade, os quais foram utilizados para realização da reatividade vascular em banho de órgãos isolados. Para os protocolos experimentais, curvas de concentraçãoresposta (0,1-100μM) foram feitas para a estrona ou para o 17β-estradiol e as tensões isométricas gravadas. Os mecanismos envolvidos no efeito induzido pela estrona foram determinados através da incubação de inibidores farmacológicos e comparado ao efeito do 17β-estradiol, que tem seu efeito vascular bem caracterizado. Observou-se que a estrona promove efeito vasorelaxante em aorta torácica de ratos, e que o relaxamento vascular promovido por ela é dependente do endotélio e do receptor de estrogênios. Após ativação do receptor de estrogênios, este ativa as vias de sinalização PI3K e Ca2+-CaM que posteriormente ativam a via NO/GMPc. Estes resultados contribuem para o melhor entendimento do papel da estrona em preparações de estrogênios conjugados equinos (CEE), que pode estar associado aos efeitos de benefícios dos estrogênios na terapia CEE.

O câncer de mama é a doença maligna mais comum e a principal causa de morte entre as mulheres. Sua complexa etiologia envolve múltiplos fatores de risco, a maioria deles relacionada aos níveis cumulativos de exposição da mama aos estrógenos. A maioria de suas ações é mediada pela ligação a seus receptores ER e ER que são fatores de transcrição. Outro fator que exerce um controle extraordinário no comportamento celular, regulando a transcrição gênica e influenciando diversos processos biológicos, e que, quando alterado, é associado ao processo de tumorigênese da mama é a matriz extracelular. A laminina, um dos principais componentes da matriz extracelular, interage com as células através das integrinas e está relacionada ao fenótipo maligno, atuando na adesão, migração, proliferação, diferenciação e sobrevivência celular. Nosso grupo identificou diversos genes diferencialmente expressos em células de câncer de mama ER+ na presença ou ausência de uma monocamada de laminina utilizando a técnica DDRT-PCR. Dois dos genes identificados, DDEF2 e PHLDA1, estão associados à adesão; DDEF2 envolvido na sinalização das integrinas e PHLDA1 relacionado com apoptose por perda de adesão. Nosso objetivo foi investigar os efeitos do 17-estradiol e da laminina na regulação da expressão dos genes DDEF2 e PHLDA1 nas linhagens celulares MCF-7, MDA-MB-231 e posteriormente S30, utilizando a técnica RT-PCR em tempo real. O gene PHLDA1 foi induzido pelo E2 via ER nas células MCF-7 e pela laminina nas células S30, e o gene DDEF2 foi reprimido pelo E2 e induzido pela laminina nas células S30 / The breast cancer is the most common malignant disease and the leading cause of death among women. Its complex etiology involves multiple risk factors, most of them related to the levels of cumulative breast exposure to estrogen. Most of its actions is mediated by binding to its receptor ER and ER that are transcription factors. Another factor that has a tremendous control in cell behavior, regulating the gene transcription and influencing various biological processes, which when altered, is attached to the process of tumorigênese of the breast is the extracellular matrix (ECM). The laminin, one of the main components of the ECM, interacts with the cells through integrins and is related to the malignant phenotype, acting in adhesion, migration, proliferation, differentiation and cell survival. Our group identified several genes diferentialy expressed in breast cancer cells ER + in the presence or absence of a laminin monolayer using the technique DDRT-PCR. Two of the genes identified, DDEF2 and PHLDA1, are associated with adhesion; DDEF2 is involved in the integrins signaling and PHLDA1 is related with apoptosis by loss of adhesion. Our goal was to investigate the effects of 17-estradiol and laminin in regulating the expression of the genes DDEF2 and PHLDA1 in MCF-7, MDA-MB-231 and later S30 cell lines, using the real time RT-PCR technique. The gene PHLDA1 was induced by E2 via ER in MCF-7 cells and the laminin in S30 cells, and the gene DDEF2 was suppressed by E2 and induced by laminin in S30 cells

Um dos grandes problemas da engenharia ambiental é a contaminação dos corpos hídricos. Os sanitaristas têm se preocupado com os hormônios sexuais, notadamente os estrógenos, compostos extremamente ativos biologicamente, que têm sido referidos como agentes etiológicos de feminilização e de vários tipos de cânceres. Os estrógenos naturais 17 \'beta\'-estradiol (E2), estriol (E3), estrona (E1) e o sintético 17 \'alfa\'-etinilestradiol (EE2), desenvolvidos para uso médico em terapias de reposição hormonal feminina e métodos contraceptivos, são os que despertam maiores preocupações, pela contínua introdução ao ambiente; hormônios que possuem a melhor conformação reconhecida pelos receptores que resultam respostas máximas, por isso são considerados responsáveis pela maioria dos efeitos disruptores desencadeados pela disposição de efluentes. A mudança de padrões quanto à atividade sexual dos jovens e a preocupação com o planejamento familiar, levaram ao grande consumo de contraceptivos que, através da urina, são levados pela rede coletora aos corpos de água. O indiscriminado uso desses hormônios na bovinocultura, suinocultura, avicultura e aqüicultura são responsáveis por parte considerável desse contaminante nos mananciais. Os hormônios excretados através da urina e fezes e agentes provenientes das indústrias de processamento de alimentos preocupam os sanitaristas porque o lançamento de efluentes in natura ou tratados, são as principais vias de contaminação do ambiente aquático, quer pelo déficit de infra-estrutura em saneamento, quer pela ineficiência tecnológica e/ou operacional na remoção desses compostos nas estações de tratamento de água ou de efluentes. Apesar de possuírem meia-vida relativamente curta, quando comparados a outros compostos orgânicos como praguicidas, os estrógenos naturais são continuamente introduzidos no ambiente, o que lhes confere caráter cumulativo. A proposta desta pesquisa foi verificar a presença de estrógenos na água bruta que chega à Estação de Tratamento de Água, após seu tratamento, em água tratada por osmose reversa e por tecnologia Milli Q (marca registrada). Para verificar e quantificar presença desses hormônios estrógenos em água de abastecimento de São Carlos-SP, foram realizadas exames através de imunoensaio quimiluminescente e radioimunoensaios. Os resultados apontaram que a ETA não possui solução eficiente para a remoção dos analitos de interesse dessa pesquisa, uma vez que na água tratada foram encontrados valores semelhantes aos da água bruta. / One of the major problems of environmental engineering is the water contamination. The sanitary persons have been concerned with the gonadal hormones, notably the estrogen, biologically active compounds extremely, which have been referred to as etiologic agents of feminization and several types of cancers. The natural estrogen 17 \'beta\'-estradiol (E2), estriol (E3), estrone (E1) and synthetic 17 \'alpha\'-ethinylestradiol (EE2), developed for medical use of hormone replacement therapy in women and contraceptive methods, are those that attract larger concerns by the continuous introduction into the environment; hormones that have the best conformation recognized by receptors that result answers maximum, so they are considered responsible for most of the effects disruptors triggered by the wastewater disposal. The change in patterns on the sexual activity of young people and the concern with family planning, led to the large consumption of contraceptives that, in the urine, are led by the distribution net to water. The indiscriminate use of these hormones in cattle, pigs, poultry and aquaculture are responsible for part of this contaminant in the source. The hormones excreted in the urine and feces and agents from the food processing industries in the sanitary concern that the launch of effluents in nature or treated, are the main routes of contamination of the aquatic environment, either by lack of infrastructure, sanitation, or by inefficiency technological and/or operating in the removal of these compounds in the treatment plants, water or effluent. Despite having relatively short stocking-lige when compared to other organics such as pesticides, natural estrogens are continuously released into the environment, which gives them character cumulative. The proposal of this research was to verify the presence of estrogen in the gross water that arrives at the water treatment plant, after the treatment, in water treated by reverse osmosis and by Milli Q technology. To verify and quantify presence of these hormones estrogen in water supply of San Carlos-SP, examinations were conducted through immunoassay chemiluminescent and radioimunoassays. The results showed that the ETA has no efficient solution for removal of interest analytes of this research, because in the treated water were found values similar to crude water.

L’objectif de ce travail de thèse est d’évaluer le potentiel de nouveaux bio-essais in vitro et in vivo basés sur le poisson zèbre pour la biosurveillance de la contamination chimique de l’eau par les xeno-estrogènes. Pour cela, les bio-essais ont été appliqués pour évaluer les effets de polluants aquatiques environnementaux seuls, mais aussi en mélanges simples (reconstitués) et complexes (échantillons environnementaux). L’évaluation d’échantillons d’eau à travers les bioessais in vitro humain (MELN) et poisson zèbre (ZELH-zfERβ2) a montré des différences qualitatives et quantitatives de réponse, non expliquées par les molécules estrogéniques détectées. Afin de mieux comprendre ces différences, l’activité de polluants aquatiques a été caractérisée sur les différents modèles, individuellement et au sein de mélanges de 2 à 12 polluants, combinant molécules estrogéniques et non estrogéniques pour simuler des situations environnementales.Les résultats obtenus montrent que les bio-essais basés sur le poisson zèbre répondent différemment, et parfois de manière opposée, au modèle humain MELN aux mélanges reconstitués. Ces différences s’expliquent par des sensibilités différentes à certaines molécules entrainant des interactions avec la réponse aux xeno-oestrogènes spécifiques à chaque modèle biologique. Dans son ensemble, ce travail montre que les particularités biologiques de chaque bioessai peuvent influencer la réponse des xeno-estrogènes quand présents au sein de mélange avec d’autres polluants. Ces résultats sont discutés au regard de l’utilisation de bio-essais dans l’évaluation de la contamination chimique des masses d’eau. / This PhD thesis aims at assessing the potential of innovative in vitro and in vivo zebrafish based bioassays for biomonitoring of surface water contamination by xeno-estrogens. For this purpose, the bioassays were applied to assess the effect of environmentally relevant surface water pollutants, alone and in simple (artificial) and complex (environmental samples) mixtures. The screening of surface water samples in zebrafish- (ZELH-zfERβ2 cells) and human-based (MELNcells) bioassays revealed qualitative and quantitative differences which could not be entirely explained by the xeno-estrogens identified. To better understand the response of bioassays to complex environmental mixtures, the activity of environmentally relevant surface waterpollutants was characterized across the bioassays, alone and in 2 to 12-component mixtures combining estrogenic and non-estrogenic chemicals to simulate an environmental contamination.The results indicate that zebrafish-based bioassays have a different and even in some cases an opposite response to the simple mixtures compared with the human-based bioassay MELN. These differences are explained by different sensitivity to some pollutants leading to bioassay-specific interactions with estrogen receptor activation. Al together, this work shows that the biological particularities of each bioassay can influence the response to estrogenic chemicals when mixed with other environmental pollutants, opening the discussion regarding their implementation in chemical water biomonitoring.

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.