Estrogen receptor α (ERα) is a ligand activated transcription factor. Many widely used synthetic compounds and natural chemicals can activate ERα. The compounds investigated in this study include 17β-estradiol (E2), diethylstilbestrol (DES), antiestrogens ICI 182,780, 4-hydroxytamoxifen, the phytoestrogen resveratrol, and the xenoestrogens bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane (HPTE) and 2,3,4,5-tetrachlorobiphenylol-4-ol (HO-PCB-Cl4). With the exception of the antiestrogens, all the compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type ERα and a construct (pERE3) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. However, these compounds differentially activated C-terminal deletion mutants of ERα. For example, neither E2 nor DES induced transactivation in MCF-7 transfected with ERα(1-537) due to partial deletion of helix 12 of ERα; however, OP, NP, resveratrol, kepone and HPTE induced this ERα mutant, demonstrating that the estrogenic activity of these synthetic compounds do not require activation function 2 (AF-2) of ERα. This study also investigated the effects of xenoestrogens on activation of reporter gene activity in MCF-7 and MDA-MB-231 cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to the luciferase gene. In MCF-7 cells, antiestrogen-induced activation of ERα/Sp1 required the zinc finger motifs of ERα, whereas activation by estrogen and some xenoestrogens activation, such as endosulfan, NP and OP required the H12 of ERα. In contrast, xenoestrogens, such as HPTE, BPA, kepone and HO-PCBCl4, significantly induced transactivation of all four ERα deletion mutants tested in this study. Moreover, RNA interference assays demonstrated structuredependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. The in vivo activities of E2, ICI 182,780, BPA and NP were further investigated in a transgenic mouse model containing pSp13 transgene. All the compounds induced luciferase activity in the mouse uterus; however activities observed in the penis and testis of male and stomach of both male and female mice were structure-dependent,. These results demonstrate that various ER ligands differentially activate ERα in breast cancer cells and transgenic mice, and their activities are dependent on ERα variants, promoter-, cell-context and selective use of different Sp proteins, suggesting these structurally diverse compounds are selective ER modulators (SERMs).

Environmental pollution and its effect on the living organisms has attracted lots of attention recently. There is a growing body of evidence that we are exposed to environmental pollutants at low concentrations in everyday life. The cells and organisms have tools to identify, neutralize and excrete the majority of the toxic compounds. The most dangerous are those that can escape this process or act at low trace concentrations. Endocrine disruptors (EDs) belong to the latter group. Endocrine disruptors can be of natural and anthropogenic origin. EDs target corresponding hormonal receptors and can act at low concentrations. A wide family of nuclear receptors recognize steroid hormones. The majority of EDs can pass through the cytoplasmic membrane, use the hydrophobic nature of the receptor-ligand binding, trigger hormone response and change the expression of the sensitive genes. By interfering with estrogen and androgen signaling, EDs can have effect on the whole organism, but the reproductive system is influenced most. In the present work, our aim was to develop the methods for ED detection and monitoring, analyze the estrogenic potency of EDs, and evaluate the effects of natural estrogens and EDs on male reproductive functions, including sperm and testicular physiology and endocrine functions. First, we...

The aim The aim of my study was to determine whether the selected endocrine disruptors (EDs) affect reproductive parameters of sperm. Due to two experimental models, in which was tested the influence of selected Eds, my work is divided into two parts: A) On the boar model (Sus scrofa) was tested effect of selected EDs (zearalenone (ZEN), di(2-ethylhexyl) phthalate (DEHP), vinclozolin (VIN) on the course of capacitation, acrosome responses in vitro and changes in the detection and localization of selected proteins. B) On the fish model (Perca fluviatilis L.) was tested the influence of the selected ED- bisphenol A (BPA) on the motility and velocity of fish sperm movement. Results Mammalian model In mammalian model the course of capacitation and acrosome reaction was significantly influencing by the examined EDs in comparision with control samples. The increase of the number of marked cells during capacitation and reduce of the number of marked cells after AR compared with the differences between experimental and control samples was confirmed by the flow cytometry method. At higher concentrations EDs clearly inhibited acrosome response and thus reduce the number of sperm capable of fertilization. The relevancy of the results of detection acrosome reaction was confirmed by using various methodological...

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Three experiments were conducted to 1) develop appropriate methods for livestock manure estrogen analysis; 2) determine estrogen removal in different manure treatment systems; and 3) determine estrogen removal from dairy manure in pilot scale reactors. In Experiment I, the recoveries of 17à -estradiol (E2) and estriol (E3) were evaluated in double distilled water and dairy manure after a base extraction and analysis of estrogens by enzyme-linked immunoassay (ELISA) and yeast estrogen screen (YES) assay. The recoveries of E2 were 104% (ELISA) and 97% (YES) in double distilled water. 112% of E2 and 79% of E3 in flushed dairy manure and 118% of E2 in anaerobic digester effluent were recovered with ELISA. 67% and 140% of E2 in flushed manure and anaerobic digester effluent, respectively, were recovered with YES assay. In Experiment II, samples were collected from a full-scale manure handling system incorporating separation and aeration (Separation/Aeration), an anaerobic digester receiving dairy manure (Anaerobic Digester), and four conventional dairy and swine manure storages. 70% of E2 (230 vs. 769 μg/cow/day) and 86% of E3 (78 vs. 552 μg/cow/day) mass were removed from the Separation/Aeration system when the effluent was compared to the influent; the ratio of E2 to total estrogenicity (E2-eq) averaged 76%. In the Anaerobic Digester, 38% of E2 (592 vs. 954 μg/cow/day) and 30% of E3 (338 vs. 483 μg/cow/day) mass were removed; E2 contributed more to E2-eq in the influent than in the effluent (43 vs. 26%). There was no significant difference for E2-eq (431 vs. 284 ng/g of total solids) and E2 (248 vs. 73 ng/g of total solids) concentrations between barn and pti in conventional dairy manure storages; E2 contributed more to E2-eq in barn manure than in pit manure (54 vs. 30%). In swine manure storages, both E2-eq (2852 vs. 1551 vs. 148 ng/g of total solids) and E2 (1933 vs. 808 vs. 89 ng/g of total solids) concentrations decreased (barn vs. primary lagoon vs. secondary lagoon; no significance analysis); the change of E2 ratio to E2-eq was not consistent between barn and lagoon manures between farms. In Experiment III, samples were collected from six pilot scale reactors: two aerated reactors (60% and 100% aeration; AER60 and AER 100), a nitrifying/denitrifying reactor (NDN), an enhanced biological phosphorus removal reactor (EBPR), an anaerobic digester (AD), and a nitrifying reactor (NI) following AD. The influent had higher mass of E2 and E2-eq than the effluent with all reactors. Estrogen removal efficiencies were expressed in two ways: % and %/aerobic hour (or hour) of the influent mass. Higher ammonia nitrogen removing reactors had higher E2 and E2-eq removal in %, higher E2 removal in %/aerobic hour, and the same E2-eq removal in %/aerobic hour compared to those with lower ammonia nitrogen removal. Estrogen removal efficiencies (both in % and %/aerobic hour) were similar in nitrifying and denitrifying reactors. Reactors with aeration supported greater estrogen removal than those without. Reactors with influent anaerobic digestion pretreatment had the same E2 and E2-eq removal in % but higher E2 and E2-eq removal in %/aerobic hour compared to those without. In conclusion, the aerobic treatment system removed more estrogens than the anaerobic one, which means aerobic conditions support more estrogen degradation than anaerobic conditions. The change of the ratios of E2 to E2-eq varied in different livestock manure treatment systems, which reflected different removal rates of E2 and other estrogenic compounds. The pilot scale reactors significantly removed E2 and E2-eq in dairy manure. Ammonia nitrogen removal rates and aeration are the two main factors influencing E2 and E2-eq removal. / Ph. D.

ZIEL Die Auswirkungen von Endokrinen Disruptoren (ED) auf die Reproduktionsorgane sind umfangreich untersucht, da eine Vielzahl endokrin wirksamer Substanzen östrogene und antiandrogene Wirkungen haben. Da sowohl Östrogen- als auch Androgenrezeptoren auch am Herzen nachgewiesen werden konnten, wurde in der vorliegenden Arbeit der Frage nachgegangen, ob ED auch direkte Wirkungen auf den Herzmuskel haben. METHODEN Die Untersuchungen erfolgten am Rattenmodell. Als Kontrollgruppe dienten orchidektomierte männliche Ratten. Getestet wurden folgende Substanzen, die den Tieren über das Futter zugesetzt wurden: Die Phytoöstrogene Genistein (Gen), Resveratrol (Res) und 8-Prenylnaringenin (8-PN), die UV-Filter-Substanzen Benzophenon-2 (BP-2), 4-Methylbenzyliden-Camphor (4-MBC) und Octylmethoxycinnamat (OMC), denen eine östrogene Wirkung zugeschrieben wird, sowie die die Steroidhormone Östradiol (E2) und Testposteron (T). Weiterhin wurden die Pestizide Linuron (Lin) und Procymidon (Pro) untersucht, die dagegen antiandrogene Wirkung haben und deshalb an intakten Tieren getestet wurden. Untersucht wurden einerseits die Herzmuskelfaserfläche mittels histomorphometrischer Auswertung, andererseits die Anzahl der proliferierenden Zellen mittels immunzytologischer Färbung mit Proliferating Cell Nuclear Antigen (PCNA). ERGEBNISSE Die Orchidektomie führte zu einer signifikanten Verkleinerung der Herzmuskelfaserfläche sowie einer Erhöhung der proliferierenden Zellen. Unter den östrogen wirksamen Substanzen führten - bis auf 4-MBC und OMC - alle Testsubstanzen sowie die E2 und T zu einer signifikanten Vergrößerung der Herzmuskelfaserfläche , wobei Res den stärksten Effekt hatte. Alle Testsubstanzen außer 8-PN erzeugten außerdem eine Verringerung der proliferierenden Zellen. Hier zeigte sich der kleinste Anteil an proliferierenden Zellen bei 4-MBC. Bei den Substanzen mit bekannter antiandrogener Wirkung, Lin und Pro, bewirkte lediglich Lin eine signifikante Vergrößerung der Herzmuskelfaserfläche. Auch der Anteil an proliferierenden Zellen wurde durch die Gabe von Lin und Pro kaum beeinflusst. SCHLUSSFOLGERUNG Alle untersuchten Endokrinen Disruptoren hatten Auswirkungen auf das Herz. Einige Wirkungen der Substanzen mit bekannter östrogener Wirkung entsprechen nicht denen von E2, weshalb hier von anderen, Östrogenrezeptor-unabhängigen Wirkmechanismen ausgegangen werden muss. Die beiden antiandrogenen Substanzen Lin und Pro zeigten keine wesentliche Wirkung auf den Herzmuskel der intakten Tiere. Zur Klärung der Frage, ob die Veränderungen am Herzmuskel gesundheitsschädlich für den Menschen sind, sind weitere Untersuchungen nötig.

Human pharmaceuticals have been shown to be entering the aquatic environment in quantities sufficient to produce adverse effects to aquatic organisms, particularly fish. The impacts of synthetic oestrogens have been well documented, but other groups of steroidal pharmaceuticals have not yet been studied. Hence, the present research was designed to study synthetic glucocorticoids (GCs), which are used in large amounts as immunosuppressive and anti-inflammatory drugs. This study involved different approaches, including in silico, in vitro, in vivo and genomics, to assess the effects of GCs on fish. Using reliable data on consumption of GCs in the UK and the LF2000-WQX hydrological model, mean concentrations of GCs in the river Thames were predicted to be in the range from 30 ng/L to 850 ng/L. Mammalian cell lines were transiently transfected with trout corticosteroid receptors (GR1, GR2 and MR) and the transactivation abilities of ten of the most prescribed GCs in the UK were measured in vitro. All tested GCs showed significantly higher activity with GR2 than with GR1. In order to assess the impact of low concentrations of GCs in vivo, two chronic exposure experiments were conducted with adult fathead minnows (Pimephales promelas). Both experiments showed potency-related and concentration-related impacts on various endpoints. There was a concentration-related increase in plasma glucose concentrations and a decrease in blood lymphocyte count. Induction of secondary sexual characters in females suggests a concentration-related masculinisation of fathead minnows. There was a decreasing trend in plasma vitellogenin concentrations in female fish with increasing exposure concentration of GCs. Expression profiles of selected genes (PEPCK, GR and Vtg) in liver also demonstrated concentration-related effects at all three tested concentrations. Hence, it was not possible to define a no effect concentration for the tested GCs. This study probably provides reliable estimates of the likely range of concentrations of GCs in a typical river, impacted by effluent from many sewage treatment plants. The in vitro results indicate that all tested GCs bind to fish GR in a similar manner to that reported for mammalian receptors. The in vivo results suggest that GCs could cause effects at very low (as low as 100 ng/L) concentrations that could be environmentally-relevant. The immunosuppresive effects could make fish susceptible to disease and the reproductive effects may have population-level impacts. It is very likely that the effects of different GCs will be additive, as has been shown for oestrogenic chemicals. Therefore, this study warrants further environmental risk assessment of GCs, especially in mixture scenarios.

Os metais podem causar efeitos adversos de grande amplitude na função reprodutiva de animais, principalmente em organismos aquáticos. Eles podem alterar o sistema endócrino, atuando na esteroidogênese, afetando o processo reprodutivo dos peixes. Nosso objetivo foi investigar os efeitos dos metais alumínio (Al) e manganês (Mn) em pH ácido sobre a esteroidogênese de machos de Astyanax bimaculatus, sexualmente maduros, após uma exposição aguda de 96 horas e avaliar se os animais foram capazes de recuperar dos possíveis efeitos destes metais em água livre de metais. Esses animais foram expostos a uma concentração nominal de 0,5 mg.L-1 de Al e Mn (isolados ou combinados), e os grupos experimentais foram mantidos em pH ácido (5,5) para manter os metais biodisponíveis. Foi realizada uma exposição aguda de 96 horas, com amostragens em 24h e 96h, e depois também um período de 96h em água livre de metais, com amostragens em 120h e 192h, a partir do o início do experimento. Foram determinadas as concentrações plasmáticas de testosterona (T), 11-cetotestosterona (11-KT), 17β-estradiol (E2) e cortisol (C) por ELISA. Além disso, foram realizadas análises histológicas dos testículos e avaliação da concentração espermática. Os metais estudados, além do pH ácido, foram capazes de aumentar as concentrações plasmáticas dos androgénos T e 11-KT. Também foi observado um aumento transitório de E2 em 24h, mas apenas em animais expostos ao Mn e depois uma diminuição em 96h. A exposição ao pH ácido e metais, sozinhos ou combinados não provocou alterações nos níveis de C. Sendo assim, Al e Mn , assim como a acidez da água podem atuar como desregulador endócrino em machos de A. bimaculatus, principalmente por estimulação da síntese de androgénos, causando alterações no sistema fisiológico. No entanto, 96 horas não foram suficientes para que os animais se recuperassem. Os testículos encontravam-se em estágio avançado de maturação, enquanto que a concentração espermática não apresentou alterações significativas que indicassem a atuação dos metais em conjunto ao pH ácido. Táticas reprodutivas podem ter sido utilizadas pela espécie para desencadear alterações na esteroidogênese testicular, principalmente acelerando o processo de espermatogênese e espermiogênese, o que pode interferir com a dinâmica reprodutiva / Metals can cause adverse wide range effects on reproductive function of animals, mostly in aquatic organisms. They can alter the endocrine system, acting on gonadal steroidogenesis, affecting the reproductive process of fish. We aimed to investigate the effects of the metals aluminum (Al) and manganese (Mn) in acidic pH on the steroidogenesis of Astyanax bimaculatus males, sexually mature, after an acute exposure of 96 hours and evaluate if the animals were able to recover of the possible effects of these metals in the water free of metals. Mature males were exposed to a concentration of 0.5 mg.l-1 of Al and Mn (isolated or combined) and the experimental groups were maintained at acidic pH (5.5) to keep the metals bioavailable. We performed an acute exposure of 96 hours, with samplings at 24h and 96h, and then also a period of 96h in water free of metals, with samplings at 120h and 192h from the beginning of the experiment. We measured the plasma levels of testosterone (T), 11-ketotestosterone (11-KT), 17?-estradiol (E2) and cortisol (C) by elisaimmunoassay. Furthermore, histological analysis of testes and evaluation of the sperm concentration were performed. The metals studied, in addition to acidic pH, were able to alter the plasma concentration of the androgens T and 11KT. A transitory increase (24h) of E2 levels was also observed, but only in animals exposed to Mn and then a decrease in 96h occurred. Exposure to acidic pH and metals, alone or combined did not trigger changes in C plasma levels. Generally, Al and Mn as well as the acidity of water can act as endocrine disruptor in A. Bimaculatus males, mainly by stimulating the androgens synthesis, causing changes on physiological system. However, 96 hours in water free of metals were not enough for the animals to recover from the effects of the metals. The testes were in the advanced stage of maturation, while sperm concentration was not significantly changed to suggest any influence of metals together with acidic pH in altering this variable. Reproductive tactic could be used by the species to trigger changes in testicular steroidogenesis, mainly accelerating the process of spermatogenesis and spermiogenesis, what may interfere with the reproductive dynamics

Os disruptores endócrinos são substâncias químicas, sintéticas ou naturais, capazes de interferir no sistema endócrino de seres humanos e animais. Pertencem à classe dos micro poluentes, pois mesmo em concentrações de μg e ng.L-1 causam efeitos adversos aos organismos a eles expostos. Os hormônios destacam-se dentre os disruptores endócrinos por serem compostos potencialmente ativos no sistema biológico e estão relacionados à origem de diversos tipos de cânceres. Esta dissertação teve por objetivo desenvolver e validar metodologia para determinar hormônios, considerados disruptores endócrinos, em amostras de água bruta e tratada da região da Bacia Hidrográfica do Rio Paraíba do Sul utilizando a técnica de cromatografia gasosa com detecção por espectrometria de massas. A população atendida pelas unidades da SABESP dessa região é de aproximadamente 11 milhões de habitantes. Através do processo de validação, a metodologia desenvolvida pode ser considerada seletiva, robusta, exata, linear e precisa para a análise proposta. Aplicando o procedimento analítico validado nas amostras coletadas, alguns dos compostos estudados puderam ser detectados tanto em água bruta como em água tratada permanecendo abaixo do limite de quantificação, exceto a cafeína que apresentou concentração média de 0,45 μg.L-1 em água bruta e 0,17 μg.L-1 em água tratada, observando-se indícios de contaminação principalmente nas épocas secas. Este projeto de pesquisa está vinculado a uma parceria entre o Instituo de Pesquisas Energéticas e Nucleares (IPEN) e a Companhia de Saneamento Básico do Estado de São Paulo (SABESP). / The endocrine disruptors are chemicals substances, synthetic or natural, which can interfere in endocrine system from humans and animals. They belong to the class of micro pollutants, because even at concentrations of μg e ng.L-1 can cause adverse effects to organisms to them exposed. The hormones stand out as endocrine disruptors because they are potentially active compounds in biological systems and are related to the origin of several types of cancers. This work aimed to develop and validate analytical methodology to determine hormones, considered endocrine disruptors, in drinking water and raw water at region of Paraíba do Sul River, using gas chromatography with mass spectrometry detector. The population served by the units of SABESP is around 11 million inhabitants. By the validation process, the developed methodology can be considered selective, robust, accurate and linear for the proposed analysis. Applying the validated analytical procedure for the samples, some compounds could be detected at drinking water and raw water remaining below limit of quantization, except caffeine that was detected in mean concentration of 0,45 μg.L-1 in raw water and 0,17 μg.L-1 in drinking water, principally due to drought season. This Project is linked to a partnership between Instituto de Pesquisas Energéticas e Nucleares (IPEN) and Companhia de Saneamento Básico do Estado de São Paulo (SABESP).

O estudo de poluentes com características orgânicas e inorgânicas nas águas subterrâneas e superficiais de uma dada região é uma ferramenta importante na avaliação da poluição. Os interferentes endócrinos são produtos químicos sintéticos ou naturais que têm a capacidade de atuar no sistema endócrino de seres humanos e animais mimetizando hormônios naturais estimulando respostas diferentes das que originalmente seriam geradas e podem produzir efeitos adversos sobre os organismos, mesmo em concentrações de μg e ng L-1. As atividades antrópicas são a principal fonte de entrada de interferentes endócrinos no meio ambiente. O objetivo desta pesquisa foi determinar 13 compostos orgânicos (dietilftalato, dibutilftalato, nonilfenol, bisfenol A, cafeína, androstano, estrona, estradiol, etinilestradiol, progesterona, coprostanol, colesterol e colestanol) que podem estar presentes nas águas subterrâneas e superficiais da área de instalação do Reator Multipropósito Brasileiro (RMB). Este é um estudo sem precedentes e de grande importância para a região avaliada, uma vez que fornecerá orientação sobre o grau de contaminação das águas locais antes do início do projeto de construção. O estudo também permitirá verificar se o projeto de construção do reator oferecerá algum prejuízo ambiental para a área em seu entorno. Para a determinação dos compostos de interesse, foi utilizado um método analítico desenvolvido e validado para determinação dos compostos de interesse em amostras de água e particulado da coluna d\'água, consistindo na concentração das amostras por extração em fase sólida (SPE) e/ou extração ultrassônica, seguido de quantificação por cromatografia gasosa acoplada a detector de espectrometria de massa (GC/MS). Os resultados revelaram baixas concentrações de alguns dos compostos orgânicos estudados, derivados de atividades antrópicas nas proximidades do local. Os rios que atravessam a área do projeto de instalação apresentam a maioria dos valores abaixo de 0,05 μg L-1, normalmente em estação seca, exceto para cafeína, em outubro de 2016 (0,80±0,03 μg L-1 a jusante do empreendimento) e outubro de 2017 (0,22±0,01 μg L-1 a jusante do empreendimento) e para colesterol em outubro de 2017 a montante do empreendimento (6,6±0,3 μg L-1). / The study of pollutants with organic and inorganic characteristics in groundwater and surface waters of a region is an important tool in the assessment of pollution. Endocrine disrupters are synthetic or natural chemicals that have ability to act in endocrine system of humans and animals mimicking natural hormones stimulating responses different from those originally generated, and can produce adverse effects on organisms even at concentrations of μg and ng L-1. Anthropogenic activities are the main source of input of endocrine disrupters into the environment. The objective of this research was to determine 13 organic compounds (diethyl phthalate, dibutyl phthalate, nonylphenol, bisphenol A, caffeine, androstane, estrone, estradiol, ethinyl estradiol, progesterone, coprostanol, cholesterol and cholestanol) that may be present in the groundwater and surface Brazilian Multipurpose Reactor (RMB). This is an unprecedented and important study for the evaluated region, as it will provide guidance on the local water degree contamination prior to the start of the construction project. The study will also allow verifying if the construction project of the reactor will offer some environmental damage to the area in its surroundings. For the determination of the compounds of interest, an analytical method developed and validated for the determination of the compounds of interest in samples of water and particulate of the water column, consisting of the concentration of the samples by solid phase extraction (SPE) followed by quantification by gas chromatography coupled to mass spectrometry detector (GC/MS). By the results were observed low concentrations of some of the organic compounds studied, derived from anthropic activities near the site. The rivers that cross the installation project area show values below 0.05 μg L-1, usually in the dry season, except for caffeine, in October 2016 (0.80±0.03 μg L-1 downstream of the project) and October 2017 (0.22±0.01 μg L-1 downstream of the enterprise) and for cholesterol in October 2017 (6.6±0.3 μg L-1 upstream).