Bisphenol A (BPA) is an estrogen receptor binding chemical, widely used in the plastics industry, and as such commonly encountered from plastic containers etc. Even at very low doses, BPA is believed to induce obesity and to have various endocrine disruptive effects. The purpose of this study was to determine possible gene expression changes in gonadal and inguinal adipose tissue from rats perinatally exposed to BPA. The method used was quantitative real-time PCR, and genes found to be up-regulated were PLZF, adiponectin, RXRa and Tcf21, while down-regulated genes were PPARγ, Tmem26, EsR1, Resistin, LPL, Chemerin, Serpina6, TFAM and Ahr. This is so far largely unsupported by other studies, and more research is needed.

Organotin compounds are one of the most produced and most used organometallic compounds. Some of these substances are endocrine disruptors, persistent organic polutants and their high toxic effects are observed. That’s why their presence in the environment caused by human activity could endanger many organisms. The aim of this thesis is summarize their properties and their occurrence in the environment. Then the quick, easy and relatively cheap method for determination of trialkyltin compounds in heavily poluted aquatic sediments using capillary zone electrophoresis is developed.

Orientador: Mary Rosa Rodrigues de Marchi / Banca: Anderson Rodrigo Moraes de Oliveira / Banca: Marcia Cristina Bisinoti / Resumo: É consenso entre cientistas do mundo inteiro que existem moléculas com o poder de alterar o funcionamento do sistema endócrino humano e de diversas espécies de animais, conhecidas atualmente como alteradores endócrinos. Dentro deste grupo de substâncias, merece destaque as isoflavonas presentes em maior abundância nos grãos de soja, principalmente a genisteína (GEN) e a daidzeína (DAID), apontadas como responsáveis por desencadear inúmeros efeitos endócrinos em espécies aquáticas. Portanto, o objetivo do presente trabalho foi otimizar e validar um novo método para determinação de GEN e DAID em efluente de ETE, água subterrânea, potável e de rio; aplicando o método já validado nestas matrizes na cidade de Araraquara e avaliar o comportamento sazonal destes alteradores endócrinos. O método apresentado é baseado na pré-concentração e simplificação de amostra proporcionado pelo uso de SPE aplicado a 2 litros de amostras de água de rio e subterrânea e ao volume de 1,5 litro de efluente de ETE, analisados por HPLC-UV utilizando um sistema MeOH/Água como alternativa ao uso de acetonitrila. A exatidão mostrou-se na faixa de 75 a 110%, com precisão entre 0,5 e 14 %. O LQ alcançado com o referido método foi de 6,3 e 188 ng L-1 para daidzeína e 2,17 e 187 ng L-1 para a genisteína nas matrizes de água subterrânea, superficial e efluente de ETE, respectivamente. A aplicação do método nas amostras de água subterrânea e do Ribeirão das Cruzes demostrou que as concentrações de DAID e GEN situaramse abaixo dos LD. Já para as amostras de efluente de ETE, estas moléculas foram identificados em todas as amostras coletadas, com concentrações variando entre 60-306 ng L-1 para GEN e de 76 a 288 ng L-1 para DAID. Comparando-se os dados de temperatura e pluviosidade com as concentrações destes fitoestrógenos no período amostrado, percebe-se que a temperatura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is a common thought among scientists worldwide that there are molecules with the capacity of altering the functioning of the endocrine system of humans and several species of animals, currently known as endocrine modifiers. Inside this group of substances, the highlighted ones are the isoflavones more abundantly present in soybean grains, especially genistein (GEN) and daidzein (DAID), pointed as those responsible for triggering a number of endocrine effects in aquatic species. Therefore, the aim of the present work was optimize and validate a new method for the determination of GEN and DAID in an effluent of a Sewage Treatment Unit (STU), as well as subterranean, drinking and river waters, applying the method already validated in these matrixes in the city of Araraquara, and then evaluate the seasonal behavior of these endocrine modifiers. The method presented is based on the sample pre-concentration and simplification enabled by the use of SPE applied to 2 liters of samples from subterranean and river waters, and to the volume of 1.5 liter of STU effluent, analyzed through HPLC-UV using a system of MeOH/Water as an alternative to the use of acetonitrile. The accuracy was shown to be in the range of 75 to 110%, with a precision between 0.5 and 14 %. The LQ reached with the method referred was of 6.3 and 188 ng L-1 for daidzein, and 2.17 and 187 ng L-1 for genistein in the matrixes of subterranean and superficial waters, as well as STU effluent, respectively. The application of the method in the samples of subterranean water and also in that of the Cruzes stream demonstrated that the DAID and GEN are below the LD. For the samples of STU effluent, such molecules were identified in all samples collected, with concentrations ranging between 60-306 ng L-1 for GEN and 76-288 ng L-1 for DAID. Comparing the temperature and rainfall data with the concentrations of these... (Complete abstract click electronic access below) / Mestre

Accumulating evidence from epidemiological, wildlife, and laboratory studies indicates that abnormalities of reproduction, development and physiology can be ascribed to environmental contaminants with biological activities. Many such contaminants disrupt essential hormone-regulated processes by virtue of their ability to interact with nuclear hormone receptors (endocrine disrupting chemicals, EDC). Of particular concern are chemicals that mimic or block estrogen signaling (xenoestrogens, XE) through their direct interaction with estrogen receptors (ER). The current model of XE action focuses on disrupted transcriptional activity, as measured by changes in the expression of ER-regulated genes. However, transcription is tightly coupled to splicing, by which a single target gene transcript is processed to multiple structurally and functionally different mRNAs. In theory, any XE that interacts with ER to regulate transcription has the potential to disrupt splicing, thereby affecting not only mRNA quantity but also quality. To address this hypothesis, alternative splicing of the gene encoding ER alpha (esr1), itself an estrogen responsive gene, was investigated. In these studies, killifish (Fundulus heteroclitus), an environmentally relevant species, and zebrafish (Danio rerio), an advantageous laboratory fish model, were used. First, the occurrence of ER alpha splice variants in adult tissues, in developing embryos and in response to estrogens in the two species was documented. Additionally, the effects of long-term, multigenerational XE exposure on ER alpha splicing were examined in two killifish populations, one from an estrogenic (polluted) site and a second population from a reference (unpolluted) site. A subset of ER alpha variants from killifish was expressed in cell culture to document their transcriptional activities. To determine the in vivo relationship between estrogen responsiveness and an ER alpha splice variant of interest, esr1 splicing was experimentally altered in living embryos by microinjecting morpholino oligonucleotides, and changes in induction of a panel of estrogen responsive gene targets were measured as markers of effect. These results provide evidence that dysregulation of mRNA processing is also a mechanism of XE action, and suggest that resultant ER alpha splice variants mediate the short-term effects of estrogen disruption and are also part of the adaptive response to long-term, multigenerational XE exposures in the natural environment.

Made available in DSpace on 2014-06-11T19:29:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:18:55Z : No. of bitstreams: 1 cavassani_tb_me_araiq.pdf: 1899529 bytes, checksum: c90c7f090f98ce6f94ef871d0d70f937 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / É consenso entre cientistas do mundo inteiro que existem moléculas com o poder de alterar o funcionamento do sistema endócrino humano e de diversas espécies de animais, conhecidas atualmente como alteradores endócrinos. Dentro deste grupo de substâncias, merece destaque as isoflavonas presentes em maior abundância nos grãos de soja, principalmente a genisteína (GEN) e a daidzeína (DAID), apontadas como responsáveis por desencadear inúmeros efeitos endócrinos em espécies aquáticas. Portanto, o objetivo do presente trabalho foi otimizar e validar um novo método para determinação de GEN e DAID em efluente de ETE, água subterrânea, potável e de rio; aplicando o método já validado nestas matrizes na cidade de Araraquara e avaliar o comportamento sazonal destes alteradores endócrinos. O método apresentado é baseado na pré-concentração e simplificação de amostra proporcionado pelo uso de SPE aplicado a 2 litros de amostras de água de rio e subterrânea e ao volume de 1,5 litro de efluente de ETE, analisados por HPLC-UV utilizando um sistema MeOH/Água como alternativa ao uso de acetonitrila. A exatidão mostrou-se na faixa de 75 a 110%, com precisão entre 0,5 e 14 %. O LQ alcançado com o referido método foi de 6,3 e 188 ng L-1 para daidzeína e 2,17 e 187 ng L-1 para a genisteína nas matrizes de água subterrânea, superficial e efluente de ETE, respectivamente. A aplicação do método nas amostras de água subterrânea e do Ribeirão das Cruzes demostrou que as concentrações de DAID e GEN situaramse abaixo dos LD. Já para as amostras de efluente de ETE, estas moléculas foram identificados em todas as amostras coletadas, com concentrações variando entre 60-306 ng L-1 para GEN e de 76 a 288 ng L-1 para DAID. Comparando-se os dados de temperatura e pluviosidade com as concentrações destes fitoestrógenos no período amostrado, percebe-se que a temperatura... / It is a common thought among scientists worldwide that there are molecules with the capacity of altering the functioning of the endocrine system of humans and several species of animals, currently known as endocrine modifiers. Inside this group of substances, the highlighted ones are the isoflavones more abundantly present in soybean grains, especially genistein (GEN) and daidzein (DAID), pointed as those responsible for triggering a number of endocrine effects in aquatic species. Therefore, the aim of the present work was optimize and validate a new method for the determination of GEN and DAID in an effluent of a Sewage Treatment Unit (STU), as well as subterranean, drinking and river waters, applying the method already validated in these matrixes in the city of Araraquara, and then evaluate the seasonal behavior of these endocrine modifiers. The method presented is based on the sample pre-concentration and simplification enabled by the use of SPE applied to 2 liters of samples from subterranean and river waters, and to the volume of 1.5 liter of STU effluent, analyzed through HPLC-UV using a system of MeOH/Water as an alternative to the use of acetonitrile. The accuracy was shown to be in the range of 75 to 110%, with a precision between 0.5 and 14 %. The LQ reached with the method referred was of 6.3 and 188 ng L-1 for daidzein, and 2.17 and 187 ng L-1 for genistein in the matrixes of subterranean and superficial waters, as well as STU effluent, respectively. The application of the method in the samples of subterranean water and also in that of the Cruzes stream demonstrated that the DAID and GEN are below the LD. For the samples of STU effluent, such molecules were identified in all samples collected, with concentrations ranging between 60-306 ng L-1 for GEN and 76-288 ng L-1 for DAID. Comparing the temperature and rainfall data with the concentrations of these... (Complete abstract click electronic access below)

Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was...

The presence of biologically-active estrogenic endocrine disrupting compounds (EDCs) in treated effluents from biological wastewater treatment facilities has prompted wide-spread interest in the behavior of these contaminants during the activated sludge process. The yeast-estrogen screen (YES) was used to quantify the estrogenic activity of samples taken from different areas of three wastewater treatment facilities. An estrogenic mass-balance around these facilities revealed that the majority of influent estrogenic activity was removed in the activated sludge process, but the main route for EDC discharge to the natural environment was via the treated effluent. The estrogenic activity in the effluent from a membrane bioreactor (MBR) was lower compared to a fully aerobic activated sludge process using secondary clarification, suggesting that enhanced removal of particulate and colloidal material may improve EDC removal efficiency. Colloidal material was obtained from settled mixed liquor suspended solids (MLSS) collected from a pilot MBR and a full-scale activated sludge process that included anoxic and aerobic zones. The MLSS was sized fractionated by filtration, and used to quantify the sorption coefficients for pyrene, 17&#946;-estradiol (E2), and 17α-ethinylestradiol (EE2) by fluorescence quenching. The MLSS-derived colloidal organic carbon (COC) sorption coefficient (Kcoc) for pyrene ranged from (< 1 to 80) L/kgcoc, indicating a similar affinity for pyrene compared to natural organic matter. Kcoc coefficients for E2 ranged between (< 1 to 158) L/kgcoc for E2 and (< 1 to 228) L/kgcoc for EE2, and are the highest E2 and EE2 sorption coefficients reported in the literature to date. There was a strong correlation between the Kcoc coefficients and molar extinction coefficient at 280 nm (e280) for pyrene and E2, suggesting that the interaction of the π;-electrons is an important factor in determining overall sorption behavior. There was no such correlation for EE2. Based on the Kcoc coefficients and COC concentrations of the samples, between 1 and 50% of the aqueous E2 and EE2 concentrations were associated with colloidal material. In a novel application of the YES bioassay, the bioavailability of colloid-associated E2 was quantified by comparing the EC50 values of the dose-response curves generated in the presence and absence of size fractionated COC. An increase in EC50 values as a function of COC concentration was attributed to a reduction in bioavailability of E2, suggesting that MLSS-derived COC can reduce, but not eliminate, the biological impact of EDCs. However, there was a high degree of variability in the EC50 values, and estimates of the colloid-associated E2 fraction based on the Kcoc-e280 correlation were unsuccessful in accurately predicting increases in EC50 values. Nevertheless, the YES bioassay may represent a powerful tool in determining the bioavailability of EDCs in complex environmental samples. Results from this research effort suggest that the colloidal phase derived from activated sludge systems represents an important transport vehicle whereby EDCs and other trace organic compounds can enter into the natural environment. Consequently, wastewater treatment plants discharging to sensitive ecosystems or involved with direct water reuse programs should optimize the treatment process to remove colloidal material. / Ph. D.

Steroid hormones in testis play an important role in spermatogenesis, maintenance of the male reproductive tract, production of semen and the maintenance of secondary sex characteristics and libido. They are also discussed as a target for substances called endocrine disruptors (EDs). No complex study was conducted on evaluation of relationships between EDs and steroid spectrum in 2 biological fluids; seminal plasma and plasma. The aim of the PhD. thesis was to develop and validate a method for determination of bisphenol A (BPA) and steroid spectrum in plasma and seminal plasma and to shed more light into mechanisms of ED action and effects of BPA and polychlorinated biphenyls (PCBs) on human spermatogenesis and steroidogenesis. Two new liquid-chromatography mass spectrometry methods for determination of BPA and 11 steroids in plasma and seminal plasma were developed and validated. The methods were used for estimation of analyte concentrations in 191 men with a different degree of fertility. Concurrently, the levels of six congeners of PCBs, gonadotropins, selenium and zinc in plasma were estimated. Partial correlations adjusted for age and BMI were calculated to evaluate relationships between these analytes. Seminal BPA, but not plasma BPA, was negatively associated with sperm concentration...

Steroid hormones in testis play an important role in spermatogenesis, maintenance of the male reproductive tract, production of semen and the maintenance of secondary sex characteristics and libido. They are also discussed as a target for substances called endocrine disruptors (EDs). No complex study was conducted on evaluation of relationships between EDs and steroid spectrum in 2 biological fluids; seminal plasma and plasma. The aim of the PhD. thesis was to develop and validate a method for determination of bisphenol A (BPA) and steroid spectrum in plasma and seminal plasma and to shed more light into mechanisms of ED action and effects of BPA and polychlorinated biphenyls (PCBs) on human spermatogenesis and steroidogenesis. Two new liquid-chromatography mass spectrometry methods for determination of BPA and 11 steroids in plasma and seminal plasma were developed and validated. The methods were used for estimation of analyte concentrations in 191 men with a different degree of fertility. Concurrently, the levels of six congeners of PCBs, gonadotropins, selenium and zinc in plasma were estimated. Partial correlations adjusted for age and BMI were calculated to evaluate relationships between these analytes. Seminal BPA, but not plasma BPA, was negatively associated with sperm concentration...

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