Avaliação in vivo do potencial estrogênico de amostra de água para consumo humano produzida por estação de tratamento do estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da mutagenicidade e da estrogenicidade dos extratos etanólicos padronizados, frações e substâncias isoladas de Arrabidaea brachypoda (DC.) Bureau

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Potential quimioprotetor do DIM (3, 3' - Di-indolil-metano) e da Genisteína em linhagens de células tumorais prostáticas humanas LNCaP e PC-3 expostas ao Bisfenol A

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-androgenic activities of diuron and its metabolites in male Nile tilapia (Oreochromis niloticus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An integrated approach to evaluate emerging contaminants in drinking water

The chemical stock of emerging contaminants in Brazilian drinking water is of great interest due to the poor water quality at surface water intakes. In addition, little is known about the effect of some contaminants, such as endocrine disrupting chemicals (EDCs), which may be present in both raw and treated water. The aim of this work was to evaluate selected emerging contaminants in Brazilian waters using both chemical and biological analyses. Sampling sites were established in different municipalities based upon raw water quality data. Estrone, 17 beta-estradiol, estriol, 17 alpha-ethinylestradiol, bisphenol A, 4-n-octylphenol and 4-n-nonylphenol were determined in the samples by liquid chromatography-tandem mass spectrometry. A yeast assay using a Saccharomyces cerevisiae bioluminescent bioreporter was used to evaluate the estrogenic activity of the water samples. The first integrated results revealed similarities between the two individual approaches, since higher values for the bioassay were accompanied by significant concentrations of some selected compounds in surface water samples. No estrogenicity was observed for drinking water samples. Our results also indicate that the usual paradigm of evaluating water quality by measuring selected EDCs in a given water sample via chemical analysis, needs to be reviewed since the observed estrogenicity of a water sample is now a better guiding parameter to the selection of samples and substances to be chemically investigated in further analysis. So far, the data bank produced in this work, i.e., the comparison between chemical burden and observed estrogenicity, is not yet sufficiently robust to fully guide this decision. (C) 2011 Elsevier B.V. All rights reserved.

In vivo and in vitro effects of genistein as obesogenic/anti-obesogenic compound in the lipid metabolism of rainbow trout (Oncorhynchus mykiss)

Nowadays, soy is one of the most used ingredients in the formulation of fish feed, due to the ample market supply, lower market price, high protein concentration and favorable amino acid composition. Nevertheless, soybean meal products are rich and primary diet source of phytoestrogens, as genistein, which may have a potential negative impact on growth, hormonal regulation and lipid metabolism in fish. The principal aim of this study was to better understand in vivo and in vitro genistein’s effects on lipid metabolism of rainbow trout. In adipose tissue it was showed an unclear role of genistein on lipid metabolism in rainbow trout, and in liver an anti-obesogenic effect, with an up-regulation of autophagy-related genes LC3b (in adipose tissue) and ATG4b (in liver and adipose tissue), a down-regulation of apoptosis-related genes CASP3 (in adipose tissue) and CASP8 (in liver). An increase of VTG mRNA levels in liver was also observed. Genistein partially exerted these effects via estrogen- receptor dependent mechanism. In white muscle, genistein seemed to promote lipid turnover, up-regulating lipogenic (FAS and LXR) and lipolytic (HSL, PPARα and PPARβ) genes. It seemed that genistein could exert its lipolytic role via autophagic way (up-regulation of ATG4b and ATG12l), not through an apoptotic pathway (down-regulation of CASP3). The effects of genistein on lipid-metabolism and apoptosis-related genes in trout muscle were not dose-dependent, only on autophagy-related genes ATG4B and ATG12l. Moreover, a partial estrogenic activity of this phytoestrogen was also seen. Through in vitro analysis (MTT and ORO assay), instead, it was observed an anti-obesogenic effect of genistein on rainbow trout adipocytes, and this effect was not mediated by ERs. Both in vivo and in vitro, genistein exerted its effects in a dose-dependent manner.

Remoção de atividade estrogênica do hormônio 17β-Estradiol em processos de oxidação de águas para abastecimento

Muitos oxidantes químicos reativos acarretam na ruptura de estruturas moleculares complexas de vários tipos de compostos orgânicos decompondo-as em estruturas mais simples e propiciando condições melhores para uma efetiva ação de micro-organismos na degradação biológica. A presença de hormônios, entre eles o 17β-estradiol, em estações de tratamento de esgoto e em águas subterrâneas e superficiais mostra a necessidade de uma avaliação dos processos de tratamento convencionais. O objetivo deste trabalho foi a análise e remoção de hormônios através de técnicas de cloração e ozonização de amostras reais de águas de saída de filtro de estações de tratamento de água (ETAs), operadas pelo Serviço Autônomo de Água e Esgoto (SAAE) de São Carlos (ETA Centro), que capta águas dos ribeirão Feijão e córrego Espraiado e pela Sociedade de Abastecimento de Água e Saneamento S/A (SANASA) de Campinas (ETAs 3 e 4), que capta águas do rio Atibaia. Foram realizados ensaios contaminando-se as amostras com 17β-estradiol, que é o hormônio natural mais presente no meio ambiente, em concentração de 6.000 ng L-1, submetendo-se tratamento com dosagens em torno de 0,5 e 2,0 mg L-1 desses oxidantes, em tempos de contato de, respectivamente, 10 e 30 min. As amostras submetidas à contaminação e tratamento e as de controle (sem contaminação e tratamento) foram analisadas através da remoção do 17β-estradiol com a verificação da atividade estrogênica das amostras por ensaios de Sistema de Expressão de Estrogênio Induzida por Levedura Bioluminescente (BLYES), que apresentou-se como uma ferramenta simples. Os resultados apresentados neste trabalho demonstram que a oxidação por ozônio se mostrou mais eficiente do que aquela por cloro para a remoção da atividade estrogênica causada, única e exclusivamente, pelo 17β-estradiol para uma dosagem inicial desse hormônio relativamente alta (6.000 ng L-1). Todavia, em todos os ensaios a concentração final da atividade estrogênica permaneceu acima do limite de quantificação desses hormônio, indicando que a remoção não foi completa, mesmo em condições favoráveis, isto é, matriz limpa, com padrões de potabilidade para os parâmetros físico-químicos.

Endocrine-disrupting compounds: A review of their challenge to sustainable and safe water supply and water reuse

The relevance of endocrine-disrupting compounds as potential contaminants of drinking water is reviewed, particularly in the reuse of wastewater. Growing populations and increasing intensification of land and water use for industry and agriculture have increased the need to reclaim wastewater for reuse, including to supplement the drinking water supply. The variety of anthropogenic chemicals that have been identified as potential endocrine disruptors in the environment and the problems arising from their use as human and livestock pharmaceuticals, as agricultural chemicals and in industry are discussed. The potentially adverse impact of these chemicals on human health and the ecology of the natural environment are reviewed. Data for the removal of estrogenic compounds from wastewater treatment are presented, together with the comparative potencies of estrogenic compounds. The relative exposure to estrogens of women on oral contraceptives, hormone replacement therapy, and through food consumption is estimated. A brief overview of some methods available or under development for the assessment of estrogenic activity in environmental samples is provided. The review concludes with a discussion of the directions for further investigation, which include human epidemiology, methodology development, and wastewater monitoring. (C) 2006 Wiley Periodicals, Inc.

Synthesis of 5-Carbapterocarpens by α-Arylation of Tetralones Followed by One-Pot Demethylation/Cyclization with BBr3

5-Carbapterocarpens, one of them displaying estrogenic activity, were prepared from α-aryltetralones in high yields through a one-pot, BBr3-promoted O-demethylation and cyclization sequence. The key α-aryltetralone intermediates were obtained by direct α-arylation of tetralones with o-alkoxybromoarenes in the presence of Pd2(dba)3 (2.5 mol-%) and tBu3PHBF4 (10 mol-%) as catalysts, together with 2.5 equiv. of KOH in dioxane/H2O (4:1), under microwave irradiation conditions (80 W, 100 °C, 40 min), leading to α-monoaryltetralones in good yields.

Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements.

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCa) and detection capabilities (CCß) were established for both the estrogen and androgen RGAs. All samples were compliant with CCa and CCß in both bioassays. Recovery rates were 96 % for 17ß-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12–25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.

Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk

Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer.Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds.A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36pgg EEQ and has been successfully applied in testing a range of milk samples. © 2014 Elsevier Ltd.

TiO2 microcrystallized glass plate mediated photocatalytic degradation of estrogenic pollutant in water

Photocatalytic degradation of estriol (E3) in an aqueous medium was investigated in the presence of TiO2 microcrystallized glass plates. To begin with, transparent glasses associated with the composition 0.4BaO-0.4TiO(2)-B2O3 (BTBO) were fabricated by the conventional melt-quench technique and subsequently subjected to controlled heat treatment at an appropriate temperature to grow anatase TiO2 microcrystals in the glass matrix. The fabricated samples were subjected to differential scanning calorimetry. X-ray diffraction and scanning electron microscopy to obtain thermal, structural and microstructural details. The photocatalytic activity of glass samples for estriol degradation was monitored by fluorescence spectroscopy. The limit of detection for estriol using fluorescence spectroscopy was analyzed. The results showed that microcrystallized TiO2 glass composites have more photocatalytic activity than as quenched glass. The degradation rate coefficient of microcrystallized TiO2 glass composite (334.54 min(-1) m(-2)) was found to be ten times larger than that of the as-quenched BTBO glasses (37.74 min(-1) m(-2)) implying that the anatase phase of TiO2 in BTBO glasses was responsible for high photocatalytic activity of estriol degradation. (c) 2014 Elsevier B.V. All rights reserved.

Measurement of estrogen activity in two Chinese lakes

Estrogenic activities of samples from two Chinese lakes, Ya-Er Lake and Donghu Lake, were measured by in vitro recombinant yeast assay and found in both lakes polluted with industrial and domestic wastewater. In methanol extracts of lake water samples the estrogen-like activity was higher than in toluene extracts. These results have been inverted for solid samples. Furthermore, the EC50 value of the water samples was close to the original concentration in the lake.

Estrogenic endocrine disruptors present in sports supplements. A risk assessment for human health

Sports supplements are becoming a regular dietary addition for consumers who view such products as a means of improving their health and performance. Previously estrogenic endocrine disruptors (EDs) were detected in 80% of 116 sports supplements investigated by biological in vitro reporter gene assays (RGAs). The aim of this study was to quantify the hormonal activity in 50 of these sports supplement samples using a validated estrogen RGA and perform an exposure and risk assessment for human health. Results showed that 17β-estradiol equivalent levels were higher than those reported as being present in the typical human omnivore diet in 33 of the sports supplements and higher than the acceptable daily intake (ADI) in 13 of these products. The highest activity samples presented a potential to influence the human daily exposure to 17β-estradiol like activity in various risk groups with a predicted hormonal impact of greatest concern in young boys and postmenopausal women. In conclusion, consumers of sports supplements may be exposed to high levels of estrogenic EDs.

Growth factor activation of ErbB2/ErbB3 signaling pathways regulate the activity of Estrogen Receptors (ER)

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles.