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Phytoöstrogene sind in den vergangenen Jahren zunehmend in den Fokus der Wissenschaft gerückt, weil sie eine potentielle Alternative zur klassischen Hormonersatztherapie darstellen. Diese ist aufgrund von zum Teil drastischen Nebenwirkungen für den Einsatz von klimakterischen Beschwerden umstritten. In dieser Arbeit wird die dosisabhängige Wirkung des Phytoöstrogens Genistein an ausgewählten östrogenselektiven Organen näher untersucht. Die Versuche wurden am Modell der langzeitovarektomierten Maus durchgeführt. Es wurden 70 ovarektomierte Tiere in sieben Gruppen aufgeteilt und untersucht. Fünf der sieben Gruppen erhielten genisteinhaltiges Futter in verschiedenen Konzentrationen. Die anderen beiden Gruppen erhielten Estradiol als Zusatz oder sojafreie Diät und dienten jeweils als Positiv- und Negativkontrollgruppe. Zusätzlich gab es eine nicht ovarektomierte intakte Kontrollgruppe, die ebenfalls sojafreies Futter erhielt. Während des dreimonatigen Versuchszeitraums wurden regelmäßig Gewicht und Futterverbrauch der Tiere gemessen. Nach Ende des Versuchs wurden die Feuchtgewichte von Uterus und Herz bestimmt sowie die Genexpression am linken Ventrikel von IGF-1, ERα und Myocardin mittels PCR analysiert. Darüber hinaus wurde am Tibia-Knochen per pQCT die Messung der Spongiosadichte, des polaren Widerstandsmoments und des prozentualen Anteils der Trabekel an der Spongiosaquerschnittsfläche durchgeführt. Die Ergebnisse zeigen, dass Genistein direkt am Herz wirkt, indem es das relative Herzgewicht und die Genexpression am Herz erhöht. Genistein beeinflusst auch das Körpergewicht und das relative Gewicht des Uterus und die untersuchten Knochenparameter dosisabhängig. Genistein kann in höherer Dosierung am Uterus proliferierend wirken, jedoch nach derzeitigem Kenntnisstand weniger stark als der klassische Hormonersatztherapie-Wirkstoff E2. Genistein kann zukünftig nur dann eine Therapiealternative zur klassischen Hormonersatztherapie darstellen, wenn es gelingt, eine Dosis zu finden, bei der Genistein die gewünschten Wirkungen entfaltet, gleichzeitig aber die unerwünschte proliferierende Wirkung an Brust und Uterus sicher ausgeschlossen werden kann. Im Modell der ovarektomierten Maus scheint eine Dosis von 1 g/kg Genistein im Futter ein vielversprechender Ansatzpunkt für weitere Untersuchungen zu sein.

Submitted by SAMUEL RODRIGUES LOURENÇO DE MORAIS null (samuelrodrigues@foa.unesp.br) on 2016-10-04T19:15:34Z No. of bitstreams: 1 TESE - SAMUEL R L DE MORAIS.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-05T12:13:50Z (GMT) No. of bitstreams: 1 morais_srl_dr_araca.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) / Made available in DSpace on 2016-10-05T12:13:50Z (GMT). No. of bitstreams: 1 morais_srl_dr_araca.pdf: 2524842 bytes, checksum: 61664d3b0e3fff46188835de7508312b (MD5) Previous issue date: 2016-08-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A diminuição das concentrações plasmáticas de estrógeno está intimamente relacionada com o aumento do estresse oxidativo e a diminuição da massa muscular em idosos. A terapêutica hormonal estrogênica (THE) e o treinamento de força (TF) apresentam resultados efetivos sobre a manutenção do tecido muscular em idosos. No entanto, os mecanismos responsáveis pelas melhorias induzias por ambas as intervenções são pouco elucidados. Nesse sentido, avaliamos os efeitos da THE, do TF e a associação sobre a manutenção do tecido muscular esquelético de ratas periestropausadas. Ratas Wistar (18 meses) foram distribuídas em: Grupo não treinado (NT-Veh), Grupo NT tratado com a THE (NT-E2), Grupo TF (TF-Veh) e Grupo TF-E2. Os animais receberam a THE (17β estradiol; 2 x semana; 25 µg/kg/administração) e/ou praticaram TF (3 x semana; 80% sobrecarga) durante 16 semanas. A THE e o TF induzem benefícios ao tecido muscular esquelético de ratas periestropausadas, no entanto, por diferentes maneiras. Enquanto a THE induziu diminuição do estresse oxidativo muscular (Dihidroetidina), o TF resultou em melhoras significativas na função muscular, no sistema antioxidante muscular (Catalase) e na expressão de miRNAs (206, 146b e 133a). Já a interação das intervenções resultou em melhora no estado redox (Sirt1, Sirt3, PGC-1α, COXIV), na responsividade dos receptores estrogênicos (ERα, ERβ e GPR30), e atividade de vias de sinalização do tecido muscular (IGF-1/Akt-1/mTOR). Além disso, as intervenções de maneira isolada ou em associação, levaram ao aumento no percentual de fibras glicolíticas e redução das oxidativas. Sugerimos que a aderências das intervenções (associadas ou não) possam minimizar/atenuar a perda da massa muscular observada em fases tardias durante o processo de envelhecimento. / The decrease of estrogen (E2) circulating levels is strongly related to increased oxidative stress and the loss of muscle mass in elderly. The hormone replacement therapy (HRT) and strength training (ST) are the main effective interventions to prevent the loss of muscle mass, however, the mechanisms involved in interventions-induced benefits are not well elucidated. In this sense we evaluate the effect of HRT, ST and association on skeletal muscle maintenance of periestropaused rats. Female Wistar rats (18 months old) were randomly assigned into: non-exercised and non-treated group (NE-Veh), NE treated group (NE-E2), exercised and non-treated group (ST-Veh) and ST-E2 group. The animals received the HRT (17β estradiol; 2 x week; 50 µg/kg/week) and/or performed ST (3 x week, 80% overload) for 16 weeks. The HRT and ST promoted beneficial effects on skeletal muscle of periestropaused rats, however, by different manners. While HRT treatment leaves the reduction of oxidative stress (Dihidroetidine), the ST resulted in significate improvement on skeletal muscle function, in skeletal muscle antioxidant system (Catalase) and in miRNAs expression (2016, 146b and 133a). Already, the association of interventions resulted in improvement of redox state (Sirt1, Sirt3, PGC-1α, COXIV), in estrogen receptor responsiveness (ERα, ERβ and GPR30) and the activity of skeletal muscle signaling pathways (IGF-1/Akt-1/mTOR). In addition, the interventions, isolated or combinated, leaves an increase of the percentage of glycolytic fibers and reduced percentage of oxidative fibers. We suggest that the adherence to interventions (combinated or not) could minimize/attenuate the loss of skeletal muscle mass observed in later phases of aging process. / 13/18907-2

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.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Hormonaly active compounds in wastewaters represent nowdays a serious problem. Proceses currently used in watewater treatment plants (WWTP) are unefficient in removing these compounds from contaminated wastewaters. The compounds are supposed to sorb onto solid sludge elements and sediments. In this work seven endocrine disruptors were detected in the sludge samples from WWTPs. A new sensitive method for detection of seven selected endocrine disruptors (4-nonylphenol, bisphenol A, estriol, 17β-estradiol, estrone, 17α- ethynylestradiol, irgasan) was developed. The method is based on accelerated solvent extraction (ASE), gel permeation chromatography (GPC) and solid phased extraction. For final extract analysis, gas chromatography coupled with mass spectrometry (GC/MS) was used. The efficiency of this method was tested using artificially contaminated sludge and the method was used to analyse real samples from several WWTPs in Czech Republic. The effect of sludge age on detection of individual analytes was assessed as well. The concentrations of endocrine disruptors measured in the samples reached up to 1 µg/g. The results are comparable or higher then those reported in other works and they show the necessity of further research on endocrine disruptors in the environment.

The overall objective of this dissertation was to examine exposures to the estrogenic endocrine disrupting chemicals (EEDCs), phthalates, bisphenol-A (BPA), and the metalloestrogens cadmium (Cd), arsenic (As), and manganese (Mn) in an older geriatric aged-population and examine associations with brain health. Given the evidence that EEDCs affect brain health and play a role in the development of cognitive dysfunction and neurodegenerative disease, and the constant environmental exposure through foods and everyday products has led this to becoming a great public health concern. Using a bioinformatic approach to find nuclear respiratory factor 1 (NRF1) gene targets involved in mitochondrial dysfunction, that are both estrogen and EEDC-sensitive, we found several genes involved in the gene pathways of Alzheimer’s disease (AD): APBB2, EIF2S1, ENO1, MAPT, and PAXIP1. Using the Center for Disease Control and Prevention (CDC), National Health and Nutrition Examination Survey (NHANES) 2011-2014 datasets to assess EEDC bioburden and associations with surrogate indicators of brain health, which include cognitive scores, memory questions, and taste and smell data, we found phthalate bioburden to be significantly higher in those with adverse brain health vii and significantly higher in females. In our logistic regression model when controlling for all known and suspected covariates in AD, in females, the phthalates in females ECP, MBP, MOH, MZP, and MIB in males and the phthalates COP, ECP, MBP, MC1, MEP, MHH, MOH, and MIB were significantly associated with poor cognitive test scores, poor memory, and taste and smell dysfunction. Among the metalloestrogens, Cd bioburden was higher in those with poor cognitive performance, poor memory, and taste and smell dysfunction, with the trend more significant in males. Among oral contraceptive (OC) and HRT (hormone replacement therapy) use, in our logistic regression model when controlling for all known and suspected covariates in AD, past OC and HRT use was associated with better cognitive test scores. The study provides further evidence of the complex role EEDCs play in overall brain health through other biological mechanisms and fills a gap in knowledge that demonstrates EEDCs effects on brain health in a geriatric age population.

<p>The main thesis hub was on development of two electrochemical biosensors for the determination of 17&beta / -estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17&beta / -estradiol endocrine disrupting compound / consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide.</p>

<p>The main thesis hub was on development of two electrochemical biosensors for the determination of 17&beta / -estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17&beta / -estradiol endocrine disrupting compound / consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide.</p>

Magister Scientiae - MSc / The main thesis hub was on development of two electrochemical biosensors for the determination of 17β-estradiol-estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17β-estradiol-estradiol endocrine disrupting compound; consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide. / South Africa