Estrogens maintain the appropriate androgen-estrogen balance for normal regulation of the structure and function of the male reproductive tract, including the prostate gland. This research investigated viability of cells and expression of selected genes in prostate carcinoma cells (PC-3) exposed to bisphenol A (BPA), an estrogen-like substance present in a number of plastic materials. PC-3 cells are able to metabolize BPA at concentrations below 100 µM. BPA exposure at concentrations between 1nM and 100 µM does not increase or significantly reduce cell viability of these cells. Although the genes investigated in this study (GSTP1 and MGMT) did not show a significant change in expression following in vitro exposure to BPA, the positive control ethinyl estradiol (EE2) caused an increase in GSTP1 expression at mRNA level. These results indicate that BPA does not affect the viability of prostate cells, and motivate a need for further research to identify other genes that could be affected by BPA.

Bisphenol A (BPA) and phthalates are chemicals found in many consumer products including water bottles, food packaging and cosmetics. Previous research has shown that there is potential for these compounds to contribute to obesity. In this analysis, the Canadian Health Measures Survey was used to investigate possible associations between urinary concentrations of these compounds and measures of adiposity. BPA urine concentrations were found to decrease with age, and significant associations with BMI and waist circumference were found in linear regression in adults. No associations with measures of adiposity were found in logistic regression for adults and significant negative associations were found in children. A similar discrepancy was found for mono-(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate, which were significantly associated with obesity in adults, but showed several significant negative associations in children. Overall, this analysis showed that it is unlikely that BPA and phthalates are contributing to adiposity in the Canadian population.

Um die Effekte von Umweltchemikalien auf Populationen von Fließgewässerorganismen zu untersuchen, sind an der TU Dresden Fließrinnen entwickelt worden. Die Komplexität wurde bewusst reduziert, um die beobachteten Effekte besser kausal analysieren zu können. Nach einer Erweiterung des Nahrungsnetzes um Grazer (Eintagsfliegen-Larven) und der weiteren Optimierung der Methode zur Bestimmung des Aufwuchses (Periphyton) wurden 2 Experimenten mit einem Pflanzenschutzmittel (Fenoxycarb) und einer Industriechemikalie (Bisphenol A) durchgeführt, um die Effekte dieser Chemikalien auf den Aufwuchs, die Eintagsfliegen-Larven Rhithrogena semicolorata und Seratella ignita, sowie Oligochaeten der Art Lumbriculus variegatus zu erfassen. Die Wirkung von Fenoxycarb in 4 Konzentrationen von 0,05 bis 50 µg L-1 wurde über eine Dauer von 101 Tagen untersucht. Auf die physiko-chemischen Parameter, den Aufwuchs und L. variegatus wurden keine Effekte festgestellt. Bei R. semicolorata wurden Effekte auf das Überleben und die Emergenz beobachtet. Für den Endpunkt "Anzahl der toten Larven in der Fließrinne" wurde eine LC50 von 3,3 µg L-1 bestimmt. Die akute Toxizität (48 h LC50) von Fenoxycarb für S. ignita ist größer als 5 mg L-1. Bei Larven von S. ignita wurden 72 Tage nach Applikation Effekte beobachtet werden, die dem Wirkmechanismus von Fenoxycarb entsprechen. In der höchsten Konzentration zeigten ca. 80 % der emergierten Tiere morphologische Abnormalitäten an 4 Segmenten des Abdomens. Die Exposition erfolgte möglicherweise durch an Aufwuchs bzw. Detritus gebundene Substanz oder deren Metabolite, da die Substanz 19 Tage nach Applikation im Wasser nicht mehr nachweisbar war. Im zweiten Experiment wurde Wirkung von Bisphenol A in 3 Konzentrationen von 5 bis 500 µg L-1 (effektiv 0,24 bis 24,1 µg L-1) über eine Dauer von 103 Tagen untersucht. Auf die physiko-chemischen Parameter wurde kein Einfluss festgestellt. Der Aufwuchs in der höchsten Konzentration erreichte nur die Hälfte des Biomasse-Maximums der Kontrolle. Die EC10 beträgt 1,8 und die EC50 21,7 µg L-1 (effektiv). Das Populationswachstum von Lumbriculus variegatus war in den beiden höchsten Konzentrationen im Vergleich zur Kontrolle um ca. 50 % reduziert. Die akute Toxizität (LC50 96 h) für L. variegatus beträgt 5,3 mg L-1. Für das Populationswachstum in den Fließrinnen wurde eine EC10 von 0,11 µg L-1 (effektiv) ermittelt, die um den Faktor 48184 unter der akuten Toxizität liegt. - Anlage: Rohdaten (0,53 MB)- Nutzung: Referat Informationsvermittlung der SLUB"

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Exposure to endocrine-disrupting chemicals as bisphenol A (BPA) during gestation and early postnatal life is known to disrupt normal developmental processes and alter the body’s endocrine system leading to deleterious effects in the developing central nervous system (CNS). BPA is an industrial synthetic chemical commonly used in the production of a range of polymers and consumer products, despite concerns about its safety. There is therefore the need to protect the developing CNS from potential damage through the administration of neuroprotective agents. Most medicinal plants are reported to possess significant protective potential against tissue damage through different mechanisms that prevent cell death, oxidative stress, inflammation, immunodeficiency, etc. In this study, the protective effects of fermented rooibos (Aspalathus linearis) tea against the deleterious effects of BPA were investigated. Rooibos is a herbal beverage indigenous to South Africa with widely acclaimed health benefits often linked to the bioactivity of its polyphenolic compounds, especially aspalathin. The anti-allergic, cardiovascular, antioxidant and neuroprotective effects of this herb have been previously reported hence, the present study aims to investigate if regular consumption of rooibos tea during pregnancy and lactation could protect the developing brain from the deleterious effects of BPA in a Wistar rat model. A total of 40 three-month old adult female pregnant dams, with an average weight of 250g, were divided into four groups (n=10). Group 1 control rats received 9% normal saline ad libitum; group 2 rats received 400μg/kg/day BPA only; group 3 rats received 20% fermented rooibos tea as well as 400μg/kg/day BPA, while group 4 rats received ad libitum 20% fermented rooibos tea only. Offspring rats were housed in the same cages as the dams and only separated after weaning on postnatal day (PND) 21. Neurobehavioural assessment using the open field test was done on postnatal day (PND) 42 after which the final body masses were taken before the rats were decapitated under deep anaesthesia, and the desired CNS parts carefully dissected out and processed for histological, biochemical and immunohistochemical studies. The results obtained showed that there was significant impairment of neurobehavioural activity, decreased cerebral and cerebellar antioxidant enzyme activity, reduced hippocampal CA1 length, significant loss of cerebellar Purkinje cells and significant astrocyte activation demonstrated by increased glial fibrillary acidic protein (GFAP) activity in experimental rats exposed to BPA only. However, co-administration of rooibos tea significantly attenuated the BPA-induced distortions. Taken together, these findings suggest that rooibos could be a potent neuroprotective agent against BPA-induced structural, functional and biochemical alterations in the developing CNS.

In patients suffering from end-stage renal disease who are treated by hemodialysis genomic damage as well as cancer incidence is elevated. One possible cause for the increased genomic damage could be the accumulation of genotoxic substances in the blood of patients. Two possible sources for those toxins have to be considered. The first possibility is that substances from dialysers, the blood tubing system or even contaminated dialysis solutions may leach into the blood of the patients during dialysis. Secondly, the loss of renal filtration leads to an accumulation of substances which are normally excreted by the kidney. If those substances possess toxic potential, they are called uremic toxins. Several of these uremic toxins are potentially genotoxic. Within this thesis several exemplary uremic toxins have been tested for genotoxic effects (homocysteine, homocysteine-thiolactone,leptine, advanced glycated end-products). Additionally, it was analysed whether substances are leaching from dialysers or blood tubing and whether they cause effects in in vitrotoxicity testing. The focus of chemical analytisis was on bisphenol A (BPA), the main component of plastics used in dialysers and dialyser membranes. / Patienten, die an terminaler Niereninsuffizienz leiden und mittels Hämodialyse behandelt werden, weisen einen erhöhten Genomschaden auf. Dieser könnte ursächlich für die erhöhte Krebsinzidenz dieser Patientengruppe sein. Eine der möglichen Ursachen für den erhöhten Genomschaden stellt die Akkumulation genotoxischer Substanzen im Blut der Patienten dar. Diese Substanzen können prinzipiell aus zwei unterschiedlichen Quellen stammen. Erstens besteht die Möglichkeit, dass während der Dialyse Substanzen aus den Dialysatoren, dem Blutschlauchsystem oder gar aus verunreinigtem Dialysat in das Blut der Patienten übertreten. Zweitens führt der Verlust der Nierenfunktion zu einer stark verminderten Exkretion harnpflichtiger Substanzen. Diese Substanzen akkumulieren im Blut und bilden, sofern sie ein toxisches Potential besitzen, die Gruppe der so genannten urämischen Toxine. Einige dieser urämischen Toxine sind potentiell auch genotoxisch. Im Rahmen der vorliegenden Dissertation wurden exemplarische Vertreter der urämischen Toxine auf ihre genotoxische Wirkung hin untersucht (Homocstein, Homocystein-Thiolacton, Leptin, Advanced Glycation End-Products). Außerdem wurde analysiert, ob Substanzen aus Dialysatormembranen oder dem Blutschlauchsystem austreten und in in vitro-Toxizitätstests Effekte zeigen. Der Fokus der Analytik lag hierbei auf dem Nachweis von Bisphenol A, dem Hauptbestandteil verschiedener Kunststoffe die für Dialysatoren und Dialysatormembranen verwendet werden.

A exposição ao Bisfenol A (BFA) ocorre frequentemente no nosso dia a dia,uma vez que esse composto é um plastificante presente em muitos utensílios domésticos, como, por exemplo, embalagens e garrafas. No entanto, esta exposição, mesmo em baixas concentrações (ng.L -¹), pode causar sérios danos ao sistema endócrino. Esses danos devem-se ao fato do BFA ser um disruptor endócrino, onde os receptores hormonais o confundem com o estrogênio ou o estriol. Diante deste cenário, o estudo de formas de mitigação deste composto no meio ambiente faz-se necessário. Uma das alternativas para remoção deste contaminante do meio ambiente é a biodegradação por bactérias selecionadas de ambientes contaminados com BFA. Neste contexto, este trabalho visou isolar bactérias degradadoras de BFA, analisar o crescimento celular das bactérias usando o BFA como fonte de carbono e quantificar a biodegradação deste composto. Foram isolados nesse trabalho 15 cepas diferentes de bactérias aeróbias do Sistema Estuarino de Santos (SES), o qual possui forte impacto antrópico pelo grande fluxo de pessoas. Além disso, o SES abriga o maior porto da América Latina (Porto de Santos) e o Polo Industrial de Cubatão. Dos micro-organismos isolados, a cepa de Shewanella sp. foi escolhida para o estudo de biodegradação, uma vez que a mesma foi capaz de tolerar até 150 mg.L- ¹ de BFA. A linhagem de Shewanella sp. isolada foi capaz de biotransformar aproximadamente 75 mg.L- ¹ de BFA em 10 horas com um µ máx de 0,123 e um Y(x/s) de 14 % em meio de cultura líquido. / The exposure to Bisphenol A (BPA) occurs frequently since this compound is a plasticizer present in many households items such as packaging and bottles. However, even the low exposure (ng.L-¹) can cause severe damage to the endocrine system and these damages are due to endocrine disruptor-activity of BPA. The hormonal receptors mistake this compound as estrogen or estriol, thus, mitigation studies to remove this contaminant from the environment are essential. One of the alternatives to remove this xenobiotic from the environment is the bioremediation by selected bacteria isolated from a BPA impacted environment. In this context, this paper aimed to isolate BPAdegrading bacteria, analyze cellular growth and biodegradation hability using BPA as a carbon source. It have been isolated fifteen different aerobic bacteria strains from the Santos Estuary System (SES), which represents one of the most important Brazilian examples of environmental degradation due to water or atmospheric pollution of industrial origin in coastal environment. In addition, SES it is home to the largest port in South America (Port of Santos) and a large industrial pole located in Cubatão. The isolated strain Shewanella halioti was chosen to this work, once is a few explored micro-organism regarding biodegradation of hydrocarbons. Shewanella halioti was able to tolerate up to 150 mg.L-¹ of BPA and biotransform 75 mg.L-¹ in 10 hours in liquid culture medium.

Neste trabalho foram imobilizados nanotubos de carbono de parede simples sobre um eletrodo de ouro policristalino gerando uma camada de nanotubos alinhados verticalmente na superfície do eletrodo. Para isto, foi utilizado um fragmento de DNA (ssDNA tiol-terminado (5-HS-TGG-GGT-TTA-TGG-AAA-TTGGAA-3)) que foi posicionado ao redor do nanotubo de carbono com o procedimento seguinte: 1,0 mg SWCNT funcionalizado foi misturado com 1,0 mL de uma solução de ssDNA de 1,0 &micro;mol L-1, e o ssDNA foi preparado em 0,1 molL-1 de PBS contendo cloreto de sódio a 10% (v / v). Em seguida, a mistura foi sonicada usando uma sonda de ultra-som por 45 min e depois centrifugada a 10000 rpm por 30 min. Finalmente, um eletrodo de Au previamente limpo foi imerso na solução de sobrenadante e monocamadas auto-organizadas (SAM), que consistem de ssDNA/SWCNT foram formadas durante 24 h numa sala refrigerada a 4 &deg;C. As características morfológicas dos eletrodos foram determinadas por microscopia de força atômica, observando-se o alinhamento vertical, que alterou a rugosidade do eletrodo de 1,95 nm para 47,5 nm, com a altura média dos SWCNTs de 260,3 nm, com um desvio padrão relativo de 19,9%. O comportamento eletroquímico do eletrodo de ouro modificado com o hibrido ssDNA/SWCNT foi caracterizado utilizando voltametria cíclica em meio de Na2SO4 0,1 mol L-1 contendo K3Fe(CN)6 5,0 mmol L-1, com velocidade de varredura de potencial de 50 mVs-1. Observou-se que a reversibilidade do par redox Fe(CN)63-/Fe(CN)64- é maior para o eletrodo modificado com ssDNA/SWCNT (&Delta;Epico= 80 mV) quando comparado ao eletrodo de Au (&Delta;Epico = 115 mV). A modificação proporcionou uma resposta mais eletrocatalítica com um deslocamento de 43 mV para valores menos positivos do potencial de oxidação do Fe(CN)63-. A oxidação no eletrodo de Au/ssDNA/SWCNTs ocorre em +417 mV e no eletrodo de Au em +460 mV. Este aumento de reversibilidade foi quantificado por espectroscopia eletroquímica de impedância faradaica, onde se encontrou os valores de constantes de velocidade de 7.56 &times; 10-5 cm s-1 para o eletrodo modificado e apenas 3,36 &times; 10-5 cm s-1 para o de ouro puro. O efeito da modificação da superfície Au com o nanohíbrido ssDNA / SWCNT na oxidação do bisfenol-A (BPA) foi avaliado em Na2SO4 0,1 mol L-1, pH 6,0, contendo 100 &micro;mol L-1 de BPA por voltametria cíclica a 50 mV s-1. Observou-se um processo de oxidação com um pico voltamétrico anódico num valor de potencial de 510 mV. Este processo de oxidação está relacionado com a eletro-oxidação de BPA para íons fenoxeno. O processo ocorreu em um potencial menos positivo do que o valor observado para o eletrodo de Au não modificado, ou seja 720 mV. Além disso, o processo oxidativo referente à superfície modificada mostrou-se mais catalítico, proporcionando um aumento do pico de oxidação de 163%. <br /> Para a metodologia analítica, procurou-se se maximizar o sinal analítico da técnica de voltametria de pulso diferencial, DPV, assim a resposta para o eletrodo de Au/ssDNA/SWCNT foi estudada em relação ao pH, salto de potenciais e a amplitude de pulso. Os valores ótimos encontrados foram 6,0, 2 mV e 50 mV, respectivamente. Nestas condições o eletrodo de Au/ssDNA/SWCNT foi aplicado para a determinação de BPA em uma solução de Na2SO4 0,1 mol L-1, pH 6,0. A resposta analítica tem um comportamento linear na faixa entre 1,0 - 4,5 &micro;mol L-1, de acordo com a seguinte equação: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A/ &micro;molL-1) [BPA], com um coeficiente de correlação de 0,996 (n = 10) e um limite de detecção (LOD) de 11,0 nmol L-1 (2,51 &micro;g L-1) determinado de acordo com as recomendações da IUPAC. O valor obtido é menor que aqueles disponíveis na literatura. / In the present work, single-walled carbon nanotubes (SWCNT) were immobilized over top a polycrystalline gold electrode. This immobilization assembled a layer of vertically aligned nanotubes on the electrode surface. For this purpose, it was used a DNA probe (ssDNA thiolated (HS-5-TGG-TTA-TGG-GGT-AAA-TTGGAA-3)) that has been used to wrap the carbon nanotube as the following procedure: 1.0 mg of functionalized SWCNT was mixed with 1.0 mL of 1.0 &micro;mol L-1 of a ssDNA solution prepared in 0.1 mol L-1 of PBS containing 10% (v/v) of sodium chloride. Next, the mixture was sonicated using an ultrasonic horn probe and then centrifuged at 10000 rpm; each process took 45 min. Finally, a previously cleaned Au electrode was immersed in the supernatant solution. Self-assembled monolayers (SAMs) consisting of ssDNA/SWCNT were formed after 24 h in a refrigerated room at 4 &deg;C. The morphological characteristics of the electrodes were determined using atomic force microscopy. It was observed that the vertical alignment increased the electrode surface roughness of 1.95 nm to 47.5 nm. The average height of the SWCNT was calculated at 260.3 nm, with a relative standard deviation of 19.9%. The electrochemical behavior of gold electrode modified with the ssDNA/SWCNT hybrid was characterized using cyclic voltammetry (CV) in 0.1 mol L-1 of Na2SO4 containing 5.0 mmol L-1 of [K3Fe(CN)6], with a scan rate of 50 mVs-1. It was observed that the reversibility of the redox couple Fe(CN)63-/Fe(CN)64- decreased using the electrode modified with ssDNA/SWCNT (&Delta;Epeak = 80 mV), when compared with the Au electrode (&Delta;Epeak = 115 mV). The modification provided an electrocatalytic response with a shift of 43 mV to less positive values on the Fe(CN)63- oxidation potential value. The oxidation on the Au/ssDNA/SWCNT electrode occurs at +417 mV and the Au electrode at +460 mV. This improvement on the reversibility was quantified using the electrochemical impedance spectroscopy, in which it was observed an apparent constant rate at 7.56 x 10-5 cm s-1 for the modified electrode and 3.36 x 10-5 cm s-1 for pure gold. The effect of the modification of the Au surface with the nanohybrid ssDNA/SWCNT on the bisphenol A (BPA) oxidation was evaluated 0.1 mol L-1 of Na2SO4 (pH 6.0) containing 100 &micro;mol L-1 of BPA. The system was evaluated using CV at 50 mV s-1. The CV experiments showed an oxidation process with an anodic peak potential at 510 mV. This oxidation process is attributed to the electro-oxidation of the BPA forming the fenoxene ions. The process occurred at a less positive potential value when compared with the unmodified Au electrode, i.e. 720 mV. Moreover, surface modified with the nanohybrid presented more catalytic providing an increase of 163% on the oxidation current peak. For the analytical methodology, the analytical signal was maximized. For this, the differential pulse voltammetry (DPV) parameters such as: pulse amplitude and step potential and pH were optimized. The optimum values found were pH at 6.0, pulse amplitude at 50 mV and step potential at 2 mV. In these conditions, the Au/ssDNA/SWCNT electrode was applied for the BPA determination in 0.1 mol L-1 of Na2SO4. The analytical response showed a linear relationship in a range from 1.0 to 4.5 &micro;mol L-1, in accordance with the following equation: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A / &micro;mol L-1) [BPA ], with a correlation coefficient of 0.996 (n = 10). The limit of detection (LOD) of 11.0 nmol L-1 (2.51 &micro;g L-1) was determined in accordance with the IUPAC recommendations. The obtained value is smaller than those available in the literature.

雙酚-A(bisphenol-A, BPA)廣泛地應用於人類的日常生活中，它是具有雌激素活性的化學物質。近年來，它對人類健康的影響引起了廣泛的關注。研究表明，胎儿期或嬰儿期暴露於BPA中會造成女性卵巢發育紊亂、青春期提前和性早熟。除青鱂、花溪鱂、大菱鮃、金魚、鯉、褐鱒魚和斑馬魚外，BPA對其它硬骨魚生殖功能的影響鮮見報導。而BPA對硬骨魚生殖軸的影響方面更是知之甚少。此外，在斑馬魚中，生殖發育的主要事件，包括性腺分化和青春期開始時間，以及青春期腦垂體激素如GTHs（FSH和LH）的時空表達模式仍然是一個未知數。因此，弄清這些問題不僅有助於理解GTHs在早期性發育中的作用，也有利於研究BPA對斑馬魚生殖軸的影響。 / 利用組織學分析方法、雙色熒光原位雜交（FISH）技術、實時定量PCR技術、蛋白質組分析和在體（活體）試驗，本文研究了斑馬魚性別分化和青春期開始的時間、腦垂體激素FSH（fshb）和LH（lhb）亞基在個體發育過程中特別是性別分化和青春期開始階段的表達模式；同時探討了在青春期之前BPA對斑馬魚生殖軸的影響。從早期的發育階段至性成熟的各個時間點分別收集樣本，用以建立斑馬魚性別分化和青春期開始的時間表及GTHs在其個體發育過程中的表達譜。利用組織學方法檢測性腺發育階段。斑馬魚的頭部（含整個腦和腦垂體）則用於FISH分析以了解其GTHs的表達譜。為分析BPA對斑馬魚生殖功能的影響，將受精後20天的幼魚暴露於濃度為10 μM的BPA中，同時以17β-雌二醇（E2, 10 nM）和睾酮（T, 10 nM） 作為陽性對照。處理20天后分別取其腦、腦垂體、肝臟和卵巢進行組織學、原位雜交、基因表達分析和蛋白質組學分析。 / 雌性斑馬魚青春期的第一個形態學標誌是從初級生長（PG）卵泡第一次轉變為/過渡到卵黃發生階段（PV）；我們的結果表明此過程大約發生在受精後第45天。同時，青春期的啟動似乎高度依賴於身體的生長。另外，原位雜交結果顯示，fshb基因的表達遠早於lhb基因, 在受精後4天就能檢測到fshb的mRNA信號（~2-3細胞/腦垂體）；而lhb的表達則在性別分化時約受精後25天才可檢測到。有趣的是， 表達lhb的細胞數量在青春期前非常少（~5-6 細胞/腦垂體），而青春期期間及之後則大幅增加。相反，在青春期之前，大量細胞表達fshb；青春期期間，表達fshb的細胞數量僅略有增加。因此，我們的結果顯示LH在雌性斑馬魚青春期啟動中具有重要的作用。另一方面，雖然BPA和E2可促進斑馬魚卵巢的分化，但它們亦能顯著抑制卵巢的生長發育。同時，BPA和E2都能明顯抑制垂體fshb的表達，這與其對卵巢大小的抑製作用似乎有密切的相關性。T對垂體fshb的表達無明顯的影響。進一步的結果顯示，BPA和E2不會影響GTHs上游調節基因（包括kiss1, kiss2, gnrh2 和gnrh3）的表達。在肝臟中，BPA和E2顯示出不同的效應。E2能誘導斑馬魚肝增生而導致其腹部水腫；而BPA暴露處理則無此效應。這些結果表明BPA具有雌激素的作用，可影響雌性斑馬魚的生殖功能，但從本研究所使用的劑量效應來看，它卻並不完全具有E2的全部效應。 / Being an estrogenic chemical and its ubiquitous presence in our daily lives, the effects of bisphenal A (BPA) on human health have received tremendous attention in recent years. Studies on the effects of BPA on female reproductive system have shown that early exposure to BPA during the prenatal or postnatal period impairs reproductive functions, including disruption of ovarian development, advanced pubertal onset, and the induction of an early, and persistent estrus. In teleost fish, few studies have been reported on the effects of BPA on reproductive function, except in medaka, Kryptolebias marmoratus, turbot, goldfish, common carp, brown trout and zebrafish. Despite these studies, the effects of BPA on reproductive axis remain largely unknown in teleost fish. On the other hand, in the zebrafish model, the major developmental events of reproduction, including the timing of puberty onset, the spatiotemporal expression patterns of key pituitary hormones such as GTHs (FSH and LH) during gonadal differentiation and puberty development remain largely unknown. Therefore, the information on these issues in zebrafish not only is valuable for understanding the roles of GTHs in early sexual development; also facilitate our study on the effects of BPA on the reproductive axis in the zebrafish. / Using histology analysis, double-colored fluorescent in situ hybridization (FISH), real-time qPCR, proteomic analysis and in vivo treatment, this study was undertaken to explore the timing of sex differentiation and puberty onset, the ontogenic expression patterns of FSH (fshb) and LH (lhb) subunits in the zebrafish pituitary with particular emphasis on the stage of sexual differentiation and puberty onset, and the effects of BPA on the reproductive axis in zebrafish during prepubertal period. To define the timeline of sex differentiation and puberty onset, and the ontogenic expression profiles of GTHs, the zebrafish were collected at different time points from early development stage to sexual maturation. The gonadal developmental stage was analyzed by histological examination. For the expression profiles of GTHs, the head of each fish including the brain and pituitary was sampled for FISH analysis. To investigate the influence of BPA on the reproductive function, Juvenile zebrafish of 20 day post-fertilization (dpf) were exposed to BPA (10 μM) for 20 days followed by sampling the brain, pituitary, liver and ovary for histological, in situ hybridization, expression analyses and proteomic analyses at 40 dpf. 17β-estradiol (E2, 10 nM) and testosterone (T, 10 nM) were also used as a positive control. / In female zebrafish, the first morphological sign for puberty is the first wave of follicle transition from the primary growth (PG) to previtellogenic stage (PV), our results showed that it occurs around 45 day post fertilization (dpf). Meanwhile, the puberty onset was highly depending on the somatic growth. The expression of fshb was much earlier than that of lhb with its mRNA signal detectable (2-3 cells/pituitary) shortly after hatching (4 dpf). In contrast, lhb expression became detectable at the time of sex differentiation (~25 dpf). Interestingly, the number of lhb-expressing cells was very low (~5-6 cells/pituitary) before puberty but increased dramatically during and after puberty onset. In contrast, the expression of fshb was abundant before puberty with only a slight increase in cell number during puberty onset. Our result strongly suggests an important role for LH at the puberty onset of female zebrafish. On the other hand, although BPA and E2 both promoted ovarian differentiation, they significantly suppressed the ovarian growth afterwards in the zebrafish. Meanwhile, both BPA and E2, but not T, dramatically decreased the expression of fshb in the pituitary, which was well correlated with the suppression of ovarian size. However, the expression of the upstream regulators of GTHs, including kiss1, kiss2, gnrh2 and gnrh3, was not affected by BPA and E2. Interestingly, at the liver level, BPA and E2 displayed different effects. E2 induced abdominal swelling due to a significant hepatic hyperplasia. However, BPA exposure had no such effect on the liver. These results indicate that BPA has estrogenic effects on female reproduction, but it does not mimic E2 in all aspects, at least for the dose tested in the present study. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Weiting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 93-120). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract in English --- p.I / Abstract in Chinese --- p.III / Acknowledgement --- p.V / Table of contents --- p.VI / List of figures and tables --- p.IX / Symbols and abbreviation --- p.XI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Functions in reproduction --- p.2 / Chapter 1.2 --- Gonadotropins --- p.2 / Chapter 1.2.1 --- Structure --- p.2 / Chapter 1.2.2 --- Expression profile --- p.2 / Chapter 1.2.3 --- Regulation --- p.4 / Chapter 1.3 --- Gonadal development --- p.8 / Chapter 1.3.1 --- Sex differentiation --- p.8 / Chapter 1.3.2 --- Puberty initiation --- p.10 / Chapter 1.4 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Puberty Initiation is Dependent on the Body Growth but not Age in Female Zebrafish / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Animals --- p.16 / Chapter 2.2.2 --- Sampling and measurement of body weight and body length --- p.16 / Chapter 2.2.3 --- Paraffin section and H & E staining --- p.17 / Chapter 2.2.4 --- Statistical analysis --- p.17 / Chapter 2.3 --- Results --- p.17 / Chapter 2.3.1 --- Growth curve of zebrafish during gonadal differentiation and maturation --- p.17 / Chapter 2.3.2 --- Gonadal differentiation in the zebrafish --- p.17 / Chapter 2.3.3 --- Puberty onset in female zebrafish --- p.18 / Chapter 2.3.4 --- Relationship of body growth and puberty initiation in female zebrafish --- p.19 / Chapter 2.4 --- Discussion --- p.19 / Chapter Chapter 3 --- Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish / Chapter 3.1 --- Introduction --- p.29 / Chapter 3.2 --- Materials and methods --- p.30 / Chapter 3.2.1 --- Animals --- p.30 / Chapter 3.2.2 --- Sampling --- p.31 / Chapter 3.2.3 --- Histological examination --- p.31 / Chapter 3.2.4 --- Total RNA isolation and reverse transcription --- p.32 / Chapter 3.2.5 --- Fluorescent double-colored in situ hybridization --- p.32 / Chapter 3.2.6 --- Microinjection of morpholino knockdown --- p.32 / Chapter 3.2.7 --- Real-time qPCR quantification of fshb, lhb and gh expression --- p.33 / Chapter 3.2.8 --- Data analysis --- p.34 / Chapter 3.3 --- Results --- p.34 / Chapter 3.3.1 --- Detection of fshb, lhb and gh expression in the pituitary of adult zebrafish --- p.34 / Chapter 3.3.2 --- Expression of fshb, lhb and gh before gonadal differentiation (4-25 dpf) --- p.34 / Chapter 3.3.3 --- Expression of fshb, lhb and gh during gonadal differentiation (22-25 dpf) --- p.35 / Chapter 3.3.4 --- Expression of fshb, lhb and gh during puberty period (~45 dpf) --- p.35 / Chapter 3.3.5 --- Phenotypes of fshb-MO and lhb-MO zebrafish in early development36 --- p.36 / Chapter 3.4 --- Discussion --- p.37 / Chapter Chapter 4 --- Neonatal Exposure to 17β-Estradiol or Bisphenol A Promotes Ovarian Differentiation but Suppresses Its Growth Probably via Inhibiting Follicle-Stimulating Hormone Expression / Chapter 4.1 --- Introduction --- p.55 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Animals --- p.57 / Chapter 4.2.2 --- In vivo treatment and hormone replacement --- p.57 / Chapter 4.2.3 --- Sampling --- p.58 / Chapter 4.2.4 --- Total RNA isolation and reverse transcription --- p.58 / Chapter 4.2.5 --- Fluorescent double-colored in situ hybridization --- p.58 / Chapter 4.2.6 --- Real-time qPCR quantification --- p.59 / Chapter 4.2.7 --- Protein extraction and quantification --- p.59 / Chapter 4.2.8 --- Two-dimensional electrophoresis --- p.60 / Chapter 4.2.9 --- Staining --- p.61 / Chapter 4.2.10 --- In-gel digestion --- p.61 / Chapter 4.2.11 --- Mass spectrometry --- p.61 / Chapter 4.2.12 --- Data analysis --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- E2 had distinct effect on the body growth and behavior --- p.62 / Chapter 4.3.2 --- BPA and E2 increased female ratio but suppressed ovarian growth in the zebrafish --- p.62 / Chapter 4.3.3 --- BPA and E2 shut down fshb but increased lhb expression in the pituitary without altering the expression of GnRH and kisspeptin in the hypothalamus --- p.63 / Chapter 4.3.4 --- E2 but not BPA induced hepatic hyperplasia --- p.64 / Chapter 4.4 --- Discussion --- p.65 / Chapter Chapter 5 --- General Discussion

Neste trabalho foram imobilizados nanotubos de carbono de parede simples sobre um eletrodo de ouro policristalino gerando uma camada de nanotubos alinhados verticalmente na superfície do eletrodo. Para isto, foi utilizado um fragmento de DNA (ssDNA tiol-terminado (5-HS-TGG-GGT-TTA-TGG-AAA-TTGGAA-3)) que foi posicionado ao redor do nanotubo de carbono com o procedimento seguinte: 1,0 mg SWCNT funcionalizado foi misturado com 1,0 mL de uma solução de ssDNA de 1,0 &micro;mol L-1, e o ssDNA foi preparado em 0,1 molL-1 de PBS contendo cloreto de sódio a 10% (v / v). Em seguida, a mistura foi sonicada usando uma sonda de ultra-som por 45 min e depois centrifugada a 10000 rpm por 30 min. Finalmente, um eletrodo de Au previamente limpo foi imerso na solução de sobrenadante e monocamadas auto-organizadas (SAM), que consistem de ssDNA/SWCNT foram formadas durante 24 h numa sala refrigerada a 4 &deg;C. As características morfológicas dos eletrodos foram determinadas por microscopia de força atômica, observando-se o alinhamento vertical, que alterou a rugosidade do eletrodo de 1,95 nm para 47,5 nm, com a altura média dos SWCNTs de 260,3 nm, com um desvio padrão relativo de 19,9%. O comportamento eletroquímico do eletrodo de ouro modificado com o hibrido ssDNA/SWCNT foi caracterizado utilizando voltametria cíclica em meio de Na2SO4 0,1 mol L-1 contendo K3Fe(CN)6 5,0 mmol L-1, com velocidade de varredura de potencial de 50 mVs-1. Observou-se que a reversibilidade do par redox Fe(CN)63-/Fe(CN)64- é maior para o eletrodo modificado com ssDNA/SWCNT (&Delta;Epico= 80 mV) quando comparado ao eletrodo de Au (&Delta;Epico = 115 mV). A modificação proporcionou uma resposta mais eletrocatalítica com um deslocamento de 43 mV para valores menos positivos do potencial de oxidação do Fe(CN)63-. A oxidação no eletrodo de Au/ssDNA/SWCNTs ocorre em +417 mV e no eletrodo de Au em +460 mV. Este aumento de reversibilidade foi quantificado por espectroscopia eletroquímica de impedância faradaica, onde se encontrou os valores de constantes de velocidade de 7.56 &times; 10-5 cm s-1 para o eletrodo modificado e apenas 3,36 &times; 10-5 cm s-1 para o de ouro puro. O efeito da modificação da superfície Au com o nanohíbrido ssDNA / SWCNT na oxidação do bisfenol-A (BPA) foi avaliado em Na2SO4 0,1 mol L-1, pH 6,0, contendo 100 &micro;mol L-1 de BPA por voltametria cíclica a 50 mV s-1. Observou-se um processo de oxidação com um pico voltamétrico anódico num valor de potencial de 510 mV. Este processo de oxidação está relacionado com a eletro-oxidação de BPA para íons fenoxeno. O processo ocorreu em um potencial menos positivo do que o valor observado para o eletrodo de Au não modificado, ou seja 720 mV. Além disso, o processo oxidativo referente à superfície modificada mostrou-se mais catalítico, proporcionando um aumento do pico de oxidação de 163%. <br /> Para a metodologia analítica, procurou-se se maximizar o sinal analítico da técnica de voltametria de pulso diferencial, DPV, assim a resposta para o eletrodo de Au/ssDNA/SWCNT foi estudada em relação ao pH, salto de potenciais e a amplitude de pulso. Os valores ótimos encontrados foram 6,0, 2 mV e 50 mV, respectivamente. Nestas condições o eletrodo de Au/ssDNA/SWCNT foi aplicado para a determinação de BPA em uma solução de Na2SO4 0,1 mol L-1, pH 6,0. A resposta analítica tem um comportamento linear na faixa entre 1,0 - 4,5 &micro;mol L-1, de acordo com a seguinte equação: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A/ &micro;molL-1) [BPA], com um coeficiente de correlação de 0,996 (n = 10) e um limite de detecção (LOD) de 11,0 nmol L-1 (2,51 &micro;g L-1) determinado de acordo com as recomendações da IUPAC. O valor obtido é menor que aqueles disponíveis na literatura. / In the present work, single-walled carbon nanotubes (SWCNT) were immobilized over top a polycrystalline gold electrode. This immobilization assembled a layer of vertically aligned nanotubes on the electrode surface. For this purpose, it was used a DNA probe (ssDNA thiolated (HS-5-TGG-TTA-TGG-GGT-AAA-TTGGAA-3)) that has been used to wrap the carbon nanotube as the following procedure: 1.0 mg of functionalized SWCNT was mixed with 1.0 mL of 1.0 &micro;mol L-1 of a ssDNA solution prepared in 0.1 mol L-1 of PBS containing 10% (v/v) of sodium chloride. Next, the mixture was sonicated using an ultrasonic horn probe and then centrifuged at 10000 rpm; each process took 45 min. Finally, a previously cleaned Au electrode was immersed in the supernatant solution. Self-assembled monolayers (SAMs) consisting of ssDNA/SWCNT were formed after 24 h in a refrigerated room at 4 &deg;C. The morphological characteristics of the electrodes were determined using atomic force microscopy. It was observed that the vertical alignment increased the electrode surface roughness of 1.95 nm to 47.5 nm. The average height of the SWCNT was calculated at 260.3 nm, with a relative standard deviation of 19.9%. The electrochemical behavior of gold electrode modified with the ssDNA/SWCNT hybrid was characterized using cyclic voltammetry (CV) in 0.1 mol L-1 of Na2SO4 containing 5.0 mmol L-1 of [K3Fe(CN)6], with a scan rate of 50 mVs-1. It was observed that the reversibility of the redox couple Fe(CN)63-/Fe(CN)64- decreased using the electrode modified with ssDNA/SWCNT (&Delta;Epeak = 80 mV), when compared with the Au electrode (&Delta;Epeak = 115 mV). The modification provided an electrocatalytic response with a shift of 43 mV to less positive values on the Fe(CN)63- oxidation potential value. The oxidation on the Au/ssDNA/SWCNT electrode occurs at +417 mV and the Au electrode at +460 mV. This improvement on the reversibility was quantified using the electrochemical impedance spectroscopy, in which it was observed an apparent constant rate at 7.56 x 10-5 cm s-1 for the modified electrode and 3.36 x 10-5 cm s-1 for pure gold. The effect of the modification of the Au surface with the nanohybrid ssDNA/SWCNT on the bisphenol A (BPA) oxidation was evaluated 0.1 mol L-1 of Na2SO4 (pH 6.0) containing 100 &micro;mol L-1 of BPA. The system was evaluated using CV at 50 mV s-1. The CV experiments showed an oxidation process with an anodic peak potential at 510 mV. This oxidation process is attributed to the electro-oxidation of the BPA forming the fenoxene ions. The process occurred at a less positive potential value when compared with the unmodified Au electrode, i.e. 720 mV. Moreover, surface modified with the nanohybrid presented more catalytic providing an increase of 163% on the oxidation current peak. For the analytical methodology, the analytical signal was maximized. For this, the differential pulse voltammetry (DPV) parameters such as: pulse amplitude and step potential and pH were optimized. The optimum values found were pH at 6.0, pulse amplitude at 50 mV and step potential at 2 mV. In these conditions, the Au/ssDNA/SWCNT electrode was applied for the BPA determination in 0.1 mol L-1 of Na2SO4. The analytical response showed a linear relationship in a range from 1.0 to 4.5 &micro;mol L-1, in accordance with the following equation: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A / &micro;mol L-1) [BPA ], with a correlation coefficient of 0.996 (n = 10). The limit of detection (LOD) of 11.0 nmol L-1 (2.51 &micro;g L-1) was determined in accordance with the IUPAC recommendations. The obtained value is smaller than those available in the literature.

Bei Daidzein und Bisphenol A handelt es sich um zwei Vertreter einer Klasse von Stoffen, die als „Umwelthormone“ (engl. endocrine disrupter) bezeichnet werden. Aus der Gruppe der Phytoöstrogene wurde Daidzein als wichtiger Vertreter, der in hohen Konzentrationen in vielen Nutzpflanzen und Nahrungsmitteln vorkommt, ausgewählt. Sojaprodukte, die den größten Beitrag einer menschlichen Exposition gegen Daidzein liefern, werden in zunehmendem Maße auch in westlichen Ländern konsumiert. Bisphenol A wurde als Vertreter der Xenoöstrogene gewählt, da es - was Weltjahresproduktion und Verwendung angeht - die wohl wichtigste Substanz dieser Gruppe darstellt. Im ersten Teil der Arbeit wurde die Biotransformation und Toxikokinetik der beiden Verbindungen nach oraler Gabe in der Ratte aufgeklärt. Dabei konnte gezeigt werden, daß die orale Bioverfügbarkeit beider Substanzen in der Ratte sehr gering war. Maximal zehn Prozent der jeweils applizierten Dosis konnten im Urin der Tiere wiedergefunden werden. Als Hauptmetabolit wurden sowohl von Daidzein als auch von Bisphenol A das jeweilige Glucuronid-Konjugat gebildet. Bei Daidzein überwog in der männlichen Ratte zusätzlich das Sulfat-Konjugat. Der Anteil an freier, d.h. unkonjugierter Verbindung betrug im Urin der Tiere zwischen 1 und 3 Prozent der Dosis. Außer den Phase II-Konjugaten, die aufgrund ihrer mangelnden östrogenen Wirksamkeit zu einer Detoxifizierung der beiden Verbindungen führte, konnten nach Gabe von Bisphenol A in der Ratte keine weiteren Metabolite identifiziert werden. Nach Exposition mit Daidzein konnten in den Faeces der Tiere in geringem Umfang die beiden reduktiven Metabolite Equol und O-DMA gefunden werden. Diese wurden wahrscheinlich im Magen-Darm-Trakt durch die Bakterien der Darmflora gebildet. Sowohl Daidzein als auch Bisphenol A wurden bei der Ratte nur unvollständig aus dem Magen-Darm-Trakt resorbiert; der Großteil der gegebenen Dosis wurde als unveränderte Substanz in den Faeces wiedergefunden. Bei Bisphenol A wurde die Ausscheidung zudem durch einen ausgeprägten enterohepatischen Kreislauf verzögert. Im zweiten Teil der Arbeit wurden zunächst empfindliche GC/MS- und HPLC-Methoden zur Quantifizierung der Verbindungen in humanen Plasma- und Urinproben entwickelt. Danach wurden freiwillige Probanden oral mit jeweils 5 mg Daidzein bzw. d16-Bisphenol A exponiert, um Daten zur Biotransformation und Toxikokinetik der beiden Substanzen im Mensch zu erhalten. Wegen des deutlich meßbaren Hintergrundes an Bisphenol A, das in allen Kontrollproben nachweisbar war, wurde für die Humanstudie die deuterierte Verbindung gegeben, für die kein störender Hintergrund meßbar war. Die Bioverfügbarkeit der Gesamt-Substanz (freie Verbindung + Konjugate) im Menschen war in beiden Fällen deutlich höher als in der Ratte. Von Daidzein wurden 40 Prozent (Ratte 10 Prozent), von Bisphenol A > 95 Prozent (Ratte 13 Prozent) der applizierten Dosis im Urin der Probanden wiedergefunden. Dabei zeigte sich ein sehr effizienter Phase II-Metabolismus; weniger als 1 Prozent der Glucuronid-Konjugatkonzentrationen wurden als unveränderte Substanz gefunden. Das Glucuronid stellte in beiden Fällen den einzigen nachweisbaren Metaboliten dar. Die Elimination von Daidzein und Bisphenol A verlief in den beiden Studien sehr schnell nach einer Kinetik erster Ordnung. Im Gegensatz zu der Ratte konnten auch bei Bisphenol A keine Auffälligkeiten in den Ausscheidungskurven beobachtet werden, Hinweise auf einen enterohepatischen Kreislauf im Menschen wurden nicht gefunden. Im Falle von Bisphenol A wurde fast die komplette applizierte Dosis (> 95 Prozent) in Form des Glucuronides im Urin wiedergefunden. Anhand der erhobenen Daten wurde anschließend eine Beurteilung des Risikos für den Menschen abgegeben. / Daidzein and bisphenol a are two representatives of a class of substances known as endocrine disrupters. A common mark of these compounds is their affinity to at least one of two estrogen receptors in vitro. This leads to speculation on how such compounds may interfere with hormonal regulation in animals and humans. As an important representative of the group of phytoestrogens daidzein has been chosen. Daidzein occurs in high concentrations in plants like soy, thus contributing to a human exposure via food. Bisphenol a has been chosen for this thesis because it probably is the most important industrial chemical suspected of endocrine activity, considering worldwide annual production numbers. Biotransformation and kinetics of the two model substances, daidzein and bisphenol a, have been elucidated. The results showed that oral bioavailability of the two chemicals has been very low. Less than ten percent of the dose given could be recovered from urine of animals. The major metabolite in biotransformation of both daidzein and bisphenol a proved to be the glucuronide of the respective compound. Additionally, after application of daidzein to rats, daidzein sulfate could be identified specifically in male animals only. The percentage of unconjugated parent compound in both studies has been shown to be between one and three percent of the dose given. No further metabolites could be found after oral administration of bpa to rats; after oral administration of daidzein to rats, equol and o-dma could be identified as minor metabolites in feces of animals. In both studies, the major part of the administered dose could be recovered as unchanged parent compound from feces. In the case of BPA, elimination was slowed by the occurence of enterohepatic circulation. This explains why the elimination of BPA and its conjugates was slow and did not follow a first-order kinetics. Furthermore, sensitive analytical methods (HPLC and GC/MS) were developed to allow quantification of low amounts of the two model compounds in human plasma und urine samples. To obtain information on the biotransformation and toxicokinetics in humans, volunteers were given 5 mg of either daidzein or d16-bisphenol a. Because of a rather high background for bisphenol a in control samples, deuterated bisphenol a had been chosen for the human study. Bioavailability of total substance (i.e. unconjugated + conjugated compound) in humans was markedly higher than in rats. After controlled exposure to daidzein 40 per cent (as compared to 10 per cent in rats) could be recovered from urine, in the case of d16-bpa more than 95 per cent (as compared to 13 per cent in rats) could be recovered. Less than 1 per cent of the concentration of the conjugated compound could be found as unchanged parent compound. In both cases, the glucuronide has been identified as sole metabolite in human volunteers. Elimination of both substances was quick and followed a first order kinetics. In the case of d16-bisphenol a, all of the given dose could be recovered from urine. With the data gathered, an assessment of the risk posed by these chemicals to humans was given.