Metallosupramolecular chemistry involves the construction of nanoscale molecular assemblies by reacting metal atoms with bridging organic ligands. The metal atoms act as a type of molecular ‘glue’ binding together the organic ligands in specific orientations. Thus, appropriate combinations of metal ions and ligands lead to the controlled self-assembly of interesting one-, two- and three-dimensional molecular aggregates. This thesis details the preparation of a range of novel flexible bridging heterocyclic ligands using conventional organic synthesis, and then explores their reactions with a variety of transition metal precursors. By varying the nature of the organic ligand and the transition metal precursor, new and exciting supramolecular topologies and architectures can be formed. A total of forty-eight ligands were synthesised in this work, forty-seven of which are new compounds. The majority of the ligands synthesised were based around commercially available bisphenol cores. All forty-eight of the ligands had nitrogen heterocyclic groups as coordinating units. The ligands discussed in this thesis can be divided into three main sections. The first involves the synthesis and coordination chemistry of two-armed ligands based around the Bisphenol A, Bisphenol Z and Bisphenol AP cores. The second section describes the synthesis and coordination chemistry of the larger Bisphenol P and Bisphenol M based two-armed bridging ligands. The third section describes the synthesis and coordination chemistry of various multi-substituted ligands, including tripodal ligands based around a trisphenol core, four-armed ligands and six-armed ligands. The two-armed bisphenol based ligands proved very successful as synthons in metallosupramolecular chemistry and produced many products with a variety of different metal atoms. The complexes characterised included discrete dimeric products, coordination polymers and a number of helicates, including a dinuclear quadruply-stranded helicate. Multi-armed ligands are topical, because they have multiple coordination sites that are capable of binding and bridging multiple metal atoms. Such coordination can lead to the construction of cage-like species and complicated networks. A series of three-armed ligands based around a trisphenol core were synthesised with the intention to use these to form such species on coordination with appropriate metal salts. Indeed, one of the products of self-assembly was an interesting M₃L₂ cage. Various other multi-armed ligands were also investigated. The ligands and complexes in this thesis were characterised by a variety of structural techniques, such as ¹H NMR, ¹³C NMR, mass spectrometry, elemental analysis and X-ray crystallography when crystals were obtained. The crystal structures of twenty-seven ligands and forty-three complexes are described.

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"

Humans are exposed to chemicals every day in our indoor environment. Chemicals have contributed to increased prosperity but also caused negative health effects. Children are vulnerable to chemicals because of their development and they breathe and drink more in relation to their body weight. That is why it’s important to reduce chemical exposure in environment for children. Preschools have a central role when it comes to materials and products that may pose a risk for exposure. The municipality has an important role to achieve a nontoxic environment which is based on a Swedish environmental quality goal (Non-toxic environment). This study is a part of the prioritized local environmental work in the municipal of Nordmaling. An inventory has been done to find out what type of chemicals that may expose children to harmful effects in preschools of Nordmaling. Electronic as a toy, soft and smelling toys, plastic around food and drinks, foam play pads were common products found during the inventory. This materials containing phthalates, brominated flame retardants, perfluorinated compounds, bisphenol A, lead and cadmium that can expose children through leaching into the indoor environment. A guidance have been constructed to achieve a non-toxic environment based on the results from the inventory in a cost effective way. The conclusion of the report is clear, identified problem areas can quickly be accomplished with small measures and low costs. Increased awareness of harmful substances and good routines for purchases and cleaning can further reduce the exposure to chemicals in the preschools.

Résumé : La prééclampsie (PE) est un désordre de la grossesse caractérisée par une dysfonction endothéliale faisant en sorte que l’endothélium devient moins sensible aux signaux de vasodilatation. La réponse provoquée par la liaison de la sérotonine au sous-type de récepteur S[indice inférieur 2] entraîne la libération de molécules aux propriétés vasoconstrictrices, qui, par une boucle de rétroaction positive, entraîne la libération de davantage de sérotonine par les plaquettes. Cette boucle amplifie la réponse et contribue ainsi à l’hypertension présente chez les femmes ayant une PE. Précédemment, il a été démontré par notre laboratoire que le Bisphénol A (BPA) s’accumulait davantage dans le placenta des femmes avec PE en comparaison aux femmes normotensives. Cette accumulation pourrait découler d’une perturbation de sa métabolisation qui impliquerait notamment la β-glucuronidase (GUSB). Des études chez les animaux ont quant à elles démontré que le BPA pouvait inhiber l’activité de la monoamine oxydase (MAO) à forte dose. Nous avons étudié l’effet du BPA à faible concentration (10 ng/ml) sur la MAO-A des cellules placentaires et démontré que le BPA inhibait la MAO-A de façon significative sans affecter son expression protéique. Afin d’expliquer l’accumulation particulière du BPA chez les femmes PE, nous avons comparé l’activité spécifique et l’expression protéique de la β-glucuronidase (GUSB) placentaire en utilisant un devis cas-témoins. Une tendance non significative suggère que la GUSB pourrait partiellement contribuer à l’accumulation du BPA chez les femmes PE. Nous avons étudié la relation entre la concentration sérique maternelle de BPA et la concentration à laquelle le fœtus est exposé par régression linéaire et corrélation de Spearman. Un tel modèle ne pourrait être utilisé pour déterminer de façon quantitative l’exposition fœtale. En revanche, en vue de la forte corrélation entre ces deux variables, une haute concentration sérique maternelle de BPA devrait se refléter par une haute exposition fœtale. Cette corrélation implique aussi que le métabolisme placentaire ne joue pas un rôle significatif dans la protection du fœtus. Le BPA pourrait ainsi contribuer à l’hypertension chez les femmes PE présentant une dysfonction endothéliale en inhibant la MAO-A et ainsi, favorisant la hausse de sérotonine circulante. Cette étude suggère les bases d’un mécanisme par lequel le BPA s’accumulerait davantage chez les femmes PE et affecterait ainsi la MAO-A placentaire et potentiellement, la MAO-A fœtale vu ses propriétés physico-chimiques. / Abstract : Preeclampsia (PE) is an hypertensive disorder of pregnancy characterized by a generalized endothelial dysfunction where the response to vasodilatation signals is compromised. The binding of serotonin to its S[subscript 2] receptor subtype 2 releases vasoconstrictor molecules which, by a positive retroaction loop, stimulates the release of more serotonin from platelets. This positive retroaction loop stimulates the vasoconstriction of blood vessels and contributes to the hypertension in women with PE. Previously, we showed that Bisphenol A (BPA) accumulates more in the placenta of women with PE than in normotensive women. This accumulation may be the result of an impaired metabolization due to the action of the β-Glucuronidase (GUSB). Animal studies showed that BPA at high dose could lower the activity of the monoamine oxidase A (MAO-A), an enzyme implicated in the metabolism of serotonin. We studied the impact of BPA at low dose (10 ng/ml) in trophoblastic primary cells and showed that even at low dose, BPA can lower its activity without affecting the protein expression. To determine if GUSB could be the cause of the BPA accumulation in women with PE, we studied its activity and protein expression in placental biopsies from women with and without PE. A nonsignificant tendency showed that the GUSB activity and protein expression were higher in women with PE. To study the impact of placental metabolism in the fetal exposure, we studied the relation between maternal and fetal concentrations of BPA with linear regression analysis and Spearman’s correlation. We showed that maternal BPA could not precisely predict the fetal exposure and that the placental metabolism is probably limited in light of the strong correlation between both variables. This strong correlation also implied that high maternal exposure would result in high fetal exposure. This study shows that the accumulation of BPA in preeclamptic women could contribute to maternal hypertension by interacting with serotonin levels. This accumulation could partially be attributed to a higher GUSB, but other factors are probably implicated. The strong correlation between maternal and fetal exposure implies that the placental metabolism of BPA is limited and does not protect the fetus significantly. This study suggests the basis of a mechanism explaining the abnormal accumulation of BPA in the placenta in women with PE and its impact on the placental MAO-A and potentially, the foetal MAO-A because of its physico-chemical properties.

Neste estudo determinou-se o nonilfenol - um composto xenobiótico utilizados na fabricação de antioxidantes, aditivos para óleos lubrificantes e produção de surfactantes etoxilados - e o bisfenol A - um composto químico utilizado na produção de diversos produtos, como, plásticos policarbonatos, resinas epóxi e retardantes de chama. Ambos exibem atividade estrogênica em concentrações abaixo de 1ng L-1 - em cinco pontos de coleta na cidade de Americana, SP. Devido à ampla utilização, esses compostos estão presentes em esgoto industrial em quantidades substanciais. A primeira etapa deste estudo foi a validação dos método analítico seguindo o Método 8270D da United States Environmental Protection Agency (USEPA) que descreve o procedimento de validação para os compostos semivoláteis analisados por cromatografia gasosa acoplada a espectrometria de massas (GC/MS). Os limites de detecção e de quantificação do método para a determinação do nonilfenol foram, respectivamente, 1 e 5 µg L-1. O coeficiente de determinação (R2) e a recuperação média para o nonilfenol foram, respectivamente, 0,99 e 99,04%. Os limites de detecção e de quantificação do método para o bisfenol A foram, respetivamente, 1 e 5 µg L-1. O coeficiente de determinação (R2) e a recuperação média para o bisfenol A foram, respectivamente, 0,99 e 91,55%. Na segunda etapa desta pesquisa analisaram-se amostras de água coletadas na cidade de Americana para a determinação do nonilfenol e do bisfenol A. Como método de extração dos compostos, usou-se a extração em fase sólida (SPE) e como técnica analítica a cromatografia gasosa acoplada a espectrometria de massas (GC/MS). Os resultados indicaram a presença desses compostos em alguns pontos de amostragem sendo que a concentração do nonilfenol a variou de 5,028 a 10,524 µg L-1 e para o bisfenol A a concentração variou de 5,645 a 295,255 µg L-1. Foram feitos também testes ecotoxicólogicos utilizando como organismo teste o microcrustáceo Daphnia similis, porém o nonilfenol e o bisfenol A, na concentração testada que variou de 0,05 a 500 µg L-1 não apresentaram toxicidade aguda. / In this study it was determined nonylphenol - a xenobiotic compound used in the manufacture of antioxidants, additives for lubricating oils and production of ethoxylated surfactant - and bisphenol A - a chemical compound used in production of various products, such as polycarbonate plastic, epoxy resins and flame retardants. Both exhibit estrogenic activity at concentrations lower than 1 ng L-1 - in five sampling points in the city of Americana, SP. Due to the widespread use of these compounds, they are present in substantial quantities in industrial sewage. The first step of this study was to validate the analytical method following the Method 8270D of the United States Environmental Protection Agency (USEPA) that describes the validation procedure for semi-volatile compounds analized by gas chromatography-mass spectrometry (GC/MS). The limits of detection and quantification for determining the nonylphenol were, respectively, 1 and 5 µg L-1. The coefficient ofdetermination (R2) and recovery for nonylphenol were respectively 0.99 and 99.04%. The limits of detection and quantification for the bisphenol A were, respectively, 1 and 5 µg L-1. The coefficient of determination (R2) and recovery for bisphenol A were respectively 0.99 and 91.55%. In the second step of this study were analized in water samples of Americana city for the determination of nonylphenol and bisphenol A. As a method of extraction of the compounds it was used solid-phase extraction (SPE) and as analytical technique GC/MS. The results indicated the presence of such compounds in some sampling points and the concentration of nonylphenol ranged from 5.028 to 10.524 µg L-1 and bisphenol A concentration ranged from 5.645 to 295.255 µg L-1. Ecotoxicological tests were also performed using as test organism microcrustaceans Daphnia similis, but nonylphenol and bisphenol A, tested at concentration ranging from 0,05 to 500 µg L-1 showed no acute toxicity.

O bisfenol A (BPA) e o nonilfenol (NP), presentes em detergentes, pesticidas, plásticos e resinas, são conhecidos como Endocrine Disrupting-Chemicals (EDCs), Disruptores ou Desreguladores Endócrinos (DE) ou ainda Perturbadores Endócrinos ou Interferentes Endócrinos. O desregulador endócrino pode ser definido como uma substância química exógena ou mistura, que promove alterações em uma ou mais funções do sistema endócrino e na sua estrutura, causando, conseqüentemente, efeitos adversos na saúde de um organismo, ou a sua descendência. Esses compostos estão presentes nas águas de abastecimento, nos efluentes domésticos e industriais. Os desreguladores endócrinos têm ação mimética aos hormônios tanto no sítio de ligação quanto nos efeitos provocados nos seres vivos a eles expostos. Os compostos bisfenol A e nonilfenol foram identificados como desreguladores endócrinos em inúmeros trabalhos em diversos países, porém sendo pouco os efeitos em organismos tropicais. Este estudo teve como objetivo avaliar a toxicidade aguda do bisfenol A e do nonilfenol para Daphnia similis e Ceriodaphnia silvestrii, bem como, a toxicidade crônica para Ceriodaphnia silvestrii e Chironomus xanthus. Nos testes de toxicidade aguda com BPA os valores de CE(I)50; 48h foram de 10,64 mg/L para Daphnia similis e 19,9 mg/L para Ceriodaphnia silvestrii. Nos testes crônicos o valor do CEO obtido para Ceriodaphnia silvestrii foi 1,29 mg/L e para Chironomus xanthus 12 mg/L e o CENO 6 mg/L. Nos testes de toxicidade aguda feitos com NP, os valores de CE(I)50; 48h foram de 0,309 mg/L para Daphnia similis, 0,4520 mg/Lpara Ceriodaphnia silvestrii com solvente água e 0,0541 mg/L com solvente etanol, e 0,03398 mg/L para Ceriodaphnia dubia. O valor do CEO para Ceriodaphnia silvestrii foi 0,0198 mg/L e para Chironomus xanthus foi 100 \'mü\'g/g, com CENO de 50 \'mü\'g/g. Apesar das concentrações encontradas nos testes serem maiores que as encontradas nos estudos que quantificaram esses compostos no ambiente, outros estudos demonstraram que, mesmo em concentrações inferiores as obtidas nesse estudo já ocorrem problemas relacionados com o tempo da muda e com a androgenização do metabolismo de cladóceros. Além disso, a partir dos resultados obtidos com estes testes, pretende-se alertar o poder público sobre os riscos inerentes da presença destes produtos químicos nas águas e da urgência em se adotar novas técnicas no tratamento de efluentes visando à remoção eficaz desses poluentes. / Bisphenol A (BPA) and nonylphenol (NP), present in detergents, pesticides, plastics and resins, are known as Endocrine-Disrupting Chemicals (EDCs), Disruptors or Endocrine Disruptors (ED), Endocrine Disruption or Interferences Endocrine. The endocrine disruptors can be defined as an exogenous chemical substance or mixture, which causes changes in one or more functions of the endocrine system and its structure, causing, therefore, adverse effects on the health of an organism, or its progeny. These compounds are present in the water supply in domestic and industrial effluents. EDs have mimetic action to hormones in both binding site as the effects caused in living beings exposed to them. The compounds bisphenol A and nonylphenol were identified as endocrine disruptors in numerous studies in many countries, but with little effects on tropical organisms. This study aimed to evaluate the acute toxicity of bisphenol A and nonylphenol Daphnia similis and Ceriodaphnia silvestrii, as well as chronic toxicity to Ceriodaphnia silvestrii and Chironomus xanthus. In acute toxicity tests with BPA values EC(I) 50, 48 h were 10.64 mg/L for Daphnia similis and 19.9 mg/L for Ceriodaphnia silvestrii; chronic In the tests the value obtained for the CEO Ceriodaphnia silvestrii was 1.29 mg/L and 12 mg/L Chironomus xanthus CENO and 6 mg/L. In acute toxicity tests made with NP values EC(I) 50; 48h were 0.309 mg/L for Daphnia similis, 0.4520 mg/L for Ceriodaphnia silvestrii solvent and water with 0.0541 mg/L ethanol solvent and 0.03398 mg/L for Ceriodaphnia dubia. The value of the CEO to Ceriodaphnia silvestrii was 0.0198 mg/L and Chironomus xanthus was 100 \'mü\'g/g, with CENO was 50 \'mü\'g/g. Although the found concentrations in the tests are higher than those found in studies to have quantified these compounds in the environment, other studies showed that at concentrations lower than those obtained in this study have problems occur with time and the change of the metabolism of Cladocera androgenization. Also, based on the results obtained with these tests, is intended to alert the public on the risks associated of the presence of these chemicals in the water and the urgency in adopting new techniques in wastewater treatment aiming at the effective removal of these pollutants in water and in the sediment.

Bisphenol A (BPA) is a common component in plastic consumer products and epoxy resin linings. Initially developed in the 1930s-40s as a synthetic hormone treatment, it is now widely considered an endocrine disrupting chemical (EDC). A growing body of epidemiological literature suggests that ubiquitous exposures to BPA may be contributing to the global epidemic of obesity, with children a particularly vulnerable population. Obesity in children, defined by a body mass index (BMI) greater than or equal to the 95th percentile for age and sex, is an epidemic of great concern in the United States. As with other chemicals, the prenatal and early life period are critical windows of exposure to BPA; however, the mechanism by which BPA may influence the development of body size in children remains unclear. Experimental studies have found that BPA influences adipogenesis in both murine and adult human preadipocyte cell lines and BPA is hypothesized to play a role in enhancing adipogenic regulation by nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ). While the timeline of the processes involved in adipogenesis in humans is not universally agreed upon, it is accepted that PPARγ is highly expressed in adipose tissue and considered to be the master regulator of adipogenesis. To answer the question of both timing and developmental origin of BPA effects on adipogensis, we employed both an epidemiological approach, and experimental methodologies using primordial cell lines, mesenchymal stem cells (MSCs). Our study characterizes early life exposures to BPA, explores the adipogenic mechanism of BPA in human MSCs via cellular morphometrics and PPARγ gene expression, and identifies associations between early life exposure to BPA and childhood obesity and adiposity. For our epidemiological assessments, we studied a birth cohort of African American and Dominican mother and child dyads in New York City. BPA was measured in spot urine samples collected during pregnancy and at child ages 3, 5, and 7 years, from mothers and children (n=568 dyads) in the Columbia Center for Children’s Environmental Health (CCCEH). We compared BPA concentrations across paired samples. We explored relationships between BPA and the class of phthalate chemicals, another common plasticizer. BPA was detected in nearly all urine samples from prenatal third trimester and childhood ages 3 years, 5 years and 7 years. Prenatal urinary BPA concentrations were significantly lower than postnatal urinary BPA concentrations (p<0.001). BPA and phthalate metabolites were correlated prenatally and at 3, 5, and 7 years (all p-values < 0.02). BPA concentrations were correlated with phthalate metabolite concentrations prenatally, and at 3, 5 and 7 years(all p-values < 0.05). Geometric means of BPA were higher among African Americans than among Dominicans in prenatal (p<0.01), 5 year (p<0.001) and 7 year (p=0.02) samples. Postnatal BPA concentrations were significantly higher among children with mothers who had never marrried marital status and were significantly higher in summer than in all other seasons (all p-values < 0.05). These findings reveal widespread BPA exposure in an inner-city minority population. Our in vitro experiment was a feasibility study which sought to determine whether exposure to BPA by human umbilical cord mesenchymal stem cells (HUMSC) induces morphological changes and PPARγ gene expression during adipogenesis. An anonymous sample of n=18 umbilical cords was collected at delivery from mothers registered at New York-Presbyterian Sloane Hospital for Women and New York-Presbyterian Allen Hospital in New York City. HUMSCs were harvested from umbilical cords using an adhesion technique. HUMSCs were then induced in culture to differentiate into adipocytes using: a standard differentiation induction mix medium, a negative vehicle control medium, a positive control medium and experimental control media. Differences in cell surface area and cell count in all cultures were assessed using ImageJ software (version 1.49n, 2014). Gene expression of PPARγ in all cultures was evaluated by RT-PCR. Cell morphometric results were based on 11,676 cells from 3 umbilical cord samples. PPARγ1 and PPARγ2 gene expression was assessed during differntiation phase and early terminal phase adipogenesis (0 to 72 hours). Cell morphometrics were assessed during middle to late terminal phase adipogenesis (days 14 and 21). No differences in cell count were observed for experimental conditions compared to standard induction medium. A significant decrease in surface area was seen in cells exposed to 100 μM concentration of BPA as compared to exposure to standard induction medium at day 14 (t=-37.02 p=0.001). Differences in cell surface area were not observed at day 21. A twofold increased expression of PPARγ1 was observed in cells exposed to 10 μM concentration of BPA by 72 hours of adipogenic induction which was higher than the increase in expression observed for cells exposed to the positive control induction medium containing 10 μM concentration of rosiglitazone. All induction media conditions had negligible effects on PPARγ2 expression. As BPA increases expression of PPARγ1 in HUMSCs during the transition into the early terminal differentiation phase of adipogenesis, HUMSCs may be an approximate target tissue for evaluating BPA effects in adipogenesis. Finally, using a longitudinal research design, we analyzed the possible effect of prenatal and postnatal BPA exposures, measured in urine, on childhood anthropometric outcome measures. Participants in the CCCEH have been followed since the third trimester of pregnancy, providing us with anthropometric data on children from birth through the age of seven years. Available anthropometric outcome measures include body mass index z-scores (BMIZ) at 5 and 7 years, as well as fat mass index (FMI), percent body fat (%BF), and waist circumference (WC) at 7 years. Prenatal urinary BPA concentrations were positively associated with child age 7 FMI (beta=0.31 kg/m2, p-value=0.04, [95%CI 0.01, 0.60]), %BF (beta=0.79, p-value=0.04, [95%CI 0.03, 1.55]), and WC (beta=1.29 cm, p-value=0.01, [95%CI 0.29, 2.30]). Child urinary BPA concentrations were not associated with childhood BMI or other anthropometric outcomes. As the prenatal exposures were associated with childhood measures of adiposity, prenatal BPA exposure may have an effect on adiposity as children age that cannot be determined by the use of BMI alone. Our results suggest BPA may contribute to the developmental origins of obesity and adiposity.

Neste estudo determinou-se o nonilfenol - um composto xenobiótico utilizados na fabricação de antioxidantes, aditivos para óleos lubrificantes e produção de surfactantes etoxilados - e o bisfenol A - um composto químico utilizado na produção de diversos produtos, como, plásticos policarbonatos, resinas epóxi e retardantes de chama. Ambos exibem atividade estrogênica em concentrações abaixo de 1ng L-1 - em cinco pontos de coleta na cidade de Americana, SP. Devido à ampla utilização, esses compostos estão presentes em esgoto industrial em quantidades substanciais. A primeira etapa deste estudo foi a validação dos método analítico seguindo o Método 8270D da United States Environmental Protection Agency (USEPA) que descreve o procedimento de validação para os compostos semivoláteis analisados por cromatografia gasosa acoplada a espectrometria de massas (GC/MS). Os limites de detecção e de quantificação do método para a determinação do nonilfenol foram, respectivamente, 1 e 5 &micro;g L-1. O coeficiente de determinação (R2) e a recuperação média para o nonilfenol foram, respectivamente, 0,99 e 99,04%. Os limites de detecção e de quantificação do método para o bisfenol A foram, respetivamente, 1 e 5 &micro;g L-1. O coeficiente de determinação (R2) e a recuperação média para o bisfenol A foram, respectivamente, 0,99 e 91,55%. Na segunda etapa desta pesquisa analisaram-se amostras de água coletadas na cidade de Americana para a determinação do nonilfenol e do bisfenol A. Como método de extração dos compostos, usou-se a extração em fase sólida (SPE) e como técnica analítica a cromatografia gasosa acoplada a espectrometria de massas (GC/MS). Os resultados indicaram a presença desses compostos em alguns pontos de amostragem sendo que a concentração do nonilfenol a variou de 5,028 a 10,524 &micro;g L-1 e para o bisfenol A a concentração variou de 5,645 a 295,255 &micro;g L-1. Foram feitos também testes ecotoxicólogicos utilizando como organismo teste o microcrustáceo Daphnia similis, porém o nonilfenol e o bisfenol A, na concentração testada que variou de 0,05 a 500 &micro;g L-1 não apresentaram toxicidade aguda. / In this study it was determined nonylphenol - a xenobiotic compound used in the manufacture of antioxidants, additives for lubricating oils and production of ethoxylated surfactant - and bisphenol A - a chemical compound used in production of various products, such as polycarbonate plastic, epoxy resins and flame retardants. Both exhibit estrogenic activity at concentrations lower than 1 ng L-1 - in five sampling points in the city of Americana, SP. Due to the widespread use of these compounds, they are present in substantial quantities in industrial sewage. The first step of this study was to validate the analytical method following the Method 8270D of the United States Environmental Protection Agency (USEPA) that describes the validation procedure for semi-volatile compounds analized by gas chromatography-mass spectrometry (GC/MS). The limits of detection and quantification for determining the nonylphenol were, respectively, 1 and 5 &micro;g L-1. The coefficient ofdetermination (R2) and recovery for nonylphenol were respectively 0.99 and 99.04%. The limits of detection and quantification for the bisphenol A were, respectively, 1 and 5 &micro;g L-1. The coefficient of determination (R2) and recovery for bisphenol A were respectively 0.99 and 91.55%. In the second step of this study were analized in water samples of Americana city for the determination of nonylphenol and bisphenol A. As a method of extraction of the compounds it was used solid-phase extraction (SPE) and as analytical technique GC/MS. The results indicated the presence of such compounds in some sampling points and the concentration of nonylphenol ranged from 5.028 to 10.524 &micro;g L-1 and bisphenol A concentration ranged from 5.645 to 295.255 &micro;g L-1. Ecotoxicological tests were also performed using as test organism microcrustaceans Daphnia similis, but nonylphenol and bisphenol A, tested at concentration ranging from 0,05 to 500 &micro;g L-1 showed no acute toxicity.

Thousands of chemical pollutants enter the environment continuously, each with the potential to cause adverse effects in both terrestrial and aquatic organisms. As a result, organisms are often exposed to a mixture of stressors within their habitat. Populations of fish inhabiting most aquatic environments are exposed to time-varying or repeated pulses of exposure, driven by run-off events or spills, or due to their mobility between polluted and clean waters. Therefore, the sustainability of fish populations is critically dependent on their ability to adapt to frequent changes in their local environment. Despite this, legislation to protect the environment from chemical contamination are generally based on toxicological measurements following exposures to single stressors, conducted under optimal laboratory conditions, and that do not take into account the variation in susceptibility of wild populations, or the potential consequences of exposure for the susceptibility of the population during future exposures, including across generations. Increasing evidence is suggesting that a number of chemicals may interact with the epigenome, and that differential responses to pollutants may be modulated, at least in part, via epigenetic mechanisms. However, our understanding of the role of epigenetic mechanisms in normal development in fish models or its susceptibility to exposure to environmental stressors is currently very limited. This thesis aimed to document the mechanisms of genetic and epigenetic responses to industrial pollutants in fish, and to explore the extent to which differential responses can be induced in the lab following exposure during the critical window of embryonic development or in adults. To address these objectives, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. I provide evidence of the sex-specific transcription of genes involved in reproduction and their regulation by epigenetic signalling in this commonly used vertebrate model and highlight important considerations regarding the use of whole tissues comprised of multiple cell types in epigenetic and transcriptomic studies. I then investigated the potential for exposure to Bisphenol A (BPA) to cause adverse effects on reproduction and to disrupt the expression profiles and promotor DNA methylation of target genes important for reproductive function and epigenetic signalling in the zebrafish. To do this, I exposed breeding zebrafish to a range of BPA concentrations over 15 days and found that BPA disrupted reproductive processes in zebrafish, likely via estrogenic mechanisms, but only at high concentrations. Importantly, exposure to environmentally relevant concentrations of BPA resulted in altered transcription of key enzymes involved in DNA methylation maintenance, and caused changes in promoter DNA methylation. I also conducted a series of repeated exposures to copper in the three-spined stickleback to investigate the extent to which differential susceptibility can be induced in the lab. This work provides evidence that pre-exposure to copper results in differential responses in future exposure scenarios both when the initial exposure occurred in adults and during embryogenesis. For adults, fish appeared to recover completely from the initial exposure following a period of depuration of 30 days, but displayed decreased susceptibility upon re-exposure. In contrast, for fish exposed during the critical windows of embryonic development when epigenetic reprogramming are hypothesised to occur, differential copper accumulation was maintained throughout life. Importantly, the initial exposure caused increased tolerance in the offspring, which was inherited up to the F2 generation. This work provides valuable information regarding potential critical windows of development which may be more susceptible to effects associated with pre-exposure, highlighting that early life exposure to a low concentration of copper can induce differential responses to copper across generations. These data highlight the extent of differential responses to chemical stressors likely to be present in wild populations, and point towards the possibility that effective population management will likely require an in-depth understanding of the exposure history of a given population in order to manage restocking initiatives, and to inform conclusions drawn from toxicity testing studies conducted using individuals originating from wild populations. In addition, these data suggest that it is likely that both epigenetic and genetic changes can contribute to the adaptation of individual populations to their local environment. Finally, other vertebrates including humans have been shown to be exposed to the chemicals tested in this thesis. Therefore, this highlights the potential for these chemicals to also cause toxic effects in humans, potentially via (epi) genetic mechanisms, and advocate the testing of the potential for inheritable phenotypes, such as those described in this thesis, to occur in mammalian models.

While diet and sedentary lifestyle remain important factors in the development of obesity, recent findings have shown the possible involvement of environmental obesogens, chemicals that can disrupt homeostatic energy balance and increase adiposity. Bisphenol A (BPA) is a compound used in the manufacturing of plastics as a hardening agent and is ubiquitous in the environment due to its widespread use. BPA has been shown to be an endocrine disruptor through its ability to mimic estrogen, which is now known to play important roles in adipose tissue growth and metabolism. In fact, a small but compelling number of studies have shown that mice exposed to BPA in utero or postnatally are fatter as adults. We hypothesized that BPA exposure exerts effects on adipose tissue, promoting adipogenesis and inflammation, and altering energy homeostasis in a manner that promotes obesity. We tested our hypothesis using both in vitro and in vivo models. First, we found that low concentrations of BPA increased the expression of the inflammatory genes, Il-6 and Tnfa, approximately 1.5-3.0 fold in mouse adipose tissue explants. We also found a 3-fold increase in the expression of the lipogenic gene, Fasn. BPA also altered the adipose tissue metabolism, increasing the levels of a number of glycolytic and TCA cycle metabolites, suggesting that BPA may disrupt energy homeostasis. We also found that BPA exposure increased proliferation of mesenchymal stem cells approximately 1.2-fold, which are potential adipocyteprecursors. The study was expanded using two different strains of mice, C57BL/6 and DBA/2J, chronically exposing them to BPA through drinking water for six weeks. A moderate concentration of BPA increased the perigonadal fat pad mass in males. This increase in adiposity was associated with adipocyte hypertrophy and decreased serum adiponectin levels. There were also changes in the expression of some genes with BPA treatment, including a 1.4-fold increase in Leptin and decreases in some Cytochrome P450 genes; however the genes differentially expressed were different between the two strains. Our results suggest that childhood exposure to low doses of BPA, in lieu of any developmental exposure, may contribute to childhood obesity.