Made available in DSpace on 2017-07-10T14:57:32Z (GMT). No. of bitstreams: 1 camila_ machado.pdf: 1503244 bytes, checksum: 361dda1eae6076e04495b3c6a4455f6e (MD5) Previous issue date: 2016-03-14 / The triclosan (TCS) is a bactericidal agent widely used in personal hygiene products, in the context of odontology, this substance has been showing itself efficient in reducing the dental plaque and gingivitis, besides controlling the progression of periodontal disease. However, it's questionable the real benefits of using the TCS in large scale in different products, once this compound has been included on the list of endocrine disrupters. The growing observation of the prevalence of environmental contaminant with properties for the endocrine disrupting has been generating a considerable debate between the scientists, regulatory agencies and general public, about the potential risks which these substances represent to the man's and animal's productive health. Many of these compounds are present in our daily routine. The objective of this study was to evaluate the effects resulted from the exposure to TCS bactericidal, during pregnancy and lactation of mother rats, on the physical development, initial sexual and testicular function of the male son on the following phase of the development: puberty and sexual maturity. For this, 16 rats Wistarprenhes were used, separated in four experimental groups, with 4 animals each one: GI- received corn oil daily by gavage; GII - received TCS, at a dose of 75 mg/kg/day. GIII- received TCS, at the dose of 150 mg/kg/day and GIV - received TCS, at the dose 300 mg/kg/day. The rats where weighed in alternate days throughout the experimental period, to the adjustment of the dose and monitored about the birth of the offspring. At birth, the offspring was weighed and evaluated about the initial physical development: age of the ears' detachment, hair onset, and eruption of incisors and eyes opening. The male offspring were kept and monitored about the external physical parameter of initial sexual development (descended testicles age and prepuce separation). When they're 60 (puberty) days and 90 (sexual maturity) years old, they were weighed and sacrificed to the organs collecting and weighing and histological analysis of the spermatogenesis stages and Sertoli cells counting. The results showed that the gain of body weight of the female rats during the pregnancy was similar to the experimental groups. However, there was a body weight-loss in the end of the experimental period, of the rats of group GIV in comparison to 14 group GI. The pregnancy average time and size of the offspring were similar between the groups. The average weigh of the offspring treated with TCS, in different doses was smaller (p<0,05), in comparison to group GI. The initial physical development and the descended testiscles were similar between the experimental groups. The offspring exposed to TCS, during pregnancy and lactation, it was observed a delay (days) on the prepucial separation. In puberty (60 days), it was observed a meaningful weight-loss of the seminal gland on groups GIII and GIV, in comparison to group GI. When they were 90 days old, it was observed liver weight-loss and a weight gain of the prostate of the animals of group GIV, in comparison to the animals of group GI. The analysis of the stages of the seminiferous epithelium cycle of the 60 days animals showed an increase of the stages I-VI on the animals treated with TCS 300mg/kg/day in comparison to group GI (p<0,01). The 90 days animals showed an increase of the stages VII-VIII, IX-XIII and decrease on the frequency of the stage XIV of the animal spermatogenesis treated with different doses of TCS when compared to group GI (p<0,01). There was no difference on the number counting of Sertoli cells between the animals of the different experimental groups. We concluded that the maternal exposition to TCS during the gestation period and lactation causes on the male offspring, intrauterine development restriction, delay on the puberty installation, change the weight of the seminal gland in animals at 60 days, as well, changes in liver weight and prostate in animals with 90 days, and disrupting the seminiferous epithelial cycle in both age. / O triclosan (TCS) é um agente bactericida amplamente utilizado em produtos de higiene pessoal. No contexto da odontologia, esta substância tem se mostrado eficaz em reduzir a placa dentária e gengivite, além de controlar a progressão da doença periodontal crônica. Entretanto, questiona-se o real benefício da utilização em larga escala do TCS em diferentes produtos, uma vez que este composto tem sido incluído na lista dos desreguladores endócrinos (DE). A crescente observação da prevalência de contaminantes ambientais com propriedades para a desregulação endócrina tem gerado considerável debate entre os cientistas, agências regulatórias e público em geral, sobre os potenciais riscos que estas substâncias representam para a saúde reprodutiva do homem e dos animais e muitos destes compostos estão presentes em nosso cotidiano. O objetivo deste estudo foi avaliar os efeitos decorrentes da exposição ao TCS, durante a prenhez e lactação das ratas mães, no desenvolvimento físico, sexual inicial e função testicular da prole masculina na puberdade e vida adulta. Para tanto, foram utilizadas 16 ratas Wistar prenhes separadas em quatro grupos experimentais, com 4 animais em cada: GI- receberam óleo de milho diariamente por gavage; GII - receberam TCS, na dose de 75 mg/kg/dia; GIII- receberam TCS, na dose de 150 mg/kg/dia e GIV- receberam TCS, na dose de 300 mg/kg/dia. As ratas foram pesadas em dias alternados ao longo de todo o período experimental, para ajuste da dose, e monitoradas quanto ao nascimento dos filhotes. Ao nascimento, a ninhada foi pesada e avaliada quanto ao desenvolvimento físico inicial: idades de descolamento das orelhas, nascimento de pêlos, erupção dos incisivos e abertura dos olhos. Os filhotes machos foram mantidos e monitorados quanto aos parâmetros físicos externos do desenvolvimento sexual inicial (idades da descida testicular e separação prepucial). Aos 60 dias de idade (puberdade) e 90 dias de idade (maturidade sexual), foram pesados e sacrificados para a coleta e pesagem de órgãos e análise histológica dos estágios da espermatogênese e contagem de células de Sertoli. Os resultados indicaram que o ganho de peso corporal das ratas ao longo da gestação foi semelhante entre os grupos experimentais. Entretanto, houve diminuição do peso corporal, ao final do período experimental, das ratas do grupo GIV, 12 quando comparado ao grupo GI. O tempo médio da gestação e tamanho da ninhada foi semelhante entre os grupos. O peso médio das ninhadas das ratas tratadas com TCS, nas diferentes doses, foi menor (p<0,05), quando comparado ao grupo GI. O desenvolvimento físico inicial e as idades de descida testicular foram semelhantes entre os grupos experimentais. Na prole exposta ao TCS, durante a gestação e lactação, foi observado um atraso no tempo (dias) da separação prepucial. Na puberdade (60 dias), foi observada uma diminuição significativa no peso da glândula seminal nos grupos GIII e GIV, quando comparado ao grupo GI. Aos 90 dias de idade, foi observada diminuição do peso do fígado e um aumento no peso da próstata dos animais do grupo GIV, quando comparado aos animais do grupo GI. A análise dos estágios do ciclo do epitélio seminífero dos animais de 60 dias mostrou aumento dos estágios I-VI nos animais tratados com TCS 300mg/Kg/dia quando comparado ao grupo GI (p<0,01). Aos 90 dias de idade os animais mostraram aumento dos estágios VII-VIII, IX-XIII e diminuição na frequência do estágio XIV da espermatogênese nos animais tratados com diferentes doses de TCS quando comparado ao grupo GI (p<0,01). Não houve diferença na contagem do número de células de Sertoli entre os animais dos diferentes grupos experimentais. Foi possível concluir que, a exposição materna ao TCS durante a gestação e lactação causa efeitos na prole masculina, tais como: Restrição de desenvolvimento intrauterino, atraso na instalação da puberdade, alteração do peso da glândula seminal nos animais com 60 dias, como também, alteração no peso do fígado e da próstata nos animais com 90 dias e desregulação no ciclo do epitélio seminífero em ambas as idades.

<p>Persistent environmental pollutants were examined for their interaction with the glucocorticoid hormone system. The focus was placed on interference with the glucocorticoid synthesis and the glucocorticoid-signalling pathway in various <i>in vitro</i> test systems.</p><p>Several aryl methyl sulphones competitively inhibited CYP11B1 activity in mouse adrenocortical Y1 cells. The DDT metabolite, 3-methylsulphonyl-2,2’-bis(4-chlorophenyl)-1,1’-dichloroethene (3-MeSO₂-DDE) had a higher affinity to the enzyme than the endogenous substrate, 11-deoxycorticosterone. In fact, 3-MeSO₂-DDE (K_i 1.6 μM) was almost as potent as the drug metyrapone (K_i 0.8 μM), a well-known inhibitor of the enzyme. 3-MeSO₂-DDE inhibited CYP11B1 activity in human adrenocortical H295R carcinoma cells, and at higher concentrations the CYP21 activity. The human H295R cell line seems to be a useful test system for studies of enzyme activities and could be used to screen endocrine disrupting chemicals interfering with the glucocorticoid hormone synthesis.</p><p>Several chiral PCB methyl sulphones and the fungicide tolylfluanid proved to be antagonists to the glucocorticoid receptor (GR) in rat hepatoma cells and/or Chinese hamster ovary cells stable transformed with a human GR and a responsive reporter vector. The 4-methylsulphonyl-2,3,6,2’,4’,5’-hexachlorobiphenyl (4-MeSO₂-CB149) enantiomers had similar antagonistic effect on the GR. Co-exposure of substances led to additive inhibitory effects on glucocorticoid-regulated protein synthesis in rat hepatoma cells. In general, 4-substituted but not 3-substituted methylsulphonyl-PCBs interacted with the glucocorticoid hormone system.</p><p>In the environment, humans and wildlife are constantly exposed to a wide range of chemicals. Considering the effects of these substances via mechanisms of actions described in this thesis, interference of xenobiotics with the glucocorticoid hormone system deserves further attention. In conclusion, environmental pollutants can interact with the glucocorticoid hormone system <i>in vitro</i>, yet the effects of the tested substances on this hormone system remain to be established <i>in vivo.</i></p>

Persistent environmental pollutants were examined for their interaction with the glucocorticoid hormone system. The focus was placed on interference with the glucocorticoid synthesis and the glucocorticoid-signalling pathway in various in vitro test systems. Several aryl methyl sulphones competitively inhibited CYP11B1 activity in mouse adrenocortical Y1 cells. The DDT metabolite, 3-methylsulphonyl-2,2’-bis(4-chlorophenyl)-1,1’-dichloroethene (3-MeSO2-DDE) had a higher affinity to the enzyme than the endogenous substrate, 11-deoxycorticosterone. In fact, 3-MeSO2-DDE (Ki 1.6 μM) was almost as potent as the drug metyrapone (Ki 0.8 μM), a well-known inhibitor of the enzyme. 3-MeSO2-DDE inhibited CYP11B1 activity in human adrenocortical H295R carcinoma cells, and at higher concentrations the CYP21 activity. The human H295R cell line seems to be a useful test system for studies of enzyme activities and could be used to screen endocrine disrupting chemicals interfering with the glucocorticoid hormone synthesis. Several chiral PCB methyl sulphones and the fungicide tolylfluanid proved to be antagonists to the glucocorticoid receptor (GR) in rat hepatoma cells and/or Chinese hamster ovary cells stable transformed with a human GR and a responsive reporter vector. The 4-methylsulphonyl-2,3,6,2’,4’,5’-hexachlorobiphenyl (4-MeSO2-CB149) enantiomers had similar antagonistic effect on the GR. Co-exposure of substances led to additive inhibitory effects on glucocorticoid-regulated protein synthesis in rat hepatoma cells. In general, 4-substituted but not 3-substituted methylsulphonyl-PCBs interacted with the glucocorticoid hormone system. In the environment, humans and wildlife are constantly exposed to a wide range of chemicals. Considering the effects of these substances via mechanisms of actions described in this thesis, interference of xenobiotics with the glucocorticoid hormone system deserves further attention. In conclusion, environmental pollutants can interact with the glucocorticoid hormone system in vitro, yet the effects of the tested substances on this hormone system remain to be established in vivo.

Endocrine disrupters and trace organic contaminants are recently recognized contaminants in wastewaters. Current concept is the multibarier approach where the contaminants are removed from the water cycle both by water and wastewater treatment facilities, as well as natural die-away. In this thesis work LC/MS/MS determination of selected EDC compounds, namely, diltiazem, progesterone, estrone, carbamazepine, benzyl butyl phthalate and acetaminophen, at ultra trace levels, have been carried out by optimizing analytical parameters. In addition, new methods were developed for their analysis in sludge samples at sub ppb levels. Following optimization and method development, occurrence of these contaminants in wastewaters and their removal in two full-scale and two pilot-scale membrane biological reactors (MBRs) was studied. Progesterone, estrone and acetaminophen were completely removed from wastewater by biodegradation. CBZ and diltiazem were not removed at all during the study. There was little effect of flux and sludge retention times on the removal of selected EDCs in these membrane plants. In SBR combined with membrane filtration, 13 different micropollutants, including Fluoxetine (FLX), Ibuprofen (IBP), Naproxen (NPX), Diclofenac (DCF), Carbamazepine (CBZ), Trimethoprim (TMP), Roxithromycin (ROX), Erythromycin (ERY), Sulfamethoxazole (SMX), Diazepam (DZP), Galaxolide (GLX), Tonalide (TON), Celestolide (CEL). CEL, GLX, TON and FLX were removed by adsorption onto the sludge while ROX, ERY, SMX, IBP and NPX were removed by biological degradation. The CBZ, DZP, TMP and DCF were not removed by biodegradation or adsorption. Whereas, following the addition of powdered activated carbon, all these compounds were removed entirely from the wastewater stream by accumulating in sludge.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2014-12-29T19:05:54Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação-Fernando Santiago Pires-2013.pdf: 1915773 bytes, checksum: 124080395d9b5ba7bc8951e1ec35c371 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-12-29T19:06:19Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação-Fernando Santiago Pires-2013.pdf: 1915773 bytes, checksum: 124080395d9b5ba7bc8951e1ec35c371 (MD5) / Made available in DSpace on 2014-12-29T19:06:19Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação-Fernando Santiago Pires-2013.pdf: 1915773 bytes, checksum: 124080395d9b5ba7bc8951e1ec35c371 (MD5) Previous issue date: 2013-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Xenobiotics pesticides are widely used in crops or even gardening throughout the world with purpose of a broad spectrum pest control, herbicides with glyphosate (N- phosphonomethyl glycine) as an active ingredient stands out amongst that group. The glyphosate (GLI) participates in a unique metabolic pathway during the synthesis of amino acids in plants, although, in animals it was thought to be harmless. However, literature indicates toxicity from commercial solutions, but few studies done with pure GLI. Moreover, to verify toxic potential of GLI, experiments done using Nuclear Magnetic Resonance (NMR). Furthermore, the test tanks were examined the presence of GLI for a period of 96 hours. It is because the GLI, during the metabolism of prokaryotes, converts to Aminomethylphosphonic acid (AMPA). The results showed the absence of AMPA at 96 hours of exposure, whereas, any changes were attributed to GLI in these animals. Primarily, the dose of CL50-96h for the experimental animal, Poecilia reticulata (guppy), was determined. It estimated at 68.51 ± 2.2 mg/L for males and at 70.56 ± 2.96 mg/L for females. The lethal dose calculated according to the recommended guidelines from the Organisation for Economic Co-operation and Development (OECD). During these tests the animals in the experimental group (EG) exposed at concentrations of 50, 55, 60.5, 66.5 and 73.2 mg/L showed swimming changes (17.5% - lethargy, 11.1 % - irregular swimming and 45.9% - hypoxia). Respiratory changes (8.3% - swam to the tank surface, 1.8% - hypo oxygenated) and changes in color pattern, such as loss of intensity of the hues orange, red, blue, and assuming a dull coloration (14.3%) in all EG. Subsequently, male’s guppy treated with sublethal doses of pure GLI in concentrations of 17.13 mg/L (25% CL50), 34.26 mg/L (50% CL50) and 51.39 mg/L (75% CL50) as per 203 guidelines of the OECD. After euthanasia, by descerebration, the male animals were dissected and the testicles fixed in buffered paraformaldehyde, sectioned in 2.0 μm slices by an ultramicrotome and placed in histoResin®. The sections stained with hematoxylin and toluidine blue/Floxine B, then photographed under light microscope. The program Image-Pro Plus provided the images, which analyzed by the program Statistica 7.0. Consequently, the data reviewed that GLI has toxic potential. It has the potential to: cause testicular regression - morphometric measures decrease, reduce the number of the spermatic compartments, reduce the gonad index (Igs) and promote histopathology which may lead to reduction or loss of function. Thus, characterizing a process of demasculinization, which indicates endocrine disruption. / Agrotóxicos são xenobióticos utilizados largamente nos lavouras ou mesmo em jardinagem em todo mundo para o controle de pragas agrícolas, e entre eles destacam-se os herbicidas a base de Glifosato (N-fosfonometil-glicina). O Glifosato (GLI) participa de uma rota metabólica exclusiva na síntese de aminoácidos nas plantas e foi considerado inofensivo aos animais. Contudo, há relatos na literatura que indicam a toxicidade das soluções comerciais à base de GLI, mas poucos abordam a atividade do GLI puro. Dessa maneira, para se verificar o potencial toxico do GLI, primeiramente, experimentos com Ressonância Magnética Nuclear (RMN) foram realizados para verificar a presença de GLI nos aquários testes pelo tempo de 96h. Isso foi feito porque o GLI pode ser convertido a ácido aminometil fosfônico (AMPA) pelo metabolismo de procariotos. Os resultados demonstraram ausência de AMPA até 96h de exposição e dessa forma foi atribuído ao GLI qualquer alteração presente nos animais testes. Depois foi determinado o valor da CL50-96h para o animal experimental Poecilia reticulata (guarú). Essa foi estimada em 68,51 ± 2,2 mg/L para machos e 70,56 ± 2,96 mg/L para fêmeas. Os testes de toxicidade letal para o cálculo da CL50-96h foram feitos de acordo com as recomendações do guia 203 da Organização para cooperação e desenvolvimento do comércio (OECD). Durante esses testes os animais do grupo experimental (GE) expostos nas concentrações de 50; 55; 60,5; 66,5 e 73,2 mg/L apresentaram alterações comportamentais como distúrbios natatórios (17,5% – letargia; 11,1% – natação irregular e 45,9% – hipoatividade), respiratórios (8,3% – nado na superfície do aquário; 1,8% – hipóxia) e alterações no padrão da coloração (14,3%) como perda da intensidade das matizes laranja, vermelha e azul e dos tons acetinados em todos os GE. Posteriormente guarús machos foram tratados com doses subletais do GLI puro nas concentrações de 17,13 mg/L (25% da CL50), 34,26 mg/L (50% da CL50) e de 51,39 mg/L (75% da CL50) conforme o guia 203 da OECD. Após a eutanásia por descerebração, os animais foram dissecados e os testículos foram fixados em paraformaldeído tamponado, inclusos em historresina e seccionados a 2,0 μm em ultramicrótomo. Os cortes foram corados com Azul de toluidina e hematoxilina/floxina e posteriormente fotografados em fotomicroscópio. As imagens forneceram dados obtidos pelo programa Image Pro-Plus que foram analisados pelo programa Statistica 7.0. Foi demonstrado que o GLI apresenta potencial tóxico para o guarú por causar regressão testicular pela redução das medidas morfométricas, redução do número de compartimentos espermáticos, redução do Índice gonadossomático (Igs) e por promover histopatologias que podem levar a diminuição ou a perda da funcionalidade do testículo. Tal processo caracteriza a desmasculinização, um indicativo de disrupção endócrina.

Depuis plusieurs années, des agents exogènes environnementaux appelés perturbateurs endocriniens (PE), sont soupçonnés d’interférer avec les fonctions essentielles de reproduction et de développement chez de nombreux organismes vivants. Au travers de ce travail, nous avons tenté de combler certaines lacunes afin de mieux comprendre les dangers pour l’Homme posés par ces produits. Des études ont été menées visant à caractériser la toxicité testiculaire, chez le rat adulte, induite par des composés aux mécanismes d’action toxique divers (PE et non PE), ceci dans le but d’établir in fine l’existence ou non d’un comportement propre aux PE. En évaluant les effets de l’anti-androgène flutamide, nous avons identifié des changements d’expression géniques dose-reliés pour les voies métaboliques majeures associées à la lésion du testicule (ex. métabolisme des acides gras), et démontré l’existence d’une "dose sans effet moléculaire" inférieure à la NOAEL définie sur la base des changements phénotypiques adverses. Des études ont également été conduites avec le 1,3-dinitrobenzène, composé décrit dans la littérature comme n’interférant pas avec le système endocrinien et ayant pour cibles les cellules de Sertoli. Nous avons établi qu’en plus d’induire l’apoptose des cellules germinales et d’altérer la progression du cycle cellulaire, cette substance affecte aussi la stéroïdogénèse, remettant quelque peu en question l’origine de sa toxicité. En conclusion, l’ensemble de nos résultats nous ont permis d’apporter des précisions sur le mécanisme d’action toxique des deux molécules examinées et d’identifier des doses sans effet pour chacune d’entre elles. Ils contribuent également au débat portant sur la définition des critères requis pour la détermination des propriétés de PE. / For several years, certain environmental chemicals known as endocrine disrupters (ED) have been suspected to modulate essential functions like reproduction and development in intact organisms. Through this work, we have tried to fill some of the gaps in the understanding of the dangers posed by these products to man. Studies in adult rats were conducted to characterize testicular toxicity induced by compounds with different mechanisms of toxicity (ED and non-ED), in order to determine ultimately whether or not there exists a behavior specific to EDs. By assessing the effects of the anti-androgen, flutamide, we identified dose-related gene expression changes for the major metabolic pathways associated with testicular injury (eg, fatty acid metabolism) and demonstrated the existence of a "molecular no effect dose" preceding the NOAEL based on adverse phenotypic changes. Studies were also conducted with 1,3-dinitrobenzene (DNB), a compound considered as a direct-acting testicular toxicant and not as an ED. We established that, in addition to inducing germ cell apoptosis and affecting cell cycle progression, DNB interferes with the biosynthesis of steroid hormones, bringing into question the classing of this molecule as a non-ED. In conclusion, our results allowed us to address relevant issues within the field of ED, leading to new insights about the mechanisms of toxicity for the two molecules examined, and the identification of doses without effect for each of them. Our data also contribute to the debate concerning the definition of criteria required for the determination of endocrine disrupting properties.

O Bisfenol A (BPA) é usado na fabricação de plásticos de policarbonato e resinas epóxi. A exposição pré-natal a esse agente pode causar diversos efeitos, tais como: antecipação da puberdade, hiperplasia de próstata, diminuição do número de espermatozoides, diminuição dos níveis de testosterona, alteração do desenvolvimento e organização tecidual da glândula mamária, diminuição da resposta celular induzida por hormônios, câncer de mama, diabetes, doenças cardiovasculares, alterações das funções de enzimas hepáticas, além de efeitos sobre o desenvolvimento cognitivo. Poucos estudos avaliam os efeitos do BPA sobre as células neuronais, porém existem evidências de que este agente induza a apoptose. O presente trabalho tem como objetivo estudar a neurotoxicidade do BPA, avaliando vias de sinalização que levam a indução da apoptose em cultura primária de hipocampo. As células foram expostas ao BPA nas concentrações de 50, 100, 150, 200, e 250 &#181;M (0,1% DMSO v/v) pelos períodos de 6, 12, 24, e 48 horas para a realização dos ensaios da atividade mitocondrial (MTT) e citotoxicidade pela liberação da enzima Lactato Desidrogenase (LDH). A partir dos resultados de MTT e LDH, foram adotados novos horários de exposição (3, 6 e 9 horas) utilizando somente as concentrações de 200 e 250 &#181;M. Neste novo desenho experimental, foi realizada a quantificação da concentração de BPA na cultura primária por HPLC-PDA, determinação da concentração de Ca2+ intracelular pela quantificação da fluorescência do Fluo-4 AM, caracterização dos mecanismos envolvidos na morte celular por citometria de fluxo e Western Blotting, e avaliação dos receptores de estrógeno ER-&#945; e ER-&#946; por Western Blotting. Nossos resultados apontam que aproximadamente 20% de BPA na concentração de 250 &#181;M após 6 horas de exposição e 18% para a concentração de 200 &#181;M com 9 horas de exposição foram absorvidos pela cultura celular. O ensaio do MTT mostrou que as células expostas a 200 e 250 &#181;M de BPA, por 12, 24 e 48 horas, apresentaram diminuição significativa da função mitocondrial em relação ao controle. Porém, não houve liberação de LDH para o meio de cultura para nenhuma das concentrações de BPA em nenhum dos períodos de incubação, o que sugere que não houve rompimento da membrana plasmática. Foi observada atividade apoptótica somente com a concentração de 250 &#181;M no período de exposição de 6 horas por citometria de fluxo. Não foram encontradas células em necrose, nem alteração na concentração de cálcio intracelular em nenhuma das condições estudadas. Na avaliação dos marcadores de morte celular, observamos aumento da razão de Bax/Bcl-2 para a concentração de 250 &#181;M em todos os períodos de exposição e aumento das caspases 8, 9 e 3 para a concentração de 250 &#181;M no período de exposição de 6 horas, indicando que o BPA deve ativar tanto a via intrínseca como a extrínseca no processo de apoptose. Verificamos ainda, por Western Blotting, que a cultura primária de hipocampo apresenta os receptores de estrógeno ER-&#945; e ER-&#946;. A exposição ao BPA aumentou os ER-&#945; e ER-&#946; avaliados por Western Blotting para as duas concentrações estudadas no período de 6 horas de exposição e, para o período de exposição de 9 horas, houve um aumento do ER-&#945; para a concentração de 250 &#181;M e do ER-&#946; para a concentração de 200 &#181;M. É possível concluir que o BPA pode levar a morte das células neuronais hipocampais por apoptose por ambas as vias intrínseca e extrínseca, sendo o processo de morte celular mais evidente para a concentração de 250 &#181;M no período de 6 horas de exposição. Sugerimos ainda que o aumento observado em ambos os receptores de estrógeno possa representar uma tentativa de interrupção ou reversão do processo de morte celular. / Bisphenol A (BPA) is used in the manufacture of polycarbonate plastics and epoxy resins. The prenatal exposure to this agent may cause several effects, such as anticipation of puberty, prostate hyperplasia, reduced number of sperm, reduced testosterone levels, alteration in the development and tissue organization of the mammary gland, decreased cellular response induced by hormones, breast cancer, diabetes, cardiovascular disease, changes in the functions of liver enzymes, and effects on cognitive development. Few studies have evaluated the effects of BPA in neuronal cells, however there are evidences that this agent may induce apoptosis. This work aims to study the neurotoxicity of BPA, by analyzing the signaling pathways of apoptosis in hippocampus primary culture. Cells were exposed to BPA at 50, 100, 150, 200, and 250 &#181;M (0.1% DMSO v/v) for 6, 12, 24, and 48 hours for the assay of mitochondrial activity (MTT) and the release of the enzyme lactate dehydrogenase (LDH). From the results of MTT and LDH, new exposure times (3, 6 and 9 hours) and only 200 and 250 &#181;M were used. In this new experimental design we performed the quantification of the BPA concentration in the primary culture by HPLC-PDA, intracellular Ca2+ quantification by Fluo-4 AM assay and the characterization of the mechanisms involved in cell death by flow cytometry and Western Blotting assays. Furthermore, evaluation of the estrogen receptor ER-&#945; and ER-&#946; was done by Western Blotting. Our results demonstrate that about 20% of the BPA concentration of 250 &#181;M after 6 hours of exposure and 18% for the concentration of 200 &#181;M with 9 hours of exposure were absorbed by the cell culture. Cells exposed to 200 and 250 &#181;M of BPA for 12, 24 and 48 hours, showed a significant decrease in mitochondrial function, by the MTT assay, compared to control. However, there was no release of LDH into the culture medium for any of the BPA concentrations in any of incubation times studied, which suggests no rupture of the plasma membrane by BPA. Apoptotic activity was observed after 6 hours of exposure to 250&#181;M BPA by flow cytometry. It was not observed cell necrosis and changes in intracellular calcium concentration in any of the studied conditions. Regarding the cell death markers, exposure to 250 &#181;M BPA in all periods of exposure resulted in an increased Bax/Bcl-2 ratio; moreover, an increase in caspase 8, 9 and 3 was detected after exposure to 250 &#181;M BPA for 6 hours. Taken together, these findings indicate that BPA activates both the intrinsic and the extrinsic pathway during the apoptotic process. We also verified by Western Blotting the presence of the estrogen receptors ER-&#945; and ER-&#946; at the primary culture of hippocampus, and that they can be modulated by BPA. The exposure to 200 and 250 &#181;M BPA for 6 hours caused an increase of ER-&#945; and ER-&#946;, however, 9 hours of exposure to 200 &#181;M and 250 &#181;M BPA increased the expression of ER-&#945; and ER-&#946;, respectively. In conclusion, BPA can lead hippocampal neuronal cells to death by both, intrinsic and extrinsic, apoptotic pathways and this process is more evident at 250 &#181;M BPA after 6 hours of exposure. Furthermore, we suggest that the increase of both estrogen receptors might represent an attempt to interrupt or reverse the cell death process.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-06-08T18:45:11Z No. of bitstreams: 2 Dissertação - Luane Ferreira Garcia - 2016.pdf: 1247655 bytes, checksum: 7639a387a5a3d1f4157419202b05240e (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-09T11:35:52Z (GMT) No. of bitstreams: 2 Dissertação - Luane Ferreira Garcia - 2016.pdf: 1247655 bytes, checksum: 7639a387a5a3d1f4157419202b05240e (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-06-09T11:35:52Z (GMT). No. of bitstreams: 2 Dissertação - Luane Ferreira Garcia - 2016.pdf: 1247655 bytes, checksum: 7639a387a5a3d1f4157419202b05240e (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2016-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Hormones are released constantly in sewage, originated by of human/animal excreta, or waste of pharmaceutical industries, not treated satisfactorily. The eviction of these pollutants in water resources produces great environmental impact, as disruption in animals’ endocrine system. The 17α-ethinylestradiol (EE2) is the most popular synthetic estrogen, which is found in water and in considerable concentrations. Several strategies are being studied to remedy this pollutant. Enzymes as laccases, which have low specificity, are able to oxidize various pollutants, thus suggesting their potential in the treatment of effluents. Another alternative are the electrochemical processes as electro-oxidation and electrocoagulation. The aim of this study was to evaluate the removal efficiency of the EE2 for biological or/and electrochemical process. The crude extract containing the laccase from Pycnoporus sanguineus was immobilized in Ca/Cu-alginate-chitosan beads. For partial characterization were determined optimum pH and optimum temperature of enzyme activity, for free and immobilized enzyme. Biological remediation was performed in these conditions: shaking (100 rpm); temperature at 28°C (± 2); times of 4, 8 and 24 hours; EE2 solution buffered at pH 4 and 5, and EE2 solution without addition of buffer. For the electrochemical remediation: magnetic stirring; voltage of 2.5, 5 and 7.5 V; times of 10, 20 and 40 minutes; pHs 5 and 7. For bio-electrochemical remediation the best conditions were used. In the remediation assays of the EE2 with immobilized enzyme, the best result was obtained for the support Ca-alginate-chitosan with 89.81% (± 2.71) removal, in sodium acetate buffer pH 5.0 and 24 hours of treatment. Under the same conditions to the free enzyme, 91.81% (± 0.86) of removal was obtained. For electrochemical remediation with titanium electrode, 86.21% (± 9.30) was removed in pH 7 phosphate buffer and 40 minutes. For sequential bio-electrochemical remediation, EE2 concentrations were below the limit of detection of the chromatograph, with the removal by immobilized enzyme acting in unbuffered solution. It can be concluded that the two technologies are very effective for the removal of the EE2. / Hormônios são lançados constantemente em esgotos, sejam oriundos de excretas humanas ou animais, sejam de resíduos provenientes das indústrias farmacêuticas, não tratados de forma eficaz. O despejo destes micropoluentes nos recursos hídricos produz grande impacto ambiental, como desregulação do sistema endócrino em animais. O 17α-etinilestradiol (EE2) é o mais popular estrogênio sintético, sendo encontrado nos recursos hídricos em concentrações consideráveis. Diversas estratégias estão sendo estudadas para remediação deste poluente. Enzimas como as lacases, que possuem baixa especificidade, são capazes de oxidar diversos poluentes, sugerindo assim seu potencial no tratamento de efluentes. Outra alternativa são os processos eletroquímicos, como a eletro-oxidação e eletrocoagulação. Sendo assim, o objetivo deste trabalho foi avaliar a eficiência de remoção do EE2 por processo biológico ou/e eletroquímico. O extrato bruto contendo a lacase de Pycnoporus sanguineus foi imobilizado em beads de quitosana-alginato-Ca/Cu. Para caracterização parcial da enzima livre e imobilizada foram determinados pH e temperatura ótima de atividade enzimática. A remediação biológica foi realizada nas condições: agitação (100 rpm); temperatura em 28°C (± 2); tempos de 4, 8 e 24 horas; solução de EE2 tamponada em pHs 4 e 5, e solução do hormônio sem adição de tampão. A remediação eletroquímica: agitação magnética; tensão de 2,5, 5 e 7,5 V; tempos de 10, 20 e 40 minutos; solução de EE2 tamponada em pHs 5 e 7. Para remediação bio-eletroquímica, de modo sequencial, foram utilizadas as condições mais adequadas para ambas as tecnologias. Nos ensaios de remediação do hormônio EE2 com a enzima imobilizada, o melhor resultado foi obtido para beads de quitosana-alginato-Ca com remoção de 89,81% (± 2,71) em tampão acetato de sódio pH 5 e 24 horas de tratamento. Nas mesmas condições, para a enzima livre foi obtido 91,81% (± 0,86) de remoção. Para remediação eletroquímica, com eletrodo de titânio foi removido 86,21% (± 9,30) do EE2, em tampão fosfato pH 7 e 40 minutos. Para a remediação bio-eletroquímica em modo sequencial, obteve-se remoção do EE2 em concentrações abaixo do limite de detecção do cromatógrafo, com a enzima imobilizada atuando em solução não tamponada. Conclui-se que as duas tecnologias são bastante eficientes para a remoção do EE2, podendo ser utilizadas separadamente ou em conjunto.

O Bisfenol A (BPA) é usado na fabricação de plásticos de policarbonato e resinas epóxi. A exposição pré-natal a esse agente pode causar diversos efeitos, tais como: antecipação da puberdade, hiperplasia de próstata, diminuição do número de espermatozoides, diminuição dos níveis de testosterona, alteração do desenvolvimento e organização tecidual da glândula mamária, diminuição da resposta celular induzida por hormônios, câncer de mama, diabetes, doenças cardiovasculares, alterações das funções de enzimas hepáticas, além de efeitos sobre o desenvolvimento cognitivo. Poucos estudos avaliam os efeitos do BPA sobre as células neuronais, porém existem evidências de que este agente induza a apoptose. O presente trabalho tem como objetivo estudar a neurotoxicidade do BPA, avaliando vias de sinalização que levam a indução da apoptose em cultura primária de hipocampo. As células foram expostas ao BPA nas concentrações de 50, 100, 150, 200, e 250 &#181;M (0,1% DMSO v/v) pelos períodos de 6, 12, 24, e 48 horas para a realização dos ensaios da atividade mitocondrial (MTT) e citotoxicidade pela liberação da enzima Lactato Desidrogenase (LDH). A partir dos resultados de MTT e LDH, foram adotados novos horários de exposição (3, 6 e 9 horas) utilizando somente as concentrações de 200 e 250 &#181;M. Neste novo desenho experimental, foi realizada a quantificação da concentração de BPA na cultura primária por HPLC-PDA, determinação da concentração de Ca2+ intracelular pela quantificação da fluorescência do Fluo-4 AM, caracterização dos mecanismos envolvidos na morte celular por citometria de fluxo e Western Blotting, e avaliação dos receptores de estrógeno ER-&#945; e ER-&#946; por Western Blotting. Nossos resultados apontam que aproximadamente 20% de BPA na concentração de 250 &#181;M após 6 horas de exposição e 18% para a concentração de 200 &#181;M com 9 horas de exposição foram absorvidos pela cultura celular. O ensaio do MTT mostrou que as células expostas a 200 e 250 &#181;M de BPA, por 12, 24 e 48 horas, apresentaram diminuição significativa da função mitocondrial em relação ao controle. Porém, não houve liberação de LDH para o meio de cultura para nenhuma das concentrações de BPA em nenhum dos períodos de incubação, o que sugere que não houve rompimento da membrana plasmática. Foi observada atividade apoptótica somente com a concentração de 250 &#181;M no período de exposição de 6 horas por citometria de fluxo. Não foram encontradas células em necrose, nem alteração na concentração de cálcio intracelular em nenhuma das condições estudadas. Na avaliação dos marcadores de morte celular, observamos aumento da razão de Bax/Bcl-2 para a concentração de 250 &#181;M em todos os períodos de exposição e aumento das caspases 8, 9 e 3 para a concentração de 250 &#181;M no período de exposição de 6 horas, indicando que o BPA deve ativar tanto a via intrínseca como a extrínseca no processo de apoptose. Verificamos ainda, por Western Blotting, que a cultura primária de hipocampo apresenta os receptores de estrógeno ER-&#945; e ER-&#946;. A exposição ao BPA aumentou os ER-&#945; e ER-&#946; avaliados por Western Blotting para as duas concentrações estudadas no período de 6 horas de exposição e, para o período de exposição de 9 horas, houve um aumento do ER-&#945; para a concentração de 250 &#181;M e do ER-&#946; para a concentração de 200 &#181;M. É possível concluir que o BPA pode levar a morte das células neuronais hipocampais por apoptose por ambas as vias intrínseca e extrínseca, sendo o processo de morte celular mais evidente para a concentração de 250 &#181;M no período de 6 horas de exposição. Sugerimos ainda que o aumento observado em ambos os receptores de estrógeno possa representar uma tentativa de interrupção ou reversão do processo de morte celular. / Bisphenol A (BPA) is used in the manufacture of polycarbonate plastics and epoxy resins. The prenatal exposure to this agent may cause several effects, such as anticipation of puberty, prostate hyperplasia, reduced number of sperm, reduced testosterone levels, alteration in the development and tissue organization of the mammary gland, decreased cellular response induced by hormones, breast cancer, diabetes, cardiovascular disease, changes in the functions of liver enzymes, and effects on cognitive development. Few studies have evaluated the effects of BPA in neuronal cells, however there are evidences that this agent may induce apoptosis. This work aims to study the neurotoxicity of BPA, by analyzing the signaling pathways of apoptosis in hippocampus primary culture. Cells were exposed to BPA at 50, 100, 150, 200, and 250 &#181;M (0.1% DMSO v/v) for 6, 12, 24, and 48 hours for the assay of mitochondrial activity (MTT) and the release of the enzyme lactate dehydrogenase (LDH). From the results of MTT and LDH, new exposure times (3, 6 and 9 hours) and only 200 and 250 &#181;M were used. In this new experimental design we performed the quantification of the BPA concentration in the primary culture by HPLC-PDA, intracellular Ca2+ quantification by Fluo-4 AM assay and the characterization of the mechanisms involved in cell death by flow cytometry and Western Blotting assays. Furthermore, evaluation of the estrogen receptor ER-&#945; and ER-&#946; was done by Western Blotting. Our results demonstrate that about 20% of the BPA concentration of 250 &#181;M after 6 hours of exposure and 18% for the concentration of 200 &#181;M with 9 hours of exposure were absorbed by the cell culture. Cells exposed to 200 and 250 &#181;M of BPA for 12, 24 and 48 hours, showed a significant decrease in mitochondrial function, by the MTT assay, compared to control. However, there was no release of LDH into the culture medium for any of the BPA concentrations in any of incubation times studied, which suggests no rupture of the plasma membrane by BPA. Apoptotic activity was observed after 6 hours of exposure to 250&#181;M BPA by flow cytometry. It was not observed cell necrosis and changes in intracellular calcium concentration in any of the studied conditions. Regarding the cell death markers, exposure to 250 &#181;M BPA in all periods of exposure resulted in an increased Bax/Bcl-2 ratio; moreover, an increase in caspase 8, 9 and 3 was detected after exposure to 250 &#181;M BPA for 6 hours. Taken together, these findings indicate that BPA activates both the intrinsic and the extrinsic pathway during the apoptotic process. We also verified by Western Blotting the presence of the estrogen receptors ER-&#945; and ER-&#946; at the primary culture of hippocampus, and that they can be modulated by BPA. The exposure to 200 and 250 &#181;M BPA for 6 hours caused an increase of ER-&#945; and ER-&#946;, however, 9 hours of exposure to 200 &#181;M and 250 &#181;M BPA increased the expression of ER-&#945; and ER-&#946;, respectively. In conclusion, BPA can lead hippocampal neuronal cells to death by both, intrinsic and extrinsic, apoptotic pathways and this process is more evident at 250 &#181;M BPA after 6 hours of exposure. Furthermore, we suggest that the increase of both estrogen receptors might represent an attempt to interrupt or reverse the cell death process.

Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden. / Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.