Orientador: Wilson de Figueiredo Jardim / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T06:01:40Z (GMT). No. of bitstreams: 1 Raimundo_CassianaCarolinaMontagner_D.pdf: 6486329 bytes, checksum: eb2082b97701f19b66fc4e6dc033da3f (MD5) Previous issue date: 2011 / Resumo: O objetivo deste trabalho foi avaliar, através de um perfil anual, a qualidade dos mananciais e da água tratada de Campinas (SP) em relação à presença de cafeína, estrona, 17b-estradiol, estriol, progesterona, testosterona, 17a-etinilestradiol, mestranol, levonorgestrel, dietilestilbestrol, triclosan, 4-n-octilfenol, 4-n-nonilfenol, bisfenol A, fenolftaleína e atrazina empregando extração em fase sólida (SPE) e cromatografia líquida acoplada ao espectrômetro de massas (LC-MS/MS), e verificar o potencial estrogênico das amostras usando o bioensaio Bioluminesce Yeast Estrogen Screening (BLYES). Os resultados mostraram a presença de seis dos 16 compostos investigados em pelo menos uma amostra de água tratada. A estrona foi detectada apenas uma vez. Concentrações de atrazina de até 687 ng L foram determinadas nas amostras provenientes do rio Capivari, e nas amostras de água tratada provenientes do rio Atibaia as concentrações não passaram de 42 ng L. A fenolftaleína foi determinada em apenas uma campanha em todas as amostras provenientes do rio Atibaia em concentrações de até 20 ng L. As concentrações médias de bisfenol A e triclosan foram 7 e 17 ng L, respectivamente. Nos mananciais foram determinados 13 dos 16 compostos investigados. Todas as amostras de água bruta e tratada apresentaram concentrações de cafeína que variaram significativamente entre os períodos de seca e cheia dos rios. As estações de tratamento de água de Campinas, operando no sistema convencional foram capazes de remover entre 30 e 99% dos contaminantes investigados. No mais, 40 % das amostras de água tratada apresentaram atividade estrogênica positiva (entre 0,01 e 1,33 ngE2equiv L) e todas as amostras de água bruta apresentaram estrogenicidade entre 0,06 e 6,54 ngE2equiv L. Sendo que estas foram maiores nos períodos de estiagem / Abstract: The purpose of this work was to evaluate an annual profile of both source of supply and drinking water quality in the city of Campinas (São Paulo State) in relation to the presence of caffeine, estrone, 17b-estradiol, estriol, progesterone, testosterone, 17a-ethynylestradiol, mestranol, levonorgestrel, diethylstilbestrol, triclosan, 4-n-octylphenol, 4-n-nonylphenol, bisphenol A, phenolphthalein and atrazine using Solid Phase Extraction (SPE) and Liquid Chromatography with Tandem Mass Spectrometry Detection (LC-MS/MS), and make sure the estrogenic potential of samples using Bioluminesce Yeast Estrogen Screening bioassay (BLYES). Results showed the presence of six of the 16 compounds investigated in least one sample of drinking water (estrone, triclosan, bisphenol A, phenolphthalein, atrazine and caffeine). Atrazine concentrations up to 687 ng L were found in drinking water samples from the Capivari River. In drinking water samples from the Atibaia River, the concentrations did not exceed 42 ng L. Phenolphthalein was determined in only one campaign in both raw and treated water from the Atibaia River at concentrations around 20 ng L. Average concentrations of bisphenol A and triclosan in drinking water samples were 7 and 17 ng L, respectively. Thirteen of the 16 investigated compounds were determined in surface waters. All samples of raw and treated water showed concentrations of caffeine that varied significantly between dry and wet seasons. The conventional water treatment plants in Campinas were able to remove between 30 and 99 % of the investigated contaminants. The estrogenic activity was evaluated using BLYES bioassay, where 40% of drinking water samples showed positive estrogenic activity (between 0.01 and 1.33 ngE2equiv L) and all samples of surface water presented estrogenic activity between 0.06 and 6.54 ngE2equiv L. These values were higher in dry winter periods / Doutorado / Quimica Analitica / Doutor em Ciências

L’un des plus importants défis de la toxicologie est de pouvoir extrapoler les résultats issus des différentes phases de l’analyse du risque sanitaire à partir de systèmes expérimentaux vers les populations humaines. Dans ce contexte, les techniques globales dites "omiques" sont de plus en plus utilisées pour caractériser les différents états des systèmes biologiques. Ainsi, la toxicogénomique permet non seulement d’étudier les mécanismes d’action des composés, d’identifier des marqueurs d’exposition, mais aussi de générer des signatures moléculaires à potentiel prédictif. En effet, des composés ayant des signatures moléculaires semblables ont également de forts risques de présenter les mêmes effets toxicologiques. L’objectif de cette thèse est d’appliquer ce concept de manière systématique, en explorant les données publiées et disponibles dans les banques dédiées à la toxicogénomique via des modèles statistiques multivariés. Ces analyses ont pour objectif de permettre le regroupement et donc la classification des composés sur la base des gènes dont ils affectent l’expression. L’appartenance de produits toxiques bien caractérisés aux classes ainsi constituées permet alors d’émettre des hypothèses quant à la toxicité des autres composés. Un jeu de données quantitatives intégrant 18 études réalisées avec la même technologie de puce à ADN et chez une seule espèce a été assemblé. De ce jeu de données, 3022 signatures toxicogénomiques correspondant à 452 composés différents ont été obtenues. Des approches de classification non supervisées afin de définir des classes de traitements induisant des altérations transcriptionnelles proches ont été mises en place. 95 et 104 classes ont été obtenues en fonction des méthodes utilisées. Finalement, une attention toute particulière a été portée sur les potentiels nouveaux perturbateurs endocriniens et xénobiotiques reprotoxiques sur-représentés dans trois classes spécifiquement. 22 composés sont en cours de test sur une lignée cellulaire humaine exprimant les enzymes de la stéroïdogenèse (NCI-H295R) pour évaluer leur potentiel effet perturbateur endocrinien. L’ensemble de ce travail a ainsi permis de démontrer la pertinence de nos approches de toxicogénomique pour la prédiction des effets toxiques de composés testés dans d’autres organes et/ou chez d’autres espèces. Il se poursuit à l’heure actuelle par la mise en place d’une base de données, TOXsIgN, permettant l’hébergement et l’accès à l’ensemble de signatures de toxicogénomique générées dans ce projet. De même, mon travail a également permis la mise en place de plusieurs outils dédiés à la toxicologie prédictive et à la visualisation de données, tels que le navigateur de génomes comme le ReproGenomics Viewer (RGV). / The core aim of my thesis project is to develop predictive toxicology approaches based on the integration of massive toxicogenomics datasets using bioinformatics and biostatistics methodologies. Specific objectives include: (1) classification of chemicals based on toxicogenomics signatures, i.e. the set of genes whose expression is known to be positively or negatively altered after exposure to these compounds; (2) the association of the resulting classes with human disorders or deleterious phenotypes based on the well-known toxicants present in those classes; (3) the prediction of novel reprotoxicants and/or endocrine disruptors based on toxicogenomics signature similarities with known chemicals affecting testis development and function. The assembled toxicogenomics dataset contains 23,657 samples covering 7092 experimental conditions (one chemical, one dose, one exposure time, one tissue) for 541 chemicals in seven distinct tissues in the rat from 18 different studies. From this dataset, 3,022 experimental conditions corresponding to 452 distinct compounds are associated to a toxicogenomics signature containing more than ten genes showing an altered expression pattern after exposure. Using unsupervised classification methods, 95 chemical clusters were defined showing close toxicogenomics signatures. The phenotype association analysis using data extracted from de Comparative Toxicogenomics Database (CTD) allowed us to identify three clusters significantly enriched in known endocrine-disrupting chemicals. Currently, 22 compounds are being tested on a human cell line expressing the enzymes of steroidogenesis (NCI-H295R) to evaluate their potential endocrine disrupting effects. These researches allowed us to demonstrate the relevance of integrating massive toxicogenomics datasets to predict adverse effects of compounds tested in different organs. It is currently being pursued through the development of a novel repository, TOXsIgN. This resource provides a flexible environment to facilitate online submission, storage and retrieval of toxicogenomics signatures by the scientific community. Similarly, the current PhD project also yielded to the implementation of several tools dedicated to predictive toxicology and data visualization including the ReproGenomics Viewer (RGV).

Les écosystèmes aquatiques constituent la destination finale des contaminants. Les organismes aquatiques sont ainsi impactés par un cocktail de molécules, dont les produits phytosanitaires. Le caractère perturbateur endocrinien (PE) de certains d’entre eux est particulièrement préoccupant. La présente étude explore les effets de pesticides modèles à potentiel PE – la chlordécone (CLD) et le pyriproxyfène (PXF), seuls et en mélange – chez le copépode estuarien Eurytemora affinis par des approches écotoxicogénomiques innovantes. En réponse aux PE, l’accent a été porté sur les effets de ces composés sur la reproduction, la croissance ou le développement. En parallèle, l’affinité des pesticides aux récepteurs aux oestrogènes et aux androgènes humains d’une part, et aux ecdystéroïdes d’autre part, a été étudiée grâce à des outils bio analytiques afin d’évaluer la pertinence de l’utilisation du potentiel PE défini par les tests cellulaires comme critère d’exclusion dans la réglementation européenne des pesticides. Ces travaux de thèse présentent, pour la première fois, les transcriptomes et protéomes d’E. affinis après exposition aux PE. Un effet sexe-dépendant a été observé par les deux approches « omics » révélant un plus fort impact des composés sur les copépodes mâles. Des gènes et protéines, impliqués dans le processus de mue et dans la gamétogénèse ont été identifiés comme candidats pour le développement de biomarqueurs moléculaires de PE chez les crustacés. Les tests cellulaires ont permis de mettre en évidence des interactions entre les contaminants et les récepteurs hormonaux. Toutefois, le screening de molécules par les tests YES/YAS/anti-YES/anti-YAS s’est heurté à quelques limites d’interprétations. Enfin, les résultats du test S2 EcR, souligne la pertinence de l'utilisation couplée de tests spécifiques d’un système endocrinien de vertébrés et d’invertébrés dans l’évaluation du risque des composés PE. / Aquatic ecosystems constitute the chemicals’ final destination. Among the xenobiotics, endocrine disruptors (ED) are compounds of major concern. In this context, ED pesticides effects were investigated in the widespread copepod Eurytemora Affinis using ecotoxicogenomics technics. In response to PE pesticides, focus was made on reproduction, growth and development. In order to evaluate the endocrine activity, in vitro tests (YES/YAS/anti-YES/anti-YAS and S2 EcR) were used to screen the compounds alone and in mixtures. These results were discussed to evaluate the “cut off criterion” used in European assessment. This work presents for the first time, transcriptomes and proteomes of E. affinis after PXF and CLD –alone and in binary mixture- exposure. A sex dependent effect was observed by the two “omics” approaches. Male copepods were more impacted by contaminants than female copepods. Genes and proteins (e.g. chitin deacetylase, kelch protein) were identified as candidates for the development of ED molecular biomarkers. In vitro tests highlighted binding of pesticides with both vertebrate and invertebrate receptors. However, a toxicity for the highest concentrations tested and some limits for the interpretation of mixtures results were limiting in YES/YAS/anti-YES/anti-YAS assays. The last observation represents particularly a major concern for interpretation of the ED mode of action of environmental matrix. S2 EcR highlighted some complementary results about the mode of action of chemicals alone and in binary mixtures. These results accentuate the need to combine vertebrate specific test and invertebrate specific test in ED risk assessment.

La protéine kinase D1, PKD1, est une sérine/thréonine kinase activée par de nombreux facteurs mitogènes. Les études, menées jusqu’à présent sur les fonctions de PKD1, ont montré qu’elle semble jouer un rôle dans la régulation de plusieurs processus biologiques fondamentaux impliqués dans le développement des tumeurs. Cependant, le rôle précis et les cibles de PKD1 restent largement méconnus. Au cours de ce travail, nous avons tout d’abord démontré que l’inhibition de PKD1 dans les cellules de mélanome inhibe la croissance et la motilité cellulaire, induit l’expression de la E-cadhérine et une diminution de la N-cadhérine. D’autre part, nous nous sommes intéressés au rôle des perturbateurs endocriniens dans les cellules tumorales mammaires et avons démontré que PKD1 est une cible des perturbateurs endocriniens (PE). Les PE, tels que le bisphénol A (BPA) et les phtalates, sont des produits chimiques ubiquitaires de notre environnement. Leur rôle dans la croissance tumorale mammaire est bien documenté. Néanmoins, les mécanismes moléculaires précis par lesquels ces molécules agissent demeurent encore inconnus. Au cours de notre travail, nous avons démontré que ces PE induisent de façon dose-dépendante la prolifération des cellules MCF-7 (cellules d’adénocarcinome mammaire) et que ce processus biologique est dépendant de l’expression de PKD1. Ainsi, l’ensemble de ce travail fait apparaître, d’une part, que PKD1 pourrait être une nouvelle cible thérapeutique anti-tumorale potentielle dans le mélanome et que, d'autre part, PKD1est une cible moléculaire de certains PE dans le cancer du sein. / Protein kinase D1, PKD1, is a serine/threonine kinase which can be activated by mitogens and that regulates various functions involved in the development of tumors. However, its precise role and targets are still unclear. Our study demonstrates that PKD1 inhibition in melanoma cells decreased cell growth and motility, and reversed the E- to N-cadherin switch. On the other hand, we examined the role of endocrine disruptors (EDs) in breast cancer cells and identified PKD1 as a target of these compounds. EDs, such as bisphenol A (BPA) and phthalates, have been found molecular mechanisms are still ubiquitously throughout our environment. Their role in breast tumor growth has been well documented. However, their precisemolecular mechanisms are still unknown. Our study demonstrates that EDs induce dose-dependently MCF-7 cell growth and that this biological process is dependent upon PKD1expression. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target in melanoma and identifies PKD1 as a new molecular target of some EDs in breast cancer cells.

Orientador: Sebastião Roberto Taboga / Resumo: A próstata é uma glândula componente do sistema genital dos mamíferos e sua principal função prostática é secretar um fluido levemente alcalino (pH 7,29) que contribui para a formação do líquido seminal. As fêmeas em certas espécies, como o roedor gerbilo da Mongólia (Meriones unguiculatus), apresentam próstata cuja morfologia se assemelha à porção ventral do complexo prostático masculino e à próstata feminina em humanos. Nas últimas décadas tem sido relatado um constante aumento da exposição a substâncias capazes de interferir na ação hormonal, conhecidas como disruptores endócrinos. O bisfenol A (BPA) pertence a uma das categorias de disruptores endócrinos presentes no meio ambiente. Estudos demonstram que a exposição a esse composto interfere negativamente na morfofisiologia prostática, favorecendo o estabelecimento de lesões pré-malignas e malignas. Além do BPA, uma das constantes problemáticas dos dias atuais é a exposição ambiental a hormônios exógenos como o estradiol (E2). Desta forma, a administração de fármacos com estrutura semelhante a hormônios esteroides pode exercer efeitos sobre a glândula, uma vez que os mesmos se ligam a seus receptores hormonais afetando a homeostasia prostática. O presente estudo teve como objetivo verificar as alterações morfofuncionais na próstata de fêmeas de gerbilo da Mongólia após tratamento perinatal com dose diária ambiental, ou seja, dose ambiental segura de BPA (50 µg/kg) e doses de 17-β estradiol (35 µg/kg), 3 vezes por semana p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...

Les organes centraux du tractus urogénital sont le testicule et l’ovaire, qui assurent la production de gamètes et d’hormones, et donc la fertilité de l’individu. Ces deux organes, parfaitement distincts et complémentaires, ont pour origine une gonade bipotentielle qui s’engagera vers une trajectoire de différenciation masculine ou féminine au cours de la vie fœtale. Les deux gonades vont par la suite subir plusieurs phases de différenciation et de développement de leurs populations cellulaires, afin d’acquérir leurs fonctions propres qui leur permettront d’assumer leur rôle à l’âge adulte. Depuis plus d’une quinzaine d’années, le concept de syndrome de dysgénésie testiculaire fait état d’un lien entre l’exposition du fœtus à des composés environnementaux et des anomalies du tractus urogénital. Bien que sujette à de vifs débats au sein de la communauté scientifique, cette hypothèse a attiré l’attention de la recherche sur les conséquences de l’exposition des mères aux xénobiotiques sur l’enfant à naître. La différenciation et le développement des gonades fœtales sont gouvernés par des programmes d’expression spécifiques de chaque sexe, dont de nombreuses zones d’ombres subsistent, notamment concernant la fraction non-codante exprimée par le génome humain. La première partie de cette thèse de doctorat présente, pour la première fois, le paysage transcriptionnel contrôlant ces processus complexes entre la 6ième et 17ième semaine de développement chez l’Homme. Grâce à l’avènement des technologies de transcriptomique, il est désormais possible d’identifier et d’observer l’expression des gènes de manière sensible et sans a priori. Le RNA-seq m’a donc permis de décrire de manière exhaustive la dynamique d’expression des gènes, pendant les stades précoces de la différenciation sexuelle, jusqu’aux phénomènes plus tardifs conduisant aux linéages des différentes populations cellulaires spécifiques du testicule et de l’ovaire. Dans une deuxième partie, mon travail de recherche s’est attaché à étudier l’impact de deux perturbateurs endocriniens suspectés, l’ibuprofène et le chlordécone, sur le programme d’expression du testicule fœtal humain. L’utilisation du RNA-seq m’a permis de définir et de comparer la signature toxicogénomique de chaque molécule afin de contribuer à la compréhension de leur mécanisme d’action et d’identifier les populations cellulaires affectées. Enfin, face à l’essor des technologies ultra-haut-débit dans les sciences de la vie, y compris dans les domaines de la reproduction, j’ai activement participé au déploiement d’une nouvelle version du Reprogenomic Viewer dans la dernière partie de ma thèse (http://rgv.genouest.org). Cet outil nternet a pour vocation de centraliser et de rendre accessibles les données de séquençage accumulées au sein de la communauté de la reproduction via des outils de visualisation intuitifs. / Fetal life is a crucial period for sexual reproduction when bipotential gonads differentiate into either a testis or an ovary. Gaining insights into the complex molecular events underlying this process is central to a better understanding of disorders of sexual development. The present work intends to improve the knowledge on molecular pathways at play during gonad development in humans using RNA-sequencing. This project particularly seeks to identify early transcriptional events that may play critical role in the regulatory network driving human sexual differentiation. To address this issue, we defined the transcriptional landscape of fetal human gonads by sequencing total RNA extracted from testes and ovaries between 6 and 17 gestational weeks. The resulting paired-end reads were mapped on the human genome and then assembled into transcripts using the Tuxedo suite. We next defined a high-confidence set of transcripts showing differential expression across samples. Clusters of co-expressed genes were subjected to functional analysis. The analysis of this massive RNA-seq dataset has led to a high-confidence set of 35,194 assembled transcripts; among which 32,391 known and novel isoforms coding genes (mRNAs), 1,209 to long non-coding (lnc) RNAs and 318 to novel unannotated transcripts/genes (NUTs). The dynamic of transcriptional landscape occurring during human fetal gonads development has been described and new genes and interesting candidates, including new genes, have been highlighted as potential key genes governing this biological process. The second interest of this work was the study of the impact of two endocrine disruptors, ibuprofene and chlordecone, on human fetal testis using RNA-seq. The transcriptional alteration induced by these compound in the gonad allowed a deeper understanding of their mechanisms of action of endocrine disruption. The last part of this work was the development of a new version of the ReproGenomics Viewer (http://rgv.genouest.org), a web tool dedicated to the integration and accumulation of sequencing data from studies performed in the field of reproduction.

The increasing use of plastics has elevated the risk of exposure to environmental pollutants such as plasticisers in the general population, making it necessary to understand the possible long term health consequences of the same. In this study we aim to understand how DEHP affects the gene expression in mice models and if it causes disruptions to its cellular pathways. Two datasets, GSE18564 and GSE14920 comprising of 15 and 60 samples respectively were downloaded from GEO database for analysis. Quality control checks were done using Principal Component Analysis and quantile normalisation. Differentially expressed genes were found using LIMMA model, following which only top 20 genes were selected for pathway analysis using KEGG and Gene Ontology. DEHP was found to be associated with chemical carcinogenesis, including negative regulation of extrinsic apoptotic signaling pathway and fatty acid metabolism. Furthermore, it seems likely that PPAR-alpha might play a key role in DEHP related metabolic disruption. Further studies are required to better elucidate the effect of DEHP on individual metabolic pathway implicated in this thesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants suspected to act as endocrine disruptors. We tested the commercial PBDE mixture DE-71 and its in vivo metabolites for estrogenic activity. MCF-7 breast cancer cells culture, ERE-luciferase gene expression, 3H-β-estradiol displacement from recombinant ERα, and ovariectomized (OVX) mice served as bioassays. Although DE-71 did not bind ERα, it was able to increase MCF-7 cell proliferation and this was prevented by the antiestrogen fulvestrant. DE-71 co-treatment reduced the effect of estradiol in MCF-7 cells. In the OVX mouse (BALB/c) 3-day assay, DE-71 administered alone had no effect on uterine or vaginal tissues but when administered subcutaneously potentiated estradiol’s effect on uterine weight in a dose-dependent manner. DE-71 administered SQ to BALB/c mice for 34 days slightly increased uterine epithelial height (UEH), vaginal epithelial thickness (VET) and mammary ductal lumen area, and attenuated the estradiol-induced increase in UEH; these effects were not seen in C57BL/6 mice. DE-71 increased liver weight in BALB/c, C57BL/6 and estrogen receptor-alpha knockout (ERαKO) mice. Liver cytochrome P450 1A (CYP1A) and CYP2B activities increased 2.5-fold and 7-fold respectively when DE-71 was administered PO, but only CYP2B increased (5-fold) after SQ treatment. Six OH-PBDE metabolites were found in mice after 34-day DE-71 treatment and all were able to bind recombinant ERα. Para-hydroxylated metabolites displayed a 10- to 30-fold higher affinity for ERα compared to ortho-hydroxylated PBDEs. Para-OH-PBDEs induced ERE-luciferase and produced an additive effect when coadministered with β-estradiol. DE-71 was also additive with β-estradiol. At high concentrations (≥ 5x10-5 M), ortho-OH-PBDEs were antiestrogenic in the ERE-luciferase assay. In conclusion, DE-71 behaves as a weak estrogen in both MCF-7 breast cancer cells and ovariectomized adult mice. Mice strain, treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight, 5%-51% depending on mouse strain and treatment regime, independently of ERα. The observations that the DE-71 mixture does not displace 3H-β-estradiol from ERα while the hydroxylated metabolites do, suggest that the cellular and tissue effects were due to a metabolic activation of individual congeners.

No description available.