Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2016-05-03T17:20:12Z No. of bitstreams: 1 Tese_Eline_VersãoFinal.pdf: 24730530 bytes, checksum: 660259a520e774bdef4c1478084f292b (MD5) / Made available in DSpace on 2016-05-03T17:20:12Z (GMT). No. of bitstreams: 1 Tese_Eline_VersãoFinal.pdf: 24730530 bytes, checksum: 660259a520e774bdef4c1478084f292b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geociências-Geoquímica. Niterói, RJ / A contaminação dos ambientes aquáticos por contaminantes emergentes é, ainda, pouco investigada no Brasil. Este estudo teve como objetivo avaliar a ocorrência de fármacos de diferentes classes terapêuticas, cafeína e bisfenola, em corpos hídricos de diferentes escalas no Estado do Rio de Janeiro. Foram eles: em micro escala, os Córregos São Domingos e Ribeirão Santíssimo (município de Santa Maria Madalena); em pequena escala, as bacias dos rios Paquequer, Guandu, Iguaçu e Sarapuí; em média escala, a bacia do médio rio Paraíba do Sul. Considerando os objetivos propostos foram analisadas 47 amostras para a determinação de 34 fármacos (acetaminofeno, ácido salicílico, codeína, diclofenaco, fenazona, ibuprofeno, ketoprofeno, meloxicam, naproxeno, piroxicam, atorvastaina, benzafibrato, gemfibrozil, alprazolam, carbamazepina, diazepam, lorazepam, venlafaxina, atenolol, metoprolol, nadolol, propanolol, sotalol, furosemida, torasemida, glibenclamida, irbesartan, losartan, valsartan, warfarina, tiabendazol, xilazina, sulfametoxazol, trimetoprim), cafeína e bisfenol-a, empregando a técnica analítica de extração em fase sólida, cromatografia a líquido de alta eficiência e espectrometria de massa tandem. Os resultados obtidos mostraram que nenhuma amostra analisada estava livre da contaminação por esses compostos e apenas dois deles (piroxicam e torasemida) não foram detectados em qualquer amostra. A freqüência com que os demais compostos foram detectados variou entre 2,1 (para a xilazina) e 100% (para ácido salicílico, velanfaxine, propanolol, tiabendazol, trimetoprim e cafeína). Uma avaliação qualitativa sobre consumo de medicamentos no município de Santa Maria Madalena mostrou uma estreita relação entre o consumo, a concentração e a frequência com que são encontrados fármacos em amostras ambientais. Os rios que apresentaram as maiores concentrações totais destes contaminantes foram (em ordem decrescente): rios Iguaçu-Sarapuí (51,8 μg L-1); córregos São Domingos e Ribeirão Santíssimo (19,0 μg L-1); rio Paquequer (18,2 μg L-1); rio Guandu (1,46 μg L-1) e rio Paraíba do Sul (1,42 μg L-1). Esses resultados mostram a importância da vazão na diluição da contaminação por fármacos, cafeína e bisfenol-a, oriundos da entrada de esgoto doméstico, o que pode ser avaliada através das cargas de contaminantes transportadas em cada bacia. A alta concentração de certos compostos (alguns quantificados neste estudo na ordem de μg L-1) não é, necessariamente, a principal preocupação ambiental, mas também, a sua persistência e atividade biológica. Contudo, para a maioria desses compostos não existem dados suficientes quanto a ocorrência, avaliação de risco e ecotoxicidade, e desta forma, é difícil prever os seus efeitos causados à saúde do homem e dos organismos aquáticos, o que torna primordial a sua investigação / The contamination of aquatic environments by emerging contaminants is still poorly investigated in Brazil. This study aimed to evaluate the occurrence of different therapeutic classes of drugs, caffeine and bisphenol-a in water bodies of different scales in the State of Rio de Janeiro. They were: in micro-scale, the streams of São Domingos and Ribeirão Santíssimo (in the city of Santa Maria Madalena); in a small-scale, the River Basins Paquequer, Guandu, Iguaçu and Sarapuí; on the medium scale, the basin of the middle Paraíba do Sul River. Considering the objectives proposed, 47 samples were analyzed for the determination of 34 pharmaceutical compounds (acetaminophen, salicylic acid, codeine, diclofenac, phenazone, ibuprofen, ketoprofen, meloxicam, naproxen, piroxicam, atorvastatin, benzafibrate gemfibrozil, alprazolam, carbamazepine, diazepam, lorazepam, venlafaxine, atenolol, metoprolol, nadolol, propranolol, sotalol, furosemide, torasemide, glibenclamide, irbesartan, losartan, valsartan, warfarin, thiabendazole, xylazine, sulfamethoxazole, trimethoprim), caffeine and bisphenol-a, using the analytical technique of Solid Phase Extraction, High Performance Liquid Chromatography and Mass Spectrometry tandem. The results showed that any sample evaluated was free from contamination by these compounds; only two compounds (piroxicam and torasemide) were not detected in any sample. The frequency of compounds detected ranged from 2.1 (for xylazine) and 100% (for salicylic acid, velanfaxine, propranolol, thiabendazole, trimethoprim and caffeine). A qualitative assessment about drug use in the municipality of Santa Maria Madalena showed a close relationship between the consumption, the concentration and the frequency wherein pharmaceutical compounds are found in environmental samples. The rivers that had the highest total concentrations of these contaminants were (in descending order): Iguaçu and Sarapuí rivers, (51.8 mg L-1), and the streams São Domingos and Ribeirão Santíssimo (19.0 mg L-1); Paquequer River (18.2 mg L- 1); Guandu River (1.46 mg L-1) and Paraíba do Sul River (1.42 mg L-1). These results show the importance of flow in the dilution of the contamination by pharmaceutical compounds, caffeine and bisphenol-a, from the entry of domestic sewage, which can be evaluated through loads of contaminants carried in each basin. The high concentration of certain compounds (some quantified in this study in order mg L-1) is not necessarily the main environmental concern, but also its persistence and biological activity. However, for most of these compounds there are insufficient data regarding the occurrence, risk assessment and ecotoxicology, and thus it is difficult to predict the health effects in humans and aquatic organisms, which makes its primary research

L'eau potable comme source d'exposition aux perturbateurs endocriniens (PE) est peu étudiée, notamment chez les personnes vulnérables comme les femmes enceintes, dont les modes de consommation d'eau évoluent pendant la grossesse. Les objectifs de la thèse étaient i) déterminer s'il existe une relation entre l'exposition prénatale hydrique aux PE, et la prévalence d'enfants ayant un petit poids de naissance pour leur âge gestationnel (PPAG) ou prématurés tenant compte des facteurs socio-économiques ; ii) estimer pour la première fois en France les usages de l'eau potable pendant la grossesse.Trois études épidémiologiques issues d'une cohorte rétrospective, ont été réalisées auprès de 13654 femmes enceintes ayant accouché dans les Deux-Sèvres entre 2005 et 2010. Une exposition hydrique aux doses moyennes de nitrates au deuxième trimestre augmentait le risque de PPAG, particulièrement chez les femmes résidant dans des quartiers favorisés. En revanche, aucune relation significative entre une exposition prénatale hydrique à la 2-hydroxyatrazine et le risque de prématurité n'a été montrée.Les usages de l'eau potable ont été évalués par un questionnaire administré en face-à-face auprès de 132 femmes enceintes françaises de la cohorte prospective EDDS. La consommation d'eau pendant la grossesse était stable et comprenait plus d'eau du robinet que d'eau embouteillée. Afin de reconstituer la dose absorbée en PE, ces résultats devront être couplés à des dosages environnementaux et dans les matrices biologiques.Ces travaux confirment l'intérêt de l'approche interdisciplinaire en santé environnementale et celui de l'éducation relative à la santé environnementale. / Drinking-water as a source of exposure to endocrine disruptors, particularly in pregnant women whose water-use habits change during pregnancy, has seldom been studied. Our objectives were i) to study the possible relationship between prenatal exposure to endocrine disruptors in drinking water, socioeconomic factors and prevalence of neonates born small-for-gestational-age (SGA) or preterm birth ; ii) to estimate for the first time in France the water-use habits of pregnant women throughout pregnancy.Three epidemiologic studies from a retrospective cohort were carried out on 13,654 pregnant women who gave birth in Deux-Sèvres (France) between 2005 and 2010. An exposure to moderate dose of nitrates in drinking-water increased SGM risk, particularly for women living in less deprived areas. No relationship between prenatal exposure to 2-hydroxyatrazin in drinking-water and preterm birth risk was found.Water-use habits during pregnancy were assessed with a questionnaire on 132 women from the EDDS prospective cohort. Water ingestion was stable during pregnancy and tap water predominated over bottled water. In order to reconstitute the dose of pollutant intake from water, the results of this estimation by questionnaire will need to be merged with analytical dosages in waters and biological matrices.Our study confirms the interest of an interdisciplinary approach to environmental health and the key importance of education in that field.

Les conditions de sécurité sanitaire encadrant les pratiques d'hémodialyse (HD) et d'hémodiafiltration (HDF) n'intègrent pas les risques liés à des micropolluants comme les perturbateurs endocriniens (PE). Les patients dialysés présentent un risque de surexposition au Bisphénol A (BPA), reconnu comme PE, en raison de sa présence dans les dispositifs médicaux utilisés lors de la dialyse et du risque d'accumulation liée à leur état rénal.Nos premiers travaux ont confirmé la présence du BPA dans les dialyseurs et démontré pour la 1ère fois que l'eau utilisée en HD était également une source de contamination importante en BPA, via la production du dialysat. De plus, nous avons mis en évidence dans l'eau de dialyse la présence de dérivés chlorés du BPA (ClxBPA), sous-produits de chloration de l'eau connus pour leur activité œstrogénique supérieure au BPA. Nous avons ensuite montré que l'HDF entrainait un risque d'exposition aux PE plus important que l'HD, via la contamination du liquide de substitution perfusé chez le patient. Ces résultats permettront aux industriels de prendre en compte le risque de contamination à ces PE ainsi qu'aux médecins et pharmaciens impliqués dans la prise en charge des patients dialysés.Peu de données sont disponibles concernant l'impact clinique d'une telle exposition chez le patient dialysé et aucune étude n'a intégré le risque lié aux ClxBPA. C’est pourquoi, nous avons développé des biomarqueurs d'exposition en mettant au point des méthodes de dosage ultrasensibles du BPA et des ClxBPA dans les urines et le plasma. Ces biomarqueurs permettront d'étudier l'impact des différentes techniques de dialyse sur l'exposition des patients à ces PE. / The health safety conditions for the practice of hemodialysis (HD) and hemodiafiltration (HDF) do not integrate the risks associated with micropollutants such as endocrine disruptors (ED). Dialysed patients are at risk of overexposure to Bisphenol A (BPA), a well-recognized ED, due to its occurrence in medical devices used during dialysis and to the risk of accumulation due to their renal impairment.In a first step we have confirmed BPA contamination in dialyzers and demonstrated, for the first time, that the water used in HD was a significant source of BPA contamination, via dialysate production. Furthermore, we highlighted the presence of chlorinated derivatives of BPA (ClxBPA), by-products of water chlorination known to have higher oestrogenic activity than BPA, in dialysis water. Then, We have demonstrated that HDF leads to a higher risk of exposure to ED than HD, via the contamination of the liquid of substitution perfused in patient. These results will allow manufacturers to take into account the risk of contamination to these ED as well as physicians and pharmacists involved in patient care.Very few data are available regarding the clinical impact of such exposition on dialysed patient and no study has included the risk arising from ClxBPA. Therefore, we have performed exposure biomarkers using ultra-sensitive analytical methods to determine BPA and ClxBPA concentration in urine and plasma. These biomarkers will allow studying the impact of different dialysis techniques on patient exposure to these ED.

En ny komplex aspekt i frågan om vattenkvalité är ackumuleringen av organiska föroreningar.Den begränsade utbredningen av vattenrening i Brasilien såväl som i andra utvecklingsländervisar på ett tydligt behov av utveckling av billiga och effektiva metoder för provtagning ivatten. Bristen på den här typen av metoder försvårar idag möjligheterna att kontrolleravattenkvalitén och på så vis även arbetet med att säkerställa de sanitära förhållandena ivattentillgångar. Det här examensarbetet innehåller en sammanfattande genomgång avprinciperna för den passiva provtagningsmetoden med namnet ”Diffusive Gradients in Thin‐Films (DGT)”, som direktöversatt får det svenska namnet ”Diffusiva gradienter i tunnfilmer”.När metoden används för provtagning för att upptäcka olika organiska ämnen i vattnetbenämns den som o‐DGT.Målet med detta arbete är att ta fram en metod med målet att specifikt upptäcka olika typerav nya framväxande organiska föroreningar som i de flesta fall förekommer bara i väldigt lågakoncentrationer i vår miljö. Grundliga undersökningar genomfördes för att få svar på huruvidabiokol kan fungera som den bindande agenten i DGT apparatur och verka för att kunnaupptäcka och identifiera dessa ämnen på ett effektivt sätt.Resultaten från de undersökningar som genomförts hittills visar på att biokol har godpotential att tillämpas som bindande agent i filter för bruk i o‐DGT apparatur. De organiskaföreningarna av intresse i denna studie kunde alla detekteras, kvantifieras och identifieras.För optimering av provtagningsmetoden bör vidare studier vid varierande förhållanden medhänsyn till pH, temperatur och jonkoncentration genomföras, såväl som studier av detbindande lagrets egenskaper i förhållande till koncentration av biokol och lagrets tjocklek.Detta för att kunna optimera tekniken, och förhoppningsvis bidra till ett konventionelltanvändande av biokol på adsorberande agent i o‐DGT anordningar i framtiden. / A new complex aspect in the matter of water quality is the occurrence of emerging organicpollutants and contaminants in waste water. The currently low extent to which treatment ofwaste water is performed in Brazil, and in the world as a whole, there is a considerable needfor development of cheap and accurate in‐situ sampling methods for far‐reaching studies ofsurface water quality. The lack of such methods today makes the maintenance andestablishing of sanitary safety difficult. This diploma work gives a brief introduction to thebasic principles of the passive sampling method known as Diffusive Gradient in Thin‐films(DGT). A method that could be useful for such monitoring of quality in water bodies worldwide.The aim of this study is to develop a method, for the detection of organic emerging pollutantsand contaminants – i.e. compounds, which usually are present at very low concentrationswhen found in the environment as a result of human activity. More specifically, this workinvestigates the potential and usefulness of the application of DGT devices in detection oforganic compounds that can affect human health and ecosystems, even at lowconcentrations, however, their effects still are in need of further investigations.This study focuses on both purely technical as well as practical points of views. The efficiencyof organic DGT (o‐DGT) with biochar as the adsorbing agent is examined targeting thedetection of organic pollutants and contaminants in surface water. In this sense, the specificaim of the work is to evaluate the performance of biochar as adsorbing agent. This workshowed that the performance of biochar as the adsorbing agent in binding layers in o‐DGTsample devices can be considered as satisfactory since all compounds of interest in this studywas successfully detected, quantified an identified. Further investigations in the future areneeded to determine the effects of varying pH, temperature and ion concentration in thedeployment media, as well as the properties of the binding layer in relation to concentrationof biochar and the thickness of the layer. These in order to optimize the method for in‐situwater sampling, aiming conventional use of biochar as the adsorbing agent in the future.

[pt] Ftalatos são os plastificantes mais utilizados industrialmente, estando disseminados em todos os ecossistemas do mundo. Como são compostos exclusivamente sintéticos, sua presença é indicativa da industrialização de uma região. Sua toxicidade é baixa, porém existem indícios de possíveis efeitos como interferentes endócrinos, especialmente no sistema reprodutor masculino. No Brasil, existem poucos estudos sobre a ocorrência e comportamento dos ftalatos no meio ambiente. Nesta tese foi estudada sua ocorrência em amostras de águas potáveis de diversos bairros das cidades do Rio de Janeiro e Niterói e do ecossistema da Baía de Guanabara (águas, sedimentos e mexilhões), a qual recebe inúmeros aportes industriais. Todas as amostras foram coletadas, extraídas em fase sólida e analisadas por cromatografia em fase gasosa, utilizando detecção por captura de elétrons. As da Baía foram amostradas nas estações seca e chuvosa, em diferentes anos. As extrações de todas as amostras obtiveram excelente repetibilidade e recuperações superiores a 76%. As águas potáveis do Rio de Janeiro e Niterói apresentaram teores de ftalatos em concentrações muito menores do que as informadas para cidades da Europa e Estados Unidos. Tais teores puderam ser correlacionados com as extensões das redes dos sistemas de abastecimento de ambas as cidades. Todas as amostras da Baía mostraram contaminações qualiquantitativas muito inferiores, quando comparadas a outros ambientes costeiros. O estudo do biomonitoramento ativo evidenciou a eficiência e rapidez da depuração dos ftalatos nos mexilhões mais contaminados do ecossistema da Baía de Guanabara. / [en] Phthalates are the most used plasticizers in industrial products, worldwide distributed in environments. Their presence is often used to indicate a region industrialization degree. Although their reported toxicity is low, phthalates are indicated for possible endocrine disrupting effects, specially in male reproductive system. In Brazil, there are few studies about the environmental occurrence and distribution of phthalates. This thesis studied the occurrence of those compounds in potable waters samples from residential areas of Rio de Janeiro and Niterói cities, and in samples from the ecosystem of Guanabara Bay (waters, sediments and mussels), which receives several industrial inputs. All the samples were collected, solid-phase extracted and the phthalate contents analyzed by gas chromatography with electron capture detection. The ones from the Bay were sampled during the dry and rainy seasons, in different years. All the extractions presented excellent repeatability and recoveries above 76%. The potable water samples from both cities presented phthalate values considerably lower than those reported for European and American cities. The results could be correlated with the water net distribution lengths for both cities. All results from the Bay ecosystem showed lower contamination when compared to other coastal environments. The controlled transplant proved quick and efficient phthalate depuration of contaminated organisms from Guanabara Bay.

L’homéostasie buccale conditionne fortement les perceptions gustatives ; elle repose sur un épithélium buccal sain et le bon fonctionnement des glandes salivaires, qui sont finement régulés par hormones sexuelles. Le but de cette thèse était de savoir si une exposition orale en bisphénol A, un migrant d’emballage alimentaire et de composites dentaires, et une alimentation riche en phyto-œstrogènes (soja) pouvaient modifier l’homéostasie buccale. Deux expérimentations ont été conduites : une étude dose-effet du BPA (5µg à 12,5 mg/kj/j) chez le rat adulte, et une étude d’interaction d’un régime riche en soja sur les effets du BPA. Sur la base de tests gustatifs et d’une approche histologique et moléculaire (qPCR-TR), la première étude identifie pour la première fois une action du BPA sur la sècheresse buccale. Nous avons constaté que le BPA était responsable d’une moindre consommation d’eau (p<0.01), d’une préférence augmenté au sel (p<0.05) et diminué au sucre (p<0.05), et d’une altération des sécrétions salivaires. Une réversibilité partielle des effets à l’arrêt du traitement. A contrario un régime riche en phyto-œstrogène augmente la prise d’eau (p<10-6) et diminue la préférence au sel (p<0.05) par rapport à un régime semi-synthétique, et peut s’opposer aux effets du BPA. Ces études montrent que BPA et phyto-œstrogènes exercent des effets œstrogéniques agonistes ou antagonistes en fonction de la cible biologique considérée, et qu’un régime à base de soja peut gommer la plupart des effets observés / Oral homeostasis strongly influences taste perceptions. It depends on a healthy oral epithelium and salivary gland secretions, which are both regulated by sex hormones. The aim of this thesis was to identify the effect of an oral exposure to Bisphenol A, a food packaging and dental sealer component, and of a soy-diet containing phytoestrogens on oral homeostasisTwo experiments were conducted in adult rats: a dose-response study of BPA (5μg 12.5 mg / kj / day), and a study about the impact of a soy-diet on the BPA disrupting effects. By using gustation choice tests, and histological and qPCR-TR molecular approach, we identify for the first time an action of BPA on dry mouth. We found that BPA reduced water consumption (p <0.01), increased salt intake (p <0.05) and decreased sugar intake (p <0.05), and also modulated salivary secretions. These effects were partially reversed by stopping oral exposure. In contrast, the soy-diet increased water intake (p <10-6) and decreased in salt preference (p <0.05) by comparing to the semi-synthetic diet did, and may correct the effects of BPA.These studies show that BPA and phytoestrogens exert agonist or antagonist estrogenic effects depending on the biological target, and that a soy-based diet can erase most of the observed BPA effects

Les phénols et les phtalates incluent des composés très largement utilisés dans des produits de la vie quotidienne. Une grande partie de la population générale y est donc largement exposée. Ces composés sont suspectés d’être des perturbateurs endocriniens et des effets sur la santé chez l’Homme ont été rapportés, notamment après une exposition périnatale. Les études épidémiologiques sur les effets sur la santé humaine reposent généralement sur un faible nombre de biospécimens pour estimer l’exposition. Cependant, la variabilité intra-individuelle des phénols et des phtalates est potentiellement forte, ce qui peut entraîner une mauvaise classification de l’exposition dans les études sur les effets des phénols et des phtalates et limite leurs conclusions. La variabilité intra-individuelle des phénols et des phtalates au cours de la grossesse n’est pas très bien caractérisée à l’heure actuelle.L’objectif de cette thèse est d’explorer l’exposition aux phénols et aux phtalates et plus précisément : 1) d’étudier les associations entre une telle exposition pendant la grossesse et la santé respiratoire de l’enfant au cours de ses premières années de vie ; 2) de caractériser la variabilité temporelle intra-individuelle de ces composés au cours de la grossesse ; et 3) d’évaluer l’efficacité d’une approche basée sur le pooling intra-sujet d’un nombre réduit d’échantillons journaliers pour estimer l’exposition.Les associations entre l’exposition aux phénols et phtalates et la santé respiratoire reposent sur n = 587 couples mèresenfants de la cohorte prospective française EDEN. Les développements sur l’estimation de l’exposition au cours de la grossesse s’appuient sur n = 16 femmes enceintes ayant participé à l’étude de faisabilité de la cohorte SEPAGES.Les travaux de cette thèse quantifient la variabilité intra-individuelle des concentrations urinaires des biomarqueurs d’exposition aux phénols et des phtalates au cours de la grossesse pour des échelles de temps variées (du jour à plusieurs mois). Ils confirment empiriquement que cette variabilité peut biaiser fortement les fonctions doses-réponses dans les études épidémiologiques explorant les effets de l’exposition fœtale à ces composés chez l’Homme.Les résultats de cette thèse enrichissent la littérature émergente sur les effets des expositions précoces aux phénols et phtalates sur la santé respiratoire de l’Homme. Cependant, notre étude ainsi que la plupart des recherches précédentes sont potentiellement limitées par les problématiques liées à la mesure de l’exposition. Ce travail souligne l’importance de stratégies d’échantillonnage des biomarqueurs d’exposition plus élaborées pour l’étude de ces composés dans de futures études épidémiologiques. Ces résultats sont aussi pertinents en dehors du contexte de la grossesse et pour d’autres composés non-persistants. De nouvelles approches, telles que le pooling répété pour chaque sujet d’un petit nombre de biospécimens journaliers, validé dans cette thèse, sont nécessaires pour caractériser efficacement l’impact des composés non-persistants sur la santé de l’Homme. / Phenols and phthalates include chemicals widely used in daily-life products, resulting in ubiquitous exposure of the general population. There is growing concern regarding the effects on human health of these compounds, suspected to be endocrine disruptors, particularly during early life. Epidemiological research on the health effects of phenols and phthalates in offspring generally rely on a few biospecimens to assess exposure. These studies are limited by the possibly strong within-subject variability, which may result in exposure misclassification. The within-subject variability in the context of pregnancy and its possible impact on dose-response functions are poorly characterized.The aim of this thesis was to study the exposure to several phenols and phthalates during pregnancy by: 1) investigating the possible associations between this exposure and respiratory outcomes in childhood; 2) characterizing the temporal within-subject variability of these compounds during pregnancy; and finally 3) studying the efficiency of a within-subject pooling approach using a small number of daily biospecimens for exposure assessment.Associations between exposure to phenols and phthalates and respiratory health relied on n = 587 mother-child pairs from the French EDEN prospective cohort. Developments about the assessment of exposure during pregnancy relied on n = 16 pregnant participants of the SEPAGES-feasibility study who had collected all their urine samples for three weeks.This work quantified the within-subject variability of phenol and phthalate biomarker concentrations during pregnancy over various time scales (day to months), and confirmed empirically that this variability is likely to strongly bias the doseresponse functions in human-based epidemiological studies exploring the effects of gestational exposure to these chemicals.This thesis adds to the emerging literature on respiratory health impacts of early-life exposure to several phenols and phthalates. However, as for most studies on the human health effects of phenol and phthalate exposure, it is potentially challenged by this exposure assessment issue. Thus, this work emphasizes the relevance of more elaborate sampling strategies for exposure biomarkers in future epidemiological studies. These results have relevance for studies outside the context of pregnancy, and also for other nonpersistent compounds. New designs, such as the within-subject pooling of biospecimens validated in this study, are needed so as to efficiently characterize the health impact of nonpersistent chemicals.

Cotutelle Québec-France. J'ai effectué une cotutelle de thèse entre l'Université de Montréal (Québec) et l'Université de Poitiers (France). / Les dérivés chlorés du Bisphénol A (ClxBPA) sont des polluants émergents qui possèdent des effets perturbateurs endocriniens. Lors du traitement de l’eau potable par chloration, le Bisphénol A (BPA) présent dans l’environnement peut réagir avec les molécules de chlore pour former des dérivés chlorés (ClBPA, Cl2BPA, Cl3BPA et Cl4BPA) ou ClxBPA. Ils sont présents dans divers milieux aquatiques tels que eaux usées et l’eau potable. Ces polluants émergents ont également été retrouvés chez l’humain, dans l’urine, le colostrum et le tissu placentaire. Dans une étude transversale, la présence de concentrations urinaires élevées de ClxBPA chez l’humain a été positivement associée au diabète de type 2. Les ClxBPA ont aussi été associés à l’obésité et à l’infarctus du myocarde. Dans la littérature, il n’existait que très peu de données sur la toxicocinétique des ClxBPA chez l’animal ou chez l’humain. Cette recherche doctorale vise à évaluer le risque lié à ces polluants émergents en déterminant les constantes métaboliques des ClxBPA, puis en élaborant une méthode analytique des métabolites du 3,3’-Cl2BPA par HPLC-MS/MS pour finalement valider un modèle pharmacocinétique à base physiologique (PBPK) chez l’animal. Dans un premier temps, les constantes métaboliques (Km, Vmax et la clairance intrinsèque) ont été déterminées à l’aide d’expérience in vitro avec des hépatocytes de rats et d’humain. Les essais ont révélé que les constantes métaboliques des ClxBPA peuvent varier considérablement en fonction des substances et des espèces. Ces expériences in vitro ont montré que la chloration avait un impact sur la clairance intrinsèque hépatique des ClxBPA chez les rats et les humains. Deuxièmement, nous avons développé une méthode analytique pour les métabolites du 3,3’-Cl2BPA par HPLC-MS/MS dans le plasma à l’Université de Poitiers. Nous avons pu valider cette méthode avec des échantillons de rats et d’humain. Les résultats ont montré que chez le rat Sprague-Dawley après administration i.v., le 3,3’-Cl2BPA-glucuronide est largement présent par rapport au 3,3’-Cl2BPA-sulfate. Pour l'humain, seul le 3,3’-Cl2BPA-G-d12 a été quantifié. Finalement, nous avons utilisé des méthodes in vitro (dialyse à l’équilibre) et in silico pour les paramètres physico-chimiques de chaque ClxBPA. Enfin, nous avons construit des modèles PBPK afin de simuler la pharmacocinétique i.v. chez le rat Sprague-Dawley pour chaque dérivé chloré. Deux modèles PBPK ont été développés : un avec le ClBPA, le Cl2BPA et le Cl3BPA versus le Cl4BPA avec un modèle à diffusion limitée. Il a été validé par une expérience in vivo sur 80 rats Sprague-Dawley. Le modèle calibré a prédit les concentrations mesurées des ClxBPA dans le plasma, le cerveau et les muscles après l’administration de 4 et 40 mg/kg pour ClBPA et Cl2BPA; 0,4 et 4 mg/kg pour Cl3BPA et Cl4BPA. Ce projet doctoral a contribué à 1) déterminer les constantes métaboliques (Km, Vmax, Clairance intrinsèque) des ClxBPA, 2) de développer et valider une méthode analytique par HPLC-MS/MS des métabolites du Cl2BPA dans le plasma animal et humain et 3) de valider deux modèles PBPK chez le rat afin de connaitre la distribution plasmatique et tissulaire des ClxBPA. / Chlorinated derivatives of Bisphenol A (ClxBPA) are emerging pollutants with endocrine disrupting effects. During the treatment of drinking water by chlorination, Bisphenol A (BPA) present in the environment can react with chlorine molecules to form chlorinated derivatives (ClBPA, Cl2BPA, Cl3BPA and Cl4BPA) or ClxBPA. They are present in various aquatic environments such as wastewater and drinking water. These emerging pollutants have also been found in humans, in urine, colostrum and placental tissue. In a cross-sectional study, high urinary ClxBPA levels in humans were positively associated with type 2 diabetes. ClxBPA has also been associated with obesity and myocardial infarction. In the literature, there was very limited data on the toxicokinetics of ClxBPA in animals or in humans. This doctoral research aims to assess the risk related to these emerging pollutants by determining the metabolic constants of ClxBPA and then developing an analytical method for 3,3'-Cl2BPA metabolites by HPLC-MS/MS to validate a physiologically based pharmacokinetic (PBPK) model in animals. First, metabolic constants (Km, Vmax, and intrinsic clearance) were determined using in vitro experiments with rat and human hepatocytes. The tests revealed that the metabolic constants of ClxBPA can vary considerably between substances and species. These in vitro experiments showed that chlorination impacted the intrinsic hepatic clearance of ClxBPA in rats and humans. Second, we developed an analytical method for 3,3'-Cl2BPA metabolites by HPLC-MS/MS in plasma at the University of Poitiers. We were able to validate this method with rat and human samples. The results demonstrated that in Sprague-Dawley rats after an i.v. administration, 3,3'-Cl2BPA-glucuronide is largely present compared to 3,3'-Cl2BPA-sulfate. For humans, only 3,3'-Cl2BPA-G-d12 was quantified. Finally, we used in vitro (equilibrium dialysis) and in silico methods for the physicochemical parameters of each ClxBPA. Finally, we constructed PBPK models to simulate the i.v. pharmacokinetics in Sprague-Dawley rats for each chlorinated derivative. Two PBPK models were developed: one with ClBPA, Cl2BPA and Cl3BPA versus Cl4BPA with a diffusion- limited model. It was validated by an in vivo experiment on 80 Sprague-Dawley rats. The calibrated model predicted measured ClxBPA concentrations in plasma, brain, and muscle after administration of 4 and 40 mg/kg for ClBPA and Cl2BPA: 0.4 and 4 mg/kg for Cl3BPA and Cl4BPA. This doctoral project contributed to 1) determine the metabolic constants (Km, Vmax, Intrinsic Clearance) of ClxBPA, 2) develop and validate an analytical method by HPLC-MS/MS of Cl2BPA metabolites in animal and human plasma and 3) validate two PBPK models in rats to understand the plasma and tissue distribution of ClxBPA.

Hormonell wirksame Chemikalien, wie Pflanzenschutzmittel oder Pharmaka gelangen durch Abwässer in die Umwelt und akkumulieren vor allem in Oberflächengewässern. Ein erhöhtes Augenmerk liegt auf Substanzen, die durch (anti)androgene und (anti)östrogene Wirkungsweise die Reproduktion von Tieren und Menschen beeinträchtigen. Bei den bisherigen Nachweismethoden für diese Stoffe handelt es sich um invasive Methoden, die das Töten der Tiere beinhalten. Diesen Methoden mangelt es jedoch an der nötigen Sensitivität, um umweltrelevante Konzentrationen der endokrinen Disruptoren (EDs) nach Kurzzeitexposition nachweisen zu können, sowie am Vermögen, alle vier Wirkmechanismen (androgen, antiandrogen, östrogen und antiöstrogen) mit einer einzelnen Testmethode feststellen und unterscheiden zu können. In dieser Studie wurde deshalb mit Hilfe männlicher Afrikanischer Krallenfrösche (Xenopus laevis) eine Testmethode entwickelt, bei der die Frösche verschiedenen (anti)androgenen und (anti)östrogenen EDs ausgesetzt wurden und ihr Rufverhalten untersucht wurde. Diese nicht-invasive Methode erwies sich als schnell und höchst sensitiv. Zudem war es erstmals möglich, die vier verschiedenen Wirkmechanismen allein anhand veränderter Ruftypen und Rufparameter zu bestimmen und zu unterscheiden. Darüber hinaus konnte gezeigt werden, dass bei Anwendung dieser Methode die Möglichkeit besteht, die Versuchstiere in weiteren Tests wiederzuverwenden, da die Rufparameter nach einer expositionsfreien Zeit von sechs Wochen wieder Kontrollwerte erreichten. Zusammengefasst kann die hier vorgestellte verhaltensphysiologische und damit nicht-invasive Methode als Biomarker für den Nachweis von (anti)androgenen und (anti)östrogenen EDs verwendet werden. Ferner zeigt die hohe Sensitivität des Tests, sowie die Möglichkeit der vollautomatischen Analyse enormer Datenmengen, dass dieser schnelle Verhaltenstest ein großes Potential hat, ein sensitiver, standardisierter und nicht-invasiver Biomarker zu werden. / Endocrine disrupting compounds (EDCs), such as herbicides, pesticides or pharmaceuticals enter the environment via sewage effluents and especially accumulate in surface waters. Research efforts so far mainly focused on EDCs with (anti)androgenic and (anti)estrogenic modes of action (MOAs), which can interfere with reproductive biology of vertebrates. To date, biomarkers for the assessment of such compounds are invasive techniques, which are not sensitive enough to detect EDCs after short-term exposures and which cannot distinguish between the four MOAs. Hence, in this study a non-invasive method for the assessment of EDCs was developed using male African clawed frogs (Xenopus laevis) as model species. Frogs were exposed to individual (anti)androgenic and (anti)estrogenic EDCs in the surrounding water and their calling behavior was analyzed. This non-invasive method turned out to be a fast and highly sensitive biomarker for the detection of (anti)androgenic and (anti)estrogenic EDCs. Moreover, this method was able to differentiate between the four different MOAs solely by determining affected parameters of the calling behavior. It was also shown that by using this method, it might be possible to reuse already tested experimental animals, because the measured affected parameters were reversed after a period of six weeks under control conditions. Taken together the here established non-invasive behavioral method can be used as biomarker for the detection of (anti)androgenic and (anti)estrogenic EDCs. Furthermore, the high sensitivity of this testing method, as well as the possibility of analyzing vast datasets rapidly in a completely automated fashion indicate the huge potential for this rapid behavior test to become a sensitive, standardized, non-invasive biomarker.

<p>Uvod: Poslednjih godina u fokusu istraživača je efekat sintetskih jednjenja na endokrini sistem. Estri ftalne kiseline se koriste u procesu plastifikacije, kao industrijski rastvarači, lubrikanti, aditivi u tekstilnoj industriji, u pesticidima, kozmetičkim proizvodima. Raste broj dokaza da je tiroidna žlezda podložna dejstvu endokrinih disruptora. Tiroidni hormoni imaju važnu ulogu u regulaciji rasta, tkivne diferencijacije, energetskog metabolizma, reprodukcije i formiranja centralnog nervnog sistema. Brojna istraživanja ukazala su da ftalati deluju kao EDs. Ciljevi istraživanja: 1. Procena izloženosti populacije mono-etilheksil-ftalatu (MEHP) i mono-etil-ftalatu (MEP). 2. Evaluacija razlika u nivou pokazatelja tirodine funkcije između ftalat pozitivnih i ftalat negativnih ispitanika i između gojaznih i normalno uhranjenih ftalat pozitivnih ispitanika. 3.Utvrđivanje razlika u serumskom nivou leptina gojaznih ispitanika sa i bez pozitivnih ftalatnih metabolita i procena povezanosti leptina sa MEP i MEHP i pokazateljima tiroidne funkcije. Izbor ispitanika i metod rada: Istraživanje je sprovedeno kao studija preseka, obuhvatilo je 201 ispitanika. Ispitanici su podeljeni u grupu MEP/MEHP pozitivnih i negativnih i na podgupe normalno uhranjenih i gojaznih. Od antropometrijskih mera određena je telesna visina, telesna masa, obim struka i indeks telesne mase. Laboratorijske analize: jutarnji uzorak urina za određivanje MEP i MEHP; na&scaron;te uzet uzorka venske krvi za FT4, FT3, TSH i leptin. Statististička analiza sprovedena je na softverskom paketu SPSS. Rezultati: Polovina stanovni&scaron;tva je izložena ftalatima. MEP dovodi do povi&scaron;enja FT4 samo u subpopulaciji gojaznih. Nije utvrđen statistički značajan uticaj MEP na FT3. Kod gojaznih MEP pozitivnih osoba ženskog pola povi&scaron;en je TSH. MEHP uzrokuje sniženje FT4 kod normalno uhranjenih ispitanika, a kod normalno uhranjenih mu&scaron;karaca snižava FT3. Nije utvrđen uticaj MEHP na tirotropin. U gojaznih nije ustanovljen uticaj DEHP i DEP na leptinsku sekreciju.Uočena je tendencija negativne korelacije leptina i FT4 kod gojaznih, dok uticaja na FT3 i TSH nema. Zaključak: Na&scaron;a populacija je u velikoj meri izložena ftalatima. Potvrđeno je da MEP i MEHP imaju uticaj na pojedine indikatore tiroidne funkcije. Ftalati u na&scaron;em istraživanju ne uzrokuju poremećaj leptinske skrecije, a leptin ima blag uticaj jedino na FT4.</p> / <p>Introduction: Effects of synthesized chemicals on endocrine system has been in the focus in the last years. Phthalates are used in plasticization, as industrial solvents, lubricants, textile industry additives, in pesticides and cosmetic products. Evidence for thyroid disruption is growing. Thyroid hormones (TH) have an important role in regulation of growth, tissue differentiation, energy metabolism, reproduction and central nervous system formation. Studies show phthalates can cause endocrine disruption. Aims: 1. Estimation of burden of mono-ethyl phthalate (MEP) and di-2-ethylheksyl phthalate(MEHP) in the population. 2. Evaluation of differences in TH and TSH in MEP/MEHP positive and negative participants, as in obese and lean MEP/MEHP positive participants. 3. Evaluation of differences in leptin in obese MEP/MEHP positive and negative subjects and evaluation of the connection between leptin, MEP, MEHP and thyroid indicators. Patients and methods: This was a cross-sectional study that comprised 201 subjects divided into MEP/MEHP positive and negative group, further subdivided in obese and lean. Anthropometric parameters done: body height, body weight, waist and body mass index. Laboratory tests done: morning urine sample analysis for MEP/MEHP and venous sample analysis for free thyroxine (FT4), free tri-iodothyronine (FT3), thyroid stimulating hormone (TSH) and leptin. Statistical analysis was done in SPSS. Results: Half of subjects were exposed to phthalates. MEP induced an increase in FT4 in obese participants and had no influence on FT3. TSH was increased in obese MEP positive female subjects. MEHP induced a decrease in FT4 in lean participants and a decrease of FT3 in lean males. There was no correlation between MEHP and TSH. Influence of MEP/MEHP on leptin secretion. A tendency for negative correlation between leptin and FT4 was seen. There was no influence of leptin on FT3 and TSH. Conclusion: Our population is greatly exposed to phthalates. MEP and MEHP influence certain thyroid indicators i.e. cause thyroid disruption. Phthalates do not influence leptin secretion in our study. There is a mild effect of leptin on FT4.</p>