▼ Perfluoroalkyl substances (PFASs) are widespread industrial pollutants that are extremely persistent in the environment. Animal studies have indicated that in-utero PFAS exposures can affect fetal growth, but findings from human studies are inconclusive. Few human studies have sufficient sample size to study the influence of PFASs on adverse birth outcomes. Here, we conducted a pooled analysis using data of 3,535 mothers and infant pairs using three sub-samples originating from the Danish National Birth Cohort (DNBC), and we evaluated the associations between prenatal PFASs exposures and birth outcomes. Maternal plasma concentrations of six types of PFASs in early pregnancy (around 8.7 gestational weeks) were studied. We found that each LN-ng/ml increase in PFOS, PFOA, PFNA and PFHpS was associated with a 65g, 51g, 52g or 56g decrease in birth weight. Moreover, we also found that prenatal PFOS, PFHpS, PFDA levels were associated with the risks for preterm birth (< 37 completed gestational week). Our findings strengthen the evidence that in-utero PFAS exposures may affect fetal growth. These findings raise concerns considering the ubiquitous contamination of PFASs in the environment. Public health strategies to prevent or lower PFASs exposures in pregnant women are needed.

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Interferentes endócrinos são substâncias que podem causar efeitos deletérios na saúde, crescimento e capacidade reprodutiva dos organismos, e colocam em risco a continuidade de várias espécies. Suas fontes são as mais variadas possíveis, sendo possível detectá-los em alguns alimentos, produtos farmacêuticos, produtos químicos e seus respectivos subprodutos de degradação. Também vêm sendo detectados em várias matrizes ambientais, tais como o ar, solo, água e sedimentos. Devido as suas capacidades de ação sinérgica, aditiva e antagônica é extremamente complexa a determinação da relação dose-resposta, informação esta necessária para a definição de limites de padrões de qualidade e medidas de controle e monitoramento. A partir da avaliação do estado da arte relativa ao tema foram apresentados os mais atuais instrumentos de gestão ambiental e suas respectivas aplicações. Finalmente, foi realizado teste visando detectar a eventual presença de efeito estrogênico em amostras de dois sistemas de grande importância para a Região Metropolitana de São Paulo: a água da Represa Guarapiranga e o efluente da Estação de Tratamento de Esgotos Barueri. Os testes realizados foram do tipo in vivo, onde peixes machos da espécie Cyprinus carpio foram expostos a amostras destes dois sistemas e foi investigada a presença da proteína vitelogenina no plasma sanguíneo em concentrações anômalas. Verifica-se a necessidade premente de maiores estudos que foquem o efeito dos interferentes endócrinos na saúde e no meio ambiente, através de ensaios biológicos, de forma a municiar a sociedade de informações que permitam definir medidas estratégicas de atuação para minimização do impacto desta poluição.

Endocrine disrupters are substances that can play a role of deleterious efects in organisms health, growth and reproductive capability. They can be found in food, pharmaceuticals, chemical products and by-products and in diverse environmental matrixes, as air, soil, water and sediments. Because their ability to sinergistic, additive and antagonistic action, it is difficulty to determine the dose-response relationship, which is necessary for establishment of quality standards and monitoring measures. From the assessment of state-of-art about endocrine disrupters were present the most current environmental tools and applications. Finally, one test was performed to detect the possible presence of estrogenic effect in samples of two systems located in Metropolitan Region of São Paulo, from Guarapiranga Reservoir and the effluent from Wastewater Treatment Plant in Barueri. The tests in vivo were with male fish of the species Cyprinus Carpio, and investigated the presence of protein vitellogenin in blood plasma and in anomalous concentrations. There is a need for more studies that focus on the effect of endocrine disrupters on human health and the environment, by means of biological tests, in order to arm the society with informations to set strategic measures of performance for minimizing the impact of this kind of pollution.

Advisors/Committee Members: Mucci, José Luiz Negrão.

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Recentemente, pesquisas vêm claramente demonstrando que xenobióticos podem apresentar efeitos deletérios sobre o sistema endócrino e passam a ser de grande importância toxicológica à medida que se tornam contaminantes ambientais. Tais substâncias passaram a ser denominadas de desreguladores endócrinos (DEs). Entre os diferentes DEs identificados, muitos são praguicidas, amplamente utilizados no meio agropecuário em todo o mundo. Atualmente, já são bem estabelecidas as metodologias para avaliação do potencial efeito DE de diferentes substâncias em roedores; porém uma busca na literatura deixa clara a falta de estudos e de protocolos para avaliação de DEs em animais de produção ruminantes, os quais certamente estão expostos a um grande número de agentes tóxicos com estas características. Assim, foi objetivo desta pesquisa dar início ao desenvolvimento de um protocolo para avaliação de efeitos DEs de xenobióticos em ruminantes. Para tal, estudou-se o bisfenol A (BPA), o qual é, classicamente, um DE para roedores. Além disto, avaliou-se o potencial efeito DE da I. carnea, uma planta tóxica de grande importância para ruminantes no Brasil e em outros países. O estudo foi realizado em duas etapas, na primeira avaliou-se os efeitos do BPA e da planta nos caprinos machos púberes; na segunda etapa propôs-se avaliar os efeitos tóxicos da exposição in utero de caprinos machos púberes a estas mesmas substâncias. Os resultados obtidos mostraram que os bodes púberes expostos ao BPA apresentaram redução significante dos níveis séricos de tiroxina, e da qualidade espermática. Também observou-se nestes mesmos animais degeneração vacuolar na rete testis. Já os caprinos machos púberes que receberam a I. carnea apresentaram aumento significante do numero de espermatozoides com alterações morfológicas e degeneração vacuolar testicular. Ainda, estes mesmos animais apresentaram diminuição estatisticamente significante dos níveis séricos dos hormônios tireoidianos (T3 e T4). O protocolo empregado para avaliação dos efeitos da exposição in utero a I. carnea ou ao BPA em machos púberes, não evidenciou nenhuma alteração hormonal e/ou reprodutiva. Conclui-se que o protocolo empregado para avaliação de possíveis efeitos DEs de xenobióticos, em ruminantes foi eficaz, no entanto, futuras metodologias deverão ser introduzidas neste protocolo, para detectar alterações mais sutis no sistema endócrino.

Recently, many studies clearly showed that xenobiotics may have deleterious effects on the endocrine system. These substances are termed endocrine disruptors (EDs), and have great toxicological significance when they become environmental contaminants. Many of EDs are identified as pesticides, which were widely used in agriculture and livestock worldwide. Currently, there are already well-established methodologies for assessing the potential effect of endocrine disruptor (ED) of different substances in rodents; however, a literature search shows clearly the lack of studies and protocols for assessing EDs in ruminant livestock, which are indubitably…

Advisors/Committee Members: Górniak, Silvana Lima.

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Endocrine Disrupter Compounds (EDCs) like pesticides, alkylphenols and hormones among other classes of compounds are responsible for alterations in the endocrine system functions. Endocrine disruption occurs when EDCs interact with the hormone receptors, altering the natural response patterns of the endocrine system. Aquatic organisms, such as fish, are able to accumulate EDCs residues in concentration several times higher than the surrounding water. Consequently, fish is a major source of contamination for both top predators and human consumers. This study aimed to develop and validate a fast method for the determination of 40 endocrine disrupters in fish fillet, using modified QuEChERS and Gas Chromatography coupled with Mass Spectrometry in tandem, using a triple quadrupole analyzer (GC-(TQ)MS/MS). A factorial design was performed to optimize the extraction procedure, which consist of the extraction of 10.0 g of fish fillet with 10.0 mL of acetonitrile with 1% (v/v) of acetic acid followed by manual shaking for 1 min. In the partition step 2.0 g of sodium chloride, 1.7 g of anhydrous sodium acetate and 0.3 g of anhydrous magnesium sulfate were added, followed by shaking and centrifugation at 3400 rpm for 8 min. Then the extract was transferred to a 15 mL polypropylene tube containing the C18 and PSA sorbents, beyond the anhydrous magnesium sulfate. After shaking and centrifuging, the supernatant was filtered and analyzed by GC-(TQ)MS/MS. The method was validated by spiking the blank sample at three concentration levels (10, 25 and 50 Ug kg-1) obtaining recovery values between 70.1 to 120.0% for 36 of the 40 endocrine disrupters evaluated. RSD values below 20% ensured the good precision of the method. The linearity was evaluated using the coefficient of determination (r2) with values higher than 0.996 for all compounds. The matrix effect was evaluated, and it was pretty intense for most compounds, with values exceeding 10%. This effect was compensated using analytical curves obtained with standards prepared in blank matrix extracts. This method was applied in 6 samples of fish fillet from different species. Residues of bisphenol A, chlorpyrifos and bifenthrin were detected. Therefore, the modified QuEChERS method proved to be appropriated for the determination of endocrine disrupters in fish fillet using GC-(TQ)MS/MS since it showed to be effective and can be applied to routine analysis.

Compostos conhecidos como disruptores endócrinos (DEs), tais como agrotóxicos, alquilfenóis, hormônios, dentre outras classes de substâncias são responsáveis pela alteração de funções do sistema endócrino. Esse efeito ocorre quando os DEs interagem com os receptores hormonais, alterando os padrões de resposta natural do sistema endócrimo. Os organismos aquáticos, como os peixes são capazes de acumular concentrações de DEs várias vezes superiores a água ao seu redor. Consequentemente, os peixes são uma importante fonte de contaminação para seus predadores e consumidores humanos. O presente trabalho teve como objetivo desenvolver…

Advisors/Committee Members: Carin von Mühlen, Vania Lucia Loro, Renato Zanella.

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En dépit de la pression règlementaire croissante pour mieux détecter les produits chimiques potentiellement responsables de perturbations de la fonction thyroïdienne, les moyens de les cribler restent insuffisants et imparfaits. Un tel criblage implique l’utilisation de nombreux tests in vitro ciblant chacun l’un des divers mécanismes de toxicité potentiellement impliqué, ou des tests in vivo plus longs et plus contraignants. Trois modèles d’étude ont ainsi été évalués pour leur potentiel à simplifier les procédures de criblage actuellement préconisées. Le premier modèle d’étude qui a été proposé est un modèle in vivo d’exposition à court terme par voie orale de rats mâles adultes. Le suivi de l’expression de gènes permet de réaliser un criblage de la perturbation de la fonction thyroïdienne après 7 jours d’exposition seulement. Le second modèle d’étude qui a été retenu pour ce travail est la lignée cellulaire PCCl3 de thyrocytes de rats, identifiée comme un modèle potentiel pour le criblage de la toxicité thyroïdienne. Enfin, la pertinence pressentie de l’utilisation d’un modèle d’hépatocytes cryopréservés (LiverbeadsTM) a été confirmée pour l’identification des composés toxiques thyroïdiens chez le rongeur dont l’action est médiée par le foie. Trois modèles d’étude in vitro et in vivo utilisables lors des tests de criblage des produits chimiques perturbateurs de la fonction thyroïdienne ont donc été identifiés. L’évaluation de cette perturbation est rendue possible par l’utilisation de variations d’expression de gènes comme principal paramètre d’étude

Whereas disruption of thyroid hormone signaling by environmental chemicals is a growing concern, identifying thyroid toxicants in early screening studies remains a challenge. Indeed, several different molecular events can initiate a such disruption of the Hypothalamus-Pituitary-Thyroid (HPT) axis. Thus, the detection of thyroid toxicants implies to conduct systematically an important battery of in vitro assays, or long in vivo studies. In the aim to simplify the procedure for the screening of all the possible mode of actions (MoAs) involved, we assessed three models for their suitability to detect in a short term different thyroid toxicants by using changes in gene expression. We identified the possibility of discriminating thyroid toxicants from compounds that do not alter thyroid function, by using this approach, in a 7-day adult male rat assay. Regarding in vitro testing, we have established the proof of concept of discriminating direct-acting thyroid toxicants from compounds that do not affect thyroid function, on the basis of up-regulated expression of a set of genes as criteria of positivity, in an early screening in vitro assay based on PCCl3 rat thyroid cells. We also confirmed the relevance of the use of alginate-embedded cryopreserved hepatocytes (LiverbeadsTM) for the screening of liver-mediated thyroid toxicity in rodents. The present work allowed the identification of three models suitable for the early screening of the disruption of the thyroid…

Advisors/Committee Members: Cupo, Anny (thesis director).

▼ Background: The rising incidence of breast cancer suggests lifestyle and environmental causes. During the last decade many chemicals, among which parabens and aluminium, have been shown to express estrogenic properties in in-vitro and animal studies. A causative relationship theory between chemicals and breast cancer has been proposed. Another theory has linked the use of underarm cosmetics with the increased prevalence of breast cancer in the upper outer quadrant.Purpose: We conducted this study to investigate the presence and distribution of five commonly used esters of para-hydroxybenzoic acid and aluminium, in the female breast and their possible relationship with breast cancer development. We attempted to shed light into the link hypothesis between underarm cosmetics and breast cancer. Materials and methods: Fourty breast cancer patiens who would undergo mastectomy, completed the study questionnaire with information about underarm cosmetics use and other lifestyle and epidemiological parameters. Histological information was retreived from their medical records. We obtained tissue samples from four different regions across every mastectomy specimen, from the axilla to the sternum and analysed them for parabens and aluminium.Results: Parabens and aluminium were found intact in almost all samples. The distribution in the four regions across the breast from the axilla to the sternum was homogenous and independent of the patient’s age, tumor location and hormone receptor status. There were no significant differences in the concentrations between women who had used underarm cosmetics and those who had not, or those who had used them in the past but have now stopped using them. There was no correlation between the length of underarm cosmetic use and the the concentration of parabens and aluminium across the breast.Discussion: This is the first study to demonstrate the presence of five commonly used p-hydroxybenzoic acid esters and aluminium in the healthy female breast tissue. The measured concentrations are low and the pattern of distribution is homogenous but universal in the studied population, thus allowing the expression of their weak estrogenic properties for a lifelong period. We did not find a causative relationship with breast cancer. However, our findings can be added to those from other in-vitro, animal and human studies on the presence, properties, and combined effect of several widely used chemicals with hormonal properties. In the light of this relatively recent evidence there is an urge for developing biomonitoring techniques, determining the total chemical burden, and investigating the mixture effects in humans. Prevention policies should support the reduction of exposure to artificial chemicals, especially to those categorised as endocrine disrupters and for vulnerable groups.Conclusions: Parabens and aluminium can be found intact in the healthy female breast. Underarm deodorants or antiperspirants do not constitute the main source of parabens and aluminium for the female breast and cannot be… Advisors/Committee Members: Barr, Lester.

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Os desreguladores endócrinos são compostos micropoluentes de ação deletéria que causam efeitos aos animais e aos seres humanos. Estes vêm trazendo a várias décadas, problemas relacionados à má formação ou ainda infertilidade de várias espécies. A presença destes compostos na ordem de ng L-1 já é capaz de gerar efeitos nocivos ao sistema endócrino. Este trabalho visou à determinação da presença e quantidade de alguns destes compostos como 17α-etinilestradiol, bisfenol A e estrona entre outros, nas águas do rio Arroio Fundo, localizado em Jacarepaguá no RJ que possui um índice de qualidade de água ruim. A primeira etapa deste trabalho foi a implementação e validação de metodologia para determinação do 17α-etinilestradiol por cromatografia líquida acoplada a detector de fluorescência onde foram estabelecido limite de detecção, de quantificação e linearidade conforme orientação do Inmetro (2010). Além da avaliação das amostras ambientais através da metodologia estabelecida. Na etapa subsequente foram realizados os experimentos relativos à determinação dos desreguladores endócrinos através da espectrometria de massas por detector tipo tandem quadrupolo, onde também foram realizados os procedimentos de validação e determinação dos compostos bisfenol A e estrona que não foram suscetíveis a efeito de matriz durante a ionização, que acabou impedindo a determinação de outros desreguladores endócrinos. Os resultados indicam que não há redução da presença dos desreguladores endócrinos detectados, e de outros compostos orgânicos também detectados, mas não identificados, entre os pontos a montante e a jusante da Unidade de Tratamento do Rio Arroio Fundo. Todavia em função do grande efeito de matriz oriundo do fato da amostra ser de extrema complexidade, não foi possível realizar uma identificação e quantificação de todos os compostos alvos.

Endocrine disrupters are compounds micropollutants of deleterious effects that cause effects to animals and humans has brought these problems to several decades related to poor training or infertility of various species. The presence of these compounds in the ng L-1 order is already capable of generating negative effects on the endocrine system. This work aimed at determining the presence and amount of some of these compounds as 17α-ethinylestradiol, bisphenol A and estrone among others, in the waters of the river Arroio Fundo, located in Jacarepaguá in Rio de Janeiro that has a bad water quality index. The first stage of this work was the implementation and validation of a method for determination of 17α-ethinylestradiol by liquid chromatography-fluorescence detection which were established limit of detection, quantification and linearity as directed by Inmetro (2010). Besides the evaluation of the samples through the established methodology. In subsequent step were carried out experiments on the determination of endocrine disruptors by tandem quadrupole mass spectrometry detector, were being performed validation procedures and determination of the compounds bisphenol A…

Advisors/Committee Members: Daniele Maia Bila, Eduardo Monteiro Martins, Alexsandro Araújo da Silva, Glauco Favilla Bauerfeldt, Simone Carvalho Chiapetta.

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Un certain nombre d'études ont étudié le lien entre cancer du sein et exposition aux dioxines et au cadmium. Toutefois, les résultats de ces études ne sont pas concluants et présentent des limites méthodologiques. L'objectif principal de cette thèse était de développer un indicateur spatial permettant d'estimer les expositions atmosphériques chroniques aux dioxines et au cadmium des femmes issues de la cohorte E3N en France, entre 1990 et 2008. Nous avons créé une base de données de 2620 sources de dioxines et 2700 sources de cadmium et estimé et géolocalisé leurs émissions. L'estimation des émissions à la source et leur localisation ont servi de base pour la construction de l'indicateur spatial. Nous avons identifié une sélection de paramètres permettant d'obtenir un accord « substantiel » entre les classifications des expositions des femmes de la cohorte E3N, localisées à l'adresse de résidence, estimées avec l'indicateur, et avec le modèle gaussien SIRANE. En utilisant le modèle SIRANE, nous avons également étudié la variabilité spatio-temporelle (1990-2008) des concentrations de dioxines et de cadmium sur la métropole de Lyon. Pour la première fois des concentrations modélisées ont été comparées à des mesures de concentrations de dioxines en air ambiant. L'indicateur développé dans cette thèse a été utilisé pour estimer le risque de cancer du sein associé à l'exposition atmosphérique aux dioxines dans une étude cas-témoins niché au sein de la cohorte E3N. Il est utilisé actuellement dans une étude sur le lien entre exposition au cadmium et risque de cancer du sein, et pourra être appliqué dans de futures études portant sur d'autres polluants ou d'autres pathologies

A number of studies have examined the link between breast cancer and exposure to air pollution, including dioxins and cadmium. However, the results of these studies are inconclusive and present a number of methodological limitations. The main objective of this thesis was to develop a spatial indicator to assess chronic atmospheric exposure to dioxins and cadmium of women from the E3N cohort in France between 1990 and 2008. We first performed an inventory and created a database of 2620 dioxins and 2700 cadmium emitting sources in France between 1990 and 2008 and estimated and geolocated their annual emissions. The location of the sources and their estimated emissions were used as the basis for the construction of the indicator. Combination of additional spatial parameters, allowed us to obtain a "substantial" agreement between the dioxin and cadmium exposure classifications of the E3N subjects geolocalised at their residential address, using the estimated bythe indicator and exposures estimates derived from the Gaussian model. We also evaluated the spatial-temporal variability of dioxin and cadmium concentrations over nearly two decades in the Lyon metropolitan area, taking into account a wide variety of source types. This is the first study comparing concentrations predicted by a dispersion model to dioxin concentrations measured in ambient air. The…

Advisors/Committee Members: Fervers, Béatrice (thesis director), Salizzoni, Pietro (thesis director).

▼ Thyroid hormones are essential in processes of growth and development especially in the development of the central nervous system. Malfunction in thyroid hormone synthesis or decrease in the availability of thyroid hormones can lead to diseases as for example congenital hypothyroidism. In this thesis genetic variations (mutations in the thyroid peroxidase gene, mutations in the monocarboxylate transporter 8 gene) as well as exogenic factors (endocrine disrupters) were examined, leading to dysfunction in thyroid hormone synthesis as well as malfunction in the availability of thyroid hormones. In a total of 39 patients with congenital hypothyroidism and an orthotopic thyroid gland mutations in the thyroid peroxidase (TPO) gene could be identified by single strand conformational polymorphism, denaturing high pressure liquid chromatography and sequencing. Two of these patients have a mutation in exon 14 of the TPO gene and showed an additional phenotype in developing neoplasia. Another defect affecting the availability of thyroid hormones especially in early brain development could be referred to a mutation in the thyroid hormone specific monocarboxylate transporter 8 (MCT8). MCT8 was recently identified to transport triiodthyronine (T3) into cells. The index patient is a hemizygous carrier of the mutation A150V leading to a new syndrome of X-linked mental retardation with striking biochemical thyroid hormone levels corresponding neither to congenital hypothyroidism nor to thyroid hormone resistance. Functional characterization by in-vitro T3 uptake-assays and subcellular localisation studies showed that the mutated MCT8 (A150V) transporter was no longer able to transport T3 into the cells. In addition to genetic defects in the TPO and MCT8 gene influences of exogenous factors on thyroid hormone availability were observed. The influence of endocrine disrupting agents on the function of the TPO were identified using in vitro assays with cells stably expressing recombinant human TPO. Advisors/Committee Members: n (gender), Prof. Dr. med. A. Grüters-Kieslich (firstReferee), Prof. Dr. med. Chr. Schöfl (furtherReferee), Prof. Dr. T. Schöneberg (furtherReferee).

▼ Many of the natural, pharmaceutical and industrial chemicals that are present in the aquatic environment can interfere with the endocrine system of fish. There is a demand for sensitive and robust test systems for risk assessment of the endocrine disrupting chemicals (EDC). The aim of this thesis was to develop a partial life-cycle test to be used for evaluation of EDC. The test was based on exposure of juvenile zebrafish to EDC during the period of gonad transformation and differentiation. The main endpoints studied were the induction of the estrogenic biomarker protein vitellogenin (Vtg) and gonad development. Zebrafish were exposed to single model substances to investigate if exposure during the period of gonad transformation and differentiation affects Vtg production and gonad development. Exposures to the synthetic estrogen 17α-ethinylestradiol (EE2) resulted in a dose-dependent increase in Vtg production, as well as female-biased sex ratios, including all-female stocks. Exposures to the androgens 17α-methyltestosterone (MT) and 17β-trenbolone (Tb) resulted in decreased Vtg concentrations, as well as male-biased sex ratios, including all-male stocks. In a comparative study with Japanese medaka a higher sensitivity of both Vtg and sex ratio of the zebrafish was shown after exposure to EE2 and Tb. Exposures to binary mixtures of the differently acting substances EE2 and Tb demonstrated that effects on both Vtg and gonad development can be detected. Application of this zebrafish partial life-cycle test on a chemically complex pulp mill effluent revealed both estrogenic and androgenic effects. In conclusion, the zebrafish respond to EDC at environmentally realistic concentrations. The results support that zebrafish is a suitable model species for evaluation of EDC.