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Obesity and the metabolic complications associated with it are increasing globally. Sedentary lifestyles, poor diet and genetic predisposition contribute to obesity. In addition, environmental chemicals such as Bisphenol A (BPA) may play a significant role. Exposure to BPA has been correlated with an array of adverse health effects on the endocrine system and whole-body homeostasis. This has resulted in manufacturers replacing it with structural analogues such as Tetra Methyl Bisphenol F (TMBPF), Bisphenol F (BPF), Bisphenol AP (BPAP), and fluorine-9-bisphenol (BHPF). BPA is a suspected obesogen as it can induce adipogenesis in human and murine preadipocytes. The effects of the BPA analogues listed above on adipogenesis have yet to be evaluated. The aim of this project is to investigate their adipogenic effects. For this purpose, we used 3T3-L1 mouse embryonic fibroblasts. This cell model can be differentiated into mature adipocytes with appropriate inducers including 3-isobutyl-1-methylxanthine (IBMX), insulin and dexamethasone, a synthetic steroid. To assess the effects of BPA analogues, the cells were treated with varying concentrations of TMBPF, BPF, BHPF, BPA, or BPAP in place of dexamethasone. The expression levels of mature adipocyte markers were assessed at mRNA and protein levels to determine the adipogenic potential of the analogues. Lipid accumulation was evaluated by Nile Red staining. A time course was performed to assess the expression levels of known transcriptional regulators of adipogenesis. The results indicate that TMBPF, BPF and BPA increase 3T3-L1 adipogenesis. BHPF and BPAP did not affect adipogenesis in this model. BPF appears to be at least as good as BPA at inducing adipogenesis. TMBPF, on the other hand, can induce adipogenesis to a greater extent than the other chemicals, including BPA, as evidenced by increased expression of adipogenic markers and lipid accumulation. Finally, key transcription factors C/ebpδ and C/ebpα, part of the adipogenic transcriptional cascade, were up-regulated at III two and six hours post-treatment by TMBPF. BPA also up-regulated C/ebpδ at two hours post-treatment. Though the adipogenic effects have become apparent for some of these analogues, the mechanism by which they elicit their effects remains to be discovered. More research is required to deduce the mechanism of action and to provide consensus on what the effects of these replacement bisphenols actually are.

The Assabet River located in eastern Massachusetts receives treated wastewater discharges from four major municipal wastewater treatment plants (WWTPs): Westborough, Marlborough, Hudson and Maynard. In periods of low flow, up to 95% of the Assabet River is wastewater effluent. Chemical analyses have shown that municipal wastewaters can contain estrogenic and dioxin-like compounds. Estrogenic compounds such as the natural estrogen 17β-estradiol (E2), the synthetic estrogen 17α-ethinyl estradiol (EE2), and the industrial compound nonylphenol (NP) can induce vitellogenin (VTG) and lead to feminization in male fish. CYP1A1-inducing compounds such as the polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenyl ethers (PBDEs) can produce both overt toxicity and alter reproductive function through the metabolism of natural estrogens. The purpose of the present research is to analyze the estrogenic and CYP1A1-inducing compounds in the Assabet River and their physiological effects on Japanese Medaka (Oryzias latipes). I used a bioassay that measures the induction of VTG and CYP1A1 in the livers of male Medaka and report results obtained by the USGS and EPA on analytical measurements of selected compounds. In the summers of 2010 and 2011 water samples were collected from the Assabet River, its tributaries and the four WWTPs. Male Medaka were exposed to the treatment samples as well as negative and positive controls. VTG and CYP1A1 induction were measured using real time RT-PCR. Concurrently collected samples from 2010 were analyzed by the USGS for more than 80 organic wastewater contaminants including several estrogenic EDCs and CYP1A1-inducing compounds. The USEPA also analyzed treated wastewater effluent samples collected from the four WWTPs for pharmaceuticals, hormones, nonylphenols and perfluorinated compounds. The bioassay from 2010 and 2011 reveal no statistically significant induction of VTG expression and only one significant induction of CYP1A1 expression. Few compounds were detected by the 2010 USGS and USEPA chemical analyses and the concentrations were low. Taken together the results indicate that VTG and CYP1A1 inducing compounds in the effluent-dominated Assabet River are present at low levels, which may be below the level of detection of the bioassays. In addition to the biological assay and chemical analysis Geographic Information Systems (GIS) was used to analyze land use/land cover (LU/LC) data in the Assabet River Watershed. Much of the land surrounding the Assabet River is forested but there are several LU/LC types that could negatively impact the water quality. High impact and low impact LU/LC types were differentiated in buffers around the Assabet River and six GIS sites. The composition of each site varies widely in its proportions of high and low impact land cover. The GIS analysis established locations on the Assabet River where water quality is more susceptible to degradation due to the distribution of high impact land use types.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting chemical (EDC) of concern due to its persistent nature and widespread presence in the aquatic environment. In mammals, effects of developmental EDC exposure on reproduction and behavior not only persist to adulthood after discontinued exposure, but are also inherited by several consecutive unexposed generations. The results presented in this thesis demonstrate that non-reproductive behavior in fish is highly sensitive to the influence of EE2 during development and the effects do not appear to be restored after a long recovery period in clean water. We have shown that exposure to low doses of EE2 during development results in increased anxiety in two fish species (zebrafish and guppy) and their offspring. We have also demonstrated that the effects of EE2 on anxiety are apparent in both sexes and are transgenerationally transmitted to two consecutive generations of unexposed offspring in the guppy. In order to investigate the possible biological mechanisms of the observed persistent effects on non-reproductive behavior, we also performed an RNA sequencing analysis of the whole-brain transcriptome in developmentally exposed zebrafish after remediation in clean water until adulthood. Differential expression of 33 genes in males and 62 genes in females were observed as a result of EE2 exposure, with only one gene affected in both sexes. Functional analysis revealed cholesterol biosynthesis and circadian rhythm to be the top two affected pathways in males and females, respectively. Both pathways have previously been implicated in anxiety behavior and represent possible candidates connecting the transcriptome alterations to the observed behavioral phenotype. The study represents an initial survey of the fish brain transcriptome by means of RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

Les activités d'élevage sont responsables de la dissémination dans l'environnement d'un grand nombre de polluants, dont les perturbateurs endocriniens (PE) qui affectent le système endocrinien des organismes vivants. La disruption endocrinienne se fait par la liaison des PE sur des récepteurs nucléaires spécifiques dont le récepteur aux strogènes ERα, le récepteur à la dioxine AhR, le récepteur X aux pregnanes PXR, le récepteur aux androgènes AR et le récepteur régulant la prolifération des peroxysomes, PPARγ. Les hormones stéroïdes sont particulièrement actives sur le récepteur ERα. Elles sont naturellement produites par les animaux et concentrées dans les effluents d'élevage épandus sur les sols agricoles. Ce travail de thèse s'est focalisé sur l'étude du devenir des hormones dans des systèmes de gestion des lisiers porcins, à échelle réelle ou contrôliée, en conditions anaérobies ou aérobies. Au préalable, la méthodologie pour l'analyse des hormones dans le lisier a été validée. Les résultats montrent que les hormones sont principalement contenues dans la fraction solide; dans cette fraction les mesures d'activité strogénique (ERα) ont révélé une activité non explicable par les concentrations en hormones, suggérant la présence d'autres composés ayant une activité sur ERα. La quantification des nonylphénols, actifs sur ERα n'a pas permis d'expliquer cette activité. L'étude d'autres voies de perturbation endocrinienne via l'activation des récepteurs AhR, PXR, PPARγ et AR a permis de détecter une forte activité sur le récepteur AhR. L'analyse chimique de cette fraction a montré l'absence de dioxines et d'hydrocarbures aromatiques polycycliques, suggérant l'implication d'autres PE qui restent à être identifiés. Dans les systèmes de traitement sous conditions réelles ou contrôlées, les conditions aérobies sont plus efficaces pour éliminer les hormones et les activités strogéniques et « dioxin-like » associées, bien que cette dernière semble plus récalcitrante. / Livestock activities are responsible for the release of a wide range of pollutants in the environment. Among these pollutants, the endocrine disruptors (ED) can affect the endocrine functions of living organism. The endocrine disruption occurs via the binding of ED to specific nuclear receptors such as the estrogen receptor ERα, dioxin receptor AhR, pregnane-X-receptor (xenobiotics), androgen receptor AR and peroxysome proliferator-activated receptor γ, PPARγ. Steroid hormones are particularly active on ERα receptor. Animals naturally produce high amounts of hormones which are concentrated in manure used for land spreading. This study deals with the fate of hormones in swine waste management facilities at full or pilot scale, under both anaerobic and aerobic conditions. Firstly, the analysis of hormones in such a complex matrix was validated. Hormones are mainly present in the solid fraction of the manure. At the same time, estrogenic activity measurements showed that in this solid fraction, the estrogenic activity cannot be explained by hormones; which suggest the presence of other compounds active on ERα. Nonylphenols concentrations cannot explain this additional activity. The study of the other pathways of endocrine disruption through the activation of AhR, PXR, PPARγ, and AR allowed to measure high AhR activity in the solid fraction of manure. The chemical analysis of this fraction did not show the presence of dioxins and polycyclic aromatic hydrocarbons traditionally involved in such kind of activity; it suggests the presence of other AhR ligands that remain to be identified. In swine manure treatment systems, aerobic conditions were more efficient to remove hormones and the associated estrogenic and AhR activities, even if this last seemed to be more recalcitrant.

A number of chemicals may have a negative impact on the environment and wildlife. Endocrine disruptors (EDs), which can mimic estrogen, interfere with natural hormones in organism and can have a negative effect on the reproductive system. Such substances include zearalenone (ZEA) - mycotoxin, produced by the fungi Fusarium. Despite the non-steriodal structure of its molecule, ZEA and its derivates possess potent estrogenic activity. The influence of ZEA on reproductive parameters and changes in expression of selected genes were tested in the outbred line of mice. This study showed significant effects of ZEA on number of reproductive performances. This effect was observed at lower examined dose, to which are humans normally exposed, and at higher doses, both showed changes in the tested parameters.

[pt] As bifenilas policloradas e os éteres difenílicos polibromados constituem duas classes de poluentes orgânicos persistentes (POPs), altamente lipofílicos, capazes de resistir à biodegradação e bioacumular através da cadeia alimentar. Os PCBs foram utilizados principalmente como fluidos isolantes em capacitores e transformadores, bem como em diversos equipamentos elétricos. Os PBDEs são retardantes de chamas, os quais são aplicados em computadores, televisores e aparelhos elétricos e eletrônicos em geral. As altas concentrações destes poluentes no meio ambiente ocorrem por diversos fatores, dentre os quais podemos citar o descarte de equipamentos eletrônicos obsoletos. Ambos os poluentes são interferentes endócrinos, provocam alterações reprodutivas, possuem efeitos carcinogênicos e são neurotóxicos. Os seres humanos, por ocuparem o topo da cadeia alimentar, estão sujeitos a elevado risco de contaminação por estes compostos. No presente estudo, foram determinados os níveis de PCBs e PBDEs em diferentes espécies de peixes oriundos da Baía da Ilha Grande, localizada ao Sul do Estado do Rio de Janeiro, Brasil. Os PCBs foram os poluentes predominantes e os níveis de PBDEs estão abaixo do limite de quantificação. Os PCBs foram encontrados em três espécies de peixes e as concentrações variaram entre 2,29 e 27,60 ng g(-1) em peso úmido para amostras de músculos e entre 3,41 e 34,22 ng g(-1) também em peso úmido para amostras de fígados. O PCB-87 foi o congênere dominante, onde sua concentração média foi de 34,22 ng g(-1) em peso úmido nas amostras de corvinas. Foram estabelecidas correlações entre as concentrações de PCBs e variáveis biométricas dos indivíduos, tais como comprimento, peso e teor lipídico, bem como a diferenciação estatística em função da sazonalidade. / [en] Polychlorinated biphenyls (PCBs) and Polybrominated Diphenyls Ethers (PBDEs) are two highly lipophilic classes of persistent organic pollutants (POPs) able to resist degradation and with the ability to bioaccumulate through the food chain. PCBs were primarily used as insulating fluids in capacitors and transformers, as well as in several electrical equipments. The high levels of these pollutants in the environment can occur due to several factors, among which the disposal of obsolete electronic equipment. Both pollutants are endocrine disrupters that can cause reproductive alterations. They are neurotoxic and have carcinogenic effects. Humans, by occupying the top of food chain, are subject to a high risk of contamination by these compounds. In this study, we determined the levels of PBDEs and PCBs in different fish species from the Ilha Grande Bay, located in the southern state of Rio de Janeiro, Brazil. PCBs were the predominant pollutants and PBDEs levels were below the limit of quantification. PCBs concentrations were found in the different tissues of fish at concentrations ranging from 2.29 to 27.60 ng g(-1) wet wt in muscles and from 3.41 to 34.22 ng g(-1) wet wt in livers. PCB-87 was the major congener detected, with a mean concentration of 34.22 ng g(-1) wet wt in croaker samples. Correlations were established between the concentration of PCBs and biometric variables of the fish individuals, such as length and fat content, and a statistical variation due seasonality was also observed.

Endocrine disruptors are natural or manmade substances which affect hormonal systems of organisms. Biologically relevant concentrations are commonly being detected in the environment. The effluents of wastewater treatment plant present their significant secondary source. Due to their occurence and quantity the interest in mixtures increases. Ecotoxicological assays with genetically modified Saccharomyces cerevisiae were aplied to verify reliability of predictive mathematical models for mixtures of standards (estrone, 17β-estradiol, 17α-ethinylestradiol, estriol, bisphenol A, irgasan, 4-nonylphenol). Chromatographic analysis along with yeast assays were used for the evaluation of real samples of wastewater treatment plant effluents and sediments. Schindler's predictive model and Full logistic model (FLM) were more reliable for predicting the whole dose-response curve compared to Generalized concentration addition (GCA). Predicted values of a parameter EC50 from all three models were comparable to empirical measurements. Three out of four samples exhibited estrogenic activity 0.65 - 1.70 ng/L 17β-estradiol ekvivalent (EEQ) above the limit of detection 0.13 - 0.33 ng/L EEQ. Antiestrogenic activity was detected in one of the samples. Prediction could be carried out only in the case of the sediments...

A poluição marinha por petróleo e seus derivados é uma preocupação crescente de caráter global. O litoral norte do estado de São Paulo é hoje caracterizado como a região mais altamente impactada por esses contaminantes de toda a costa Sul e Sudeste do país. Dentre as substâncias que compõem esses produtos estão os Hidrocarbonetos Aromáticos Policíclicos (HPAs), como o fenantreno, que apresentam potencial de alterar o sistema endócrino de peixes, gerando desajustes hormonais de alto risco para o processo reprodutivo dos animais expostos. Com base nestes fatos, o presente trabalho buscou verificar efeitos do fenantreno como desregulador endócrino em juvenis de E. marginatus. Essa espécie se caracteriza pelo hermafroditismo protogínico, de modo que os juvenis costumam apresentar altas concentrações de esteroides gonadais, o que gera preocupação quanto os efeitos reprodutivos decorrentes de alterações no padrão de produção de esteroides, especialmente em uma espécie que já vem sofrendo declínios populacionais por conta da sobrepesca. A CL50 encontrada para juvenis de E. marginatus foi 1,51 mg⁄L. Em seguida, foi realizado um bioensaio subletal (96h) com quatro grupos experimentais (n=5) em duplicata: grupo controle; duas concentrações distintas de fenantreno (0,1 mg⁄L e 1 mg⁄L) e grupo exposto ao veículo de diluição (0,004% de etanol). Foram dosadas as concentrações de 17β-estradiol (E2), testosterona (T) e 11-cetotestosterona (11-KT) plasmáticos por ELISA. Gônadas, fígado e baço foram processados para análise histológica. Foi conduzido bioensaio in vitro com incubação de fragmentos gonadais sob ação de fenantreno (50μM) ou etanol. Os níveis dos mesmos esteroides também foram dosados por ELISA. A exposição in vivo ao fenantreno resultou em aumento na área dos hepatócitos, bem como no número de centro melanomacrofágicos e hemossiderose no baço. O etanol induziu efeitos similares no baço. Os níveis de E2 e T não se alteraram no plasma ou no meio de incubação in vitro. O fenantreno reduziu a concentração de 11-KT in vitro e in vivo. No plasma, o etanol também causou esse decréscimo. Considerando a importância do 11-KT no desenvolvimento dos ovários em teleósteos, o fenantreno parece causar desregulação da esteroidogênese em juvenis de E. marginatus, possivelmente gerando disfunções durante a inversão sexual / Marine pollution by crude oil and its residual products is a growing global concern. The Northern coast of São Paulo is characterized as the highly impacted area by these contaminants through Brazil\'s South and Southeast coast. Polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, are the main crude oil components and have a significant toxic potential to biota, acting as endocrine disruptors (ED) and negatively impacting reproduction. This study aimed to investigate the effects of phenanthrene as ED on gonadal steroidogenesis in E. marginatus juveniles. As a hermaphrodite protogynous species, E. marginatus juveniles maintain high levels of plasma steroids as substrates for sex inversion, which causes concern about reproductive effects of disruptions in steroid production patterns, especially in an overfished species. LD50 to E. marginatus exposed to phenanthrene was established in 1.51 mg⁄L. An in vivo sublethal exposure (96 h) to phenanthrene was carried out at two concentrations (0.1mg⁄L and 1 mg⁄L); exposure to the vehicle (ethanol) was also performed. Plasma levels of 17β-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by ELISA. Gonads, liver and spleen were processed for histological analysis. In an in vitro bioassay, gonad fragments were incubated with phenanthrene (50μM) or ethanol. Steroid levels in the culture media were also measured by ELISA. The in vivo exposure to phenanthrene triggered an increase of the area of the hepatocytes, increased number of melanomacrophagic centers and hemosiderosis in the spleen. Ethanol also induced similar effects on spleen. E2 and T levels did not change neither in plasma nor in vitro media. Phenanthrene sharply reduced 11-KT levels in vitro and in vivo. In plasma, ethanol also decreased 11-KT levels. Considering the importance of 11-KT in fish developing ovaries, phenanthrene seems to disrupt steroidogenesis in juvenile grouper, possibly being able to cause dysfunctions during sex change