Orientador: Wilma De Grava Kempinas / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T22:18:22Z (GMT). No. of bitstreams: 1 Guerra_MarinaTrevizan_D.pdf: 6009222 bytes, checksum: 1ca90ae1ef82035a0e6930b277dac172 (MD5) Previous issue date: 2013 / Resumo: Os desreguladores endócrinos são substâncias químicas que podem mimetizar ou antagonizar os hormônios endógenos, alterando o equilíbrio hormonal necessário para o desenvolvimento correto. Sabe-se que a exposição de organismos a agentes hormonalmente ativos durante períodos críticos do desenvolvimento pode levar a alterações permanentes detectadas somente na vida adulta. Em várias espécies animais estes compostos podem atingir o feto via placenta ou a prole, pelo leite materno. Embora pesquisas iniciais tenham focado em estrógenos e antiandrógenos ambientais, a presença de compostos com atividade androgênica tem sido descrita como contaminante em rios e animais destinados a alimentação. Vários estudos demonstraram que a exposição de animais a altos níveis de andrógenos durante o período perinatal pode causar alterações morfológicas, bioquímicas e funcionais permanentes em vários órgãos e sistemas. A exposição perinatal a andrógenos exógenos pode causar masculinização fisiológica e comportamental em fêmeas e levar a alterações reprodutivas em machos. O objetivo deste trabalho foi avaliar os possíveis efeitos da exposição a um agente androgênico sobre o sistema genital e a função reprodutiva de animais expostos durante períodos críticos de desenvolvimento. Para tanto, ratas prenhes foram alocadas em quatro grupos experimentais: controle, que recebeu somente óleo de milho (veículo), e tratados com propionato de testosterona nas doses de 0,05mg/kg, 0,1mg/kg, ou 0,2mg/kg. Os tratamentos foram realizados por via subcutânea, do dia gestacional 12 ao final da lactação. A prole feminina foi avaliada quanto ao número de células germinativas fetais, peso corpóreo e distância anogenital no dia pós-natal 1, número de mamilos, idade de instalação da puberdade, histologia uterina e ovariana, dosagens hormonais, comportamento sexual, fertilidade, níveis de receptores esteroides uterinos através de imunohistoquímica, assim como taxa de proliferação/morte celular uterina e testes uterotróficos. Nos filhotes do sexo masculino foram avaliados, na idade adulta e meia idade, a histologia testicular e epididimária, contagens, morfologia e motilidade espermáticas, dosagens hormonais, comportamento sexual e fertilidade. Os resultados demonstraram que em fêmeas, a exposição perinatal ao propionato de testosterona ocasionou alteração na intensidade de marcação dos receptores esteroides uterinos em todas as doses testadas, diminuição nos índices de proliferação e morte celular no tecido uterino nas doses de 0,1 e 0,2mg/kg e aumentou a predisposição uterina a responder a estímulos estrogênicos na dose de 0,1mg/kg e 0,2mg/kg. Entretanto, estas alterações não foram capazes de prejudicar a diferenciação sexual feminina e a fisiologia normal do sistema genital feminino. Em relação aos filhotes do sexo masculino, a exposição perinatal a andrógenos provocou redução na produção e nas reservas espermáticas na idade adulta, sem, contudo, alterar a fertilidade destes animais após acasalamentos naturais. Podemos concluir que a exposição perinatal a andrógenos, nas doses utilizadas neste experimento, provocou o aparecimento de alterações tardias no sistema genital feminino como modificações nos receptores esteroides uterinos e anormalidade na resposta a estímulos estrogênicos e, no sistema genital masculino, alterações na produção espermática. Estas repercussões, entretanto, não foram capazes de alterar o desempenho geral de fertilidade em ambos os sexos / Abstract: Endocrine disruptors are chemicals that may mimic or antagonize endogenous hormones, altering the critical hormonal balance required for proper health and development. It is well known that exposure of organisms to some hormonally active agents during critical periods of development imprints permanent changes that can be detected later in adulthood. In many animal species these substances reach the fetus via placenta and/or the offspring via the mother's milk. Although first researches have focused on environmental estrogens and antiandrogens, androgenic activity has been described as contaminant in rivers and beef cattle. Previous studies demonstrated that exposure of experimental animals to high levels of androgens during perinatal age causes permanent morphological, biochemical, and functional alterations in several organs and systems. Perinatal exposure of female rodents to exogenous androgens results in both physiological and behavioral masculinization and causes reproductive disruption in male rodents. The aim of this study was to assess the possible effects of an androgenic compound on genital system and reproductive function of animals exposed during critical periods of development. Pregnant female rats were allocated into four experimental groups: control, which received corn oil (vehicle), and treated with testosterone propionate at doses of 0.05mg/kg, 0.1mg/kg and 0.2mg/kg. Treatments were performed subcutaneously, from gestational day 12 until the end of lactation. The female offspring was evaluated for fetal germ cell number, body weight and anogenital distance at post-natal day 1, number of nipples, puberty onset, histology of reproductive organs, hormonal levels, sexual behavior, fertility test, immunohistochemistry of steroid receptors, proliferation/apoptotic index, uterotrophic test and uterine stimulation with estrogens. On male offspring, at adult age and middle-age, histology of testis and epididymis, sperm counts, morphology and motility, hormonal levels, sexual behavior and fertility test were evaluated. Results demonstrated that, in females, the perinatal testosterone propionate exposure caused an alteration in the pattern of uterine steroidal receptors imunostaining in all tested doses, a decreased proliferation/apoptotic index in the uterine tissue at 0.1 and 0.2mg/kg and a greater predisposition from uterus in responding to estrogens stimulation at dose of 0.2mg/kg. However, these alterations were not capable of impairing female sexual differentiation and normal physiology of female genital tract. On male offspring, the perinatal androgenization provoked a reduction in sperm production and reserves at adult age, without altering fertility after natural mating. We can conclude that perinatal exposure to androgens, at doses used in this experiment, provoked the appearance of late alterations in the female genital system as modifications in the uterine steroidal receptors and abnormalities in uterine response to estrogenic stimulation, and in male genital system, as spermatic production impairment. These repercussions, however, were not capable to alter the general fertility performance in both sexes / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

No description available.

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...