Orientador: Rafael Henrique Nóbrega / Resumo: A decisão sobre se uma gônada bipotencial se desenvolverá em um testículo ou em um ovário é considerado um estágio crítico na diferenciação sexual dos vertebrados. A administração de esteróides exógenos durante este período pode afetar essa plasticidade, promovendo a diferenciação sexual na direção feminina ou masculina. Dessa forma, o objetivo desta tese foi avaliar os efeitos do tratamento de 17β-estradiol no desenvolvimento de Astyanax altiparanae (lambari), através de análises histológicas e de análises de expressão genica de possíveis genes envolvidos em vias masculinas e femininas. Para isso, larvas com gônadas indiferenciadas foram alimentadas com Artemia contendo diferentes concentrações de estradiol durante 28 dias, desde o 1 dia pós-eclosão (dpe) até o período que precede a diferenciação gonadal. Nossos resultados mostraram que o E2 modificou o fenotípo e a relação sexual histológica e, surpreendentemente, induziu intersexo com com a presença de óvulos nos testículos nas concentrações de 2 e 6 mg de E2/kg de alimento. Esses dados são uma evidência clara de que o tratamento utilizado não foi suficiente para induzir a reversão completa do sexo em A. altiparanae. Em termos de expressão gênica, o tratamento com E2 (6 mg/kg de alimento) produziu uma notável plasticidade gonadal entre machos e fêmeas aos 90 dias após a eclosão (dph). Os machos, denominados “machos resistentes ao estradiol”, superexpressaram os genes masculinos, como dmrt1, sox9 e amh. Dessa forma, nó... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The decision whether a bipotential gonad will become a testis or ovary is considered a critical stage in vertebrate sex determination. Administration of exogenous steroids can affect this plasticity by skewing the sex gonadal differentiation towards a male or female. The aim of this study is to evaluate the effects of 17β-estradiol (E2) diet on Astyanax altiparanae (lambari) development, focusing on the gonadal development and gene expression analysis of possible candidate genes involved in either male or female pathways. Larvae with undifferentiated gonads were fed with steroid diet containing different concentrations of E2 during 28 days, from the mouth opening until a period that precedes the gonadal differentiation. Animals were sampled at 90 days post-hatching (dhp) for histology and gene expression analysis. Our results showed that E2 disrupted both phenotypic and histological sex ratios, and surprisingly, induced intersex with testis-ova in the concentrations of 2 and 6 mg E2/Kg food. This data is a clear evidence that the treatment used was not enough to induce complete sex reversal in A. altiparanae. However, in terms of gene expression, E2 (6mg/Kg food) induced a remarkable gonadal plasticity between males and females at 90 dph. The males, named as E2 resistant males, overexpressed the male-biased genes, such as dmrt1, sox9 and amh. We suggested that these males were able to resist the E2-induced feminization by the expression of genes related to testis differentiat... (Complete abstract click electronic access below) / Doutor

Nomegestrolacetat (NOMAC) är ett progestin, selektivt till progesteronreceptorn, med antigonadotropiska och antiandrogena egenskaper. Det används bland annat i kombination med det naturligt förekommande östrogenet 17β-estradiol (E2) för antikonception eller vid postmenopausal behandling. Det föreligger alltid en förhöjd trombosrisk vid hormonterapi, men NOMAC/E2 tros medföra en lägre risk jämfört med andra terapier. Syftet med detta arbete var därför att undersöka om trombosrisken är högre med NOMAC/E2 vid hormonterapi jämfört med andra terapier. Metoden för arbetet var litteraturstudier. Sökningar gjordes genom PubMed i databasen Medline och sex studier valdes ut. NOMAC/E2 visade sig påverka hemostatiska biomarkörer och lipider i lägre utsträckning än levonorgestrel/etinylestradiol (LNG/EE). SHBG (könshormonbindande globulin) ökade däremot mer med NOMAC/EE än med LNG/EE. Den fördelaktiga effekt, som E2 har på endotelcellernas kväveoxidbildning, påverkades inte av NOMAC, och LNG visade liknande egenskaper. Medroxiprogesteron hämmade istället denna antitrombotiska effekt. Utifrån detta arbete kan ingen slutsats dras om trombosrisken är större eller mindre med NOMAC/E2 jämfört med andra terapier. För att kunna göra detta krävs det stora epidemiologiska studier. Däremot kan trombosrisken med NOMAC/E2 anses vara jämförbar med dagens förstahandsval vid kombinerad antikonception som är LNG/EE. Trombosrisken med NOMAC jämfört med MPA kan möjligtvis vara lägre, men detta behöver undersökas i större kliniska studier.

Objectives: Breast cancer resistance protein (BCRP) confers resistance to certain anticancer drugs such as mitoxantrone, topotecan and SN-38. A putative estrogen response element (ERE) was located in the promoter region of the BCRP gene. The present study aimed to investigate whether human BCRP expression is regulated pretranscriptionally by 17β-estradiol. Methods: Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a control constitutive cytomegalovirus (CMV) promoter, respectively, which were transfected into estrogen receptor α (ERα)-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Results: 17β-Estradiol significantly upregulated BCRP mRNA and protein expression in a dose-dependent manner, and the effect was abolished by the antiestrogen tamoxifen. Furthermore, electrophoretic mobility shift assays demonstrated that the putative ERE in the promoter region of the BCRP gene and ERα are essential for transcriptional activation of BCRP by 17β-estradiol. Conclusions: Taken together, our findings indicate that BCRP expression is upregulated by 17β-estradiol via a novel pretranscriptional mechanism which might be involved in 17β-estradiol-ER complexes binding to the ERE of BCRP promoter via the classical pathway to activate transcription of the BCRP gene.

The project involved the study of 12 Victorian municipal wastewater treatment plant discharges. These included lagoon-based plants and those with activated sludge based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected in November 2003, and then again in April and June 2004, and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and the measurement of specific hormonal concentrations (17-estradiol) using enzyme-linked immunosorbent assays (ELISA). Almost all of the effluents examined showed estrogenic activity, to a greater or lesser extent (no response to 55 ng/L 17β-estradiol equivalents). On the whole, the levels of estrogenic activity observed were to the lower end of the range observed overseas in the northern hemisphere, and comparable with that recently reported in Australia and New Zealand using similar, human-estrogen receptor based assays (no response to ~ 10 ng/L 17β-estradiol equivalents). The reassuring low/no assay response is bolstered by the chemical assessment of estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part in the range 2-5 ng/L. From an aquatic environmental perspective, it is difficult to say with any certainty what the potential risk to aquatic organisms in waters receiving these effluents will be. Typically, in environmental risk assessment one first looks to agreed national or international guideline or trigger values for the type of waters being assessed. In this case, there are as yet no guideline values. Without guideline values to drive the assessment, then one compares a chemical’s concentration in a sample (in this case a WWTP effluent) with data obtained from toxicological experiments in which the concentration known to elicit a specific effect has been determined. In this case, levels of 17β-estradiol were typically between the lowest reported level to induce the production of Female-indicative proteins in male fish (plasma vitellogen; 1 ng/L), and the lowest concentration of known to induce intersex in fish (8 ng/L). Consequently, such levels in a WWTP discharge are likely to be an environmental risk if there is little or no dilution of the discharge by the receiving water, i.e. discharge represents major component of stream flow. In short, to truly assess the risk (hormonal impact) of these WWTP effluents, in vivo testing needs to be undertaken, ideally with a representative native species but failing that with a ‘standard’ species such as the fathead minnow. When this programme began, the ‘watching brief’, being held in Australia on the topic of endocrine disrupting chemicals and their potential effects on aquatic wildlife was considered too passive by many. It still is, by some. Despite the assurance the results may provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground, reassurance research to provide data for higher-level risk assessment by industry and government agencies.

Tests were conducted for 71 pesticides in weekly bulk (wet + dry) deposition samples collected from May 25 to September 21 over two years at two sites in the City of Winnipeg, Canada. Twenty-one pesticides and their metabolites were detected in this study and 99% of samples collected contained mixtures of two or more pesticides. Malathion and glyphosate were the largest contributors to bulk deposition in 2010 and 2011, respectively. A second study examined the mineralization of 2,4-D and 17β-estradiol using a novel in-field soil microcosm study and a series of laboratory experiments under different temperature incubations. Results indicated that temperature fluctuations do not greatly affect the amount or rate of mineralization relative to the traditionally constant laboratory incubations of 20°C; however long-term freezing of soil reduced potential mineralization over time. This research advances scientific knowledge of agri-chemical fate and provides data for strengthening current environmental policy analysis in Canada.

Tests were conducted for 71 pesticides in weekly bulk (wet + dry) deposition samples collected from May 25 to September 21 over two years at two sites in the City of Winnipeg, Canada. Twenty-one pesticides and their metabolites were detected in this study and 99% of samples collected contained mixtures of two or more pesticides. Malathion and glyphosate were the largest contributors to bulk deposition in 2010 and 2011, respectively. A second study examined the mineralization of 2,4-D and 17β-estradiol using a novel in-field soil microcosm study and a series of laboratory experiments under different temperature incubations. Results indicated that temperature fluctuations do not greatly affect the amount or rate of mineralization relative to the traditionally constant laboratory incubations of 20°C; however long-term freezing of soil reduced potential mineralization over time. This research advances scientific knowledge of agri-chemical fate and provides data for strengthening current environmental policy analysis in Canada.

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No. of bitstreams: 1 Dissertação - Vanessa Ribeiro Reis.pdf: 1868086 bytes, checksum: 7062926683a60bb93f40e764881fe486 (MD5) Previous issue date: 2015-08-21 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The tambaqui Colossoma macropomum is the main farmed fish among the Brazilian native species. The female tambaqui stands out because it grows 18% more than the male in intensive farming fish system (unpublished data). When monosex cultivation is more profitable is fish industry, techniques of sex reversal can be used for directing the sexual differentiation towards to desired gender. The objective of this research was to identify the best dose of 17β-estradiol (E2) for direct feminization of tambaqui, as the first step in developing a protocol for effective sex reversal for the specie. Due to experimental control and to avoid the possible genetic effects on the results, the study was divided into two tests (September / October and November / December 2014). In every test, it was used 500 tambaqui fries (100 fries/treatment) of 30 days post hatch (dph) with an average length 14.87 ± 2.85 mm. The fries were fed diets containing different doses de E2 (0 - control, 20, 40, 80 and 120 mg/kg of E2 diet) for six weeks. After the treatments, the fish were transferred to cages where they remained growing for the sampling. Sixty days after the end of the treatment, blood sample was collected to determine the concentration of E2 in plasma. Sampling of the gonads was performed when the fish was around seven months. In the animals with standard length (SL) mean of 13.96 ± 1.29 cm it was not possible to identify the sex, due to lack of histological evidence of ovaries or testes. In the individuals in which sex could be determined, E2 demonstrated great influence on the increase in females at the highest dose. The E2 concentration in the plasma of the animals showed that this hormone was fully metabolized by the fish body, as treated (20, 40 and 80 mg/kg E2 feed) and control presented similar plasma E2 values. However, in the treatment 120 mg of E2 kg of feed the animals presented a lower plasma concentration and statistically different from the control and other treatments. This suggests that the higher dose of E2 was not only fully metabolized by the organism, but also decreased the endocrine production of this hormone (negative feed-back). The water of the treatment was analyzed before and after coloration. Results showed that there was a residual presence of E2 after the experiment which disappeared after chlorine. Our results indicate that 120 mg/kg E2 of diet administered for six weeks is the most effective treatment for tambaqui feminization from 14 mm length, and that water chloration after estradiol treatment eliminates every residue of this steroid. / O tambaqui Colossoma macropomum é o peixe mais produzido em cativeiro dentre as espécies nativas brasileiras. A fêmea se destaca por pesar cerca de 18% a mais que o macho em sistema de cultivo intensivo (dados não publicados). Quando as características zootécnicas de um gênero são mais rentáveis que de outro é comum o cultivo monosexo. As técnicas de inversão sexual podem ser utilizadas no direcionamento da diferenciação sexual para o gênero desejado. Este trabalho teve como objetivo identificar a melhor dose de 17β-estradiol (E2) para a feminização direta de tambaqui. O experimento foi dividido em dois ensaios (setembro/novembro e outubro/dezembro de 2014). Para cada ensaio utilizou-se 500 pós-larvas de tambaqui (100/tratamento), com trinta dias pós-eclosão (dpe) e com comprimento médio 14,87 mm. Foram alimentadas com dietas contendo diferentes doses de E2 na ração (0, 20, 40, 80 e 120 mg/Kg) por seis semanas. Após os tratamentos os peixes foram transferidos para tanques-rede onde permaneceram até a realização da amostragem. Sessenta dias após o tratamento foi realizada coleta de sangue. A coleta das gônadas foi realizada quando os peixes tinham entre cinco e sete meses. Nos animais com comprimento padrão (CP) médio de 13,96 ± 1,29 cm não foi possível a identificação do sexo, devido à ausência de evidências histológicas comprovatórias de ovários ou testículos. Nos animais sexados, o E2 demonstrou grande influência no aumento de fêmeas na dose mais elevada. A concentração de E2 no plasma dos animais mostrou que este esteroide foi totalmente metabolizado pelo organismo dos peixes, sendo estatisticamente igual para 20, 40 e 80 mg E2/Kg de ração e o controle. Para dose de 120 mg/Kg os animais apresentaram uma concentração plasmática de estradiol significativamente menor que o controle e demais tratamentos. Sugerindo que para essa concentração o organismo dos peixes não só metabolizou todo o esteroide consumido, mas também diminuiu a produção endógena desse hormônio. As análises da água utilizada nos tratamentos, antes e após cloração, demonstraram a eficiência deste método na eliminação total dos resquícios hormonais de estradiol. Nossos resultados mostraram que 120 mg de E2 por quilograma de ração, administrados durante seis semanas, é o tratamento mais eficaz para a feminização de larvas de tambaqui a partir de 14 mm de comprimento. E a cloração da água após o tratamento com estradiol é 100% eficaz na eliminação de resíduos deste esteroide.

Exposure to exogenous estrogens such as synthetic 17α-ethinyl estradiol (EE) occurs via multiple sources (i.e. hormonal contraceptives, environmental contamination, hormone replacement therapy). The natural estrogen, 17β-estradiol (E2), is a well-studied immunomodulatory hormone at both environmental and pharmacologic levels. Conversely, little data exist regarding the immune effects of EE at either environmentally-relevant exposure levels or at pharmacological levels. Further, EE is delivered to patients in a clinical setting via different routes of exposure (e.g. subcutaneous or oral). Many key questions in relation to potential immunological effects of EE are unanswered. Important variables in estrogen-modulation of the immune system include: (i) dose, (ii) age, (iii) gender, and (iv) route of exposure. Thus, pertinent questions emerge. Does exposure to EE at low concentrations for a subacute duration affect the immune or reproductive systems? Are the effects similar in both hormones and between sexes? Are these effects similar in juvenile and aged mice? How do the effects compare across two common routes of exposure (subcutaneous versus oral)? To address these questions, three separate studies were performed. In the first study, we investigated whether very low, but environmentally relevant, doses of EE, E2 (10 ng/kg body weight), or vehicle orally administered every other day for 21 days to young (6 week-old) and aged (>15 month-old) C57BL/6 mice had immunomodulatory effects. As expected, significant gender and age-related differences were noted with regard to thymus weight, thymocyte recovery, spleen weight, and splenocyte recovery. However, low dose treatment of either E2 or EE had no marked effects on the thymus or spleen organ to body weight ratios, cell numbers, or lymphocyte subsets. Low dose oral estrogens did not alter the ability of activated splenocytes to induce interferon-γ or nitric oxide. No effects on male reproductive organ to BW ratios of young or aged mice were found. Similarly, with the exception of E2-stimulating effects on the female reproductive tract of young mice, there were no pronounced effects in females. In separate studies, intact juvenile female and male C57BL/6 mice were given daily subcutaneous (second study) or oral (third study) doses of either EE or E2 (0.04, 0.4, or 4.0 μg per 25 g BW) for 21 days. In the subcutaneous exposure study, both EE and E2 morphologically altered uterine and seminal vesicle weights. However, EE had a more pronounced effect compared to E2, especially in males, even at the lowest dose administered. Additionally, like E2, EE induced thymic atrophy in both sexes. In female mice, thymic atrophy and thymic cellularity were significantly decreased by subcutaneous EE and E2 at doses of 0.4 and 4.0 μg/25 g body weights. EE elicited significantly more pronounced thymic atrophy-inducing effects compared to E2 at the 4.0 μg/25g dose. In males, thymocyte cellularity was decreased by both subcutaneous EE and E2 only at the highest dose tested (4.0 μg/25 g body weight), whereas only 4.0 EE significantly decreased thymus to body weight ratios. Neither splenic weights, splenic cellularity, nor splenic cell phenotype were affected by either estrogenic compound regardless of route of exposure. Oral exposure of EE or E2 did not induce marked immunological effects. Collectively, these data demonstrate that select thymic and reproductive endpoints are significantly altered following a 21-day subcutaneous exposure to either EE or E2 and that the thymus is a more sensitive target than the spleen with regard to subacute exposure to EE. In addition, EE at a comparable dose was more potent than E2 at exerting thymic and reproductive organ morphological alterations. Furthermore, route of administration is critical, as subcutaneous exposure induced far more dramatic thymic and reproductive morphological alterations than did oral administration. Future studies need to address the precise mechanism through which EE induces thymic atrophy and diminished thymus cellularity. Are these effects mediated directly through the thymus, perhaps through estrogen-induced increased thymocyte apoptosis or alterations to thymic epithelial cells? Or could EE be mediating alterations via bone marrow stem cells targeted for distribution to the thymus? Our novel findings regarding EE-induced effects on the thymus are of health significance and set the stage for future work. / Master of Science

No / Wound infection is a major clinical problem, yet understanding of bacterial host interactions in the skin remains limited. Microbe-derived molecules, known as pathogen-associated molecular patterns, are recognised in barrier tissues by pattern-recognition receptors. In particular, the pathogen-associated molecular pattern, lipopolysaccharide (LPS), a component of microbial cell walls and a specific ligand for Toll-like receptor 4, has been widely used to mimic systemic and local infection across a range of tissues. Here we administered LPS derived from Klebsiella pneumoniae, a species of bacteria that is emerging as a wound-associated pathogen, to full-thickness cutaneous wounds in C57/BL6 mice. Early in healing, LPS-treated wounds displayed increased local apoptosis and reduced proliferation. Subsequent healing progression was delayed with reduced re-epithelialisation, increased proliferation, a heightened inflammatory response and perturbed wound matrix deposition. Our group and others have previously demonstrated the beneficial effects of 17β-estradiol treatment across a range of preclinical wound models. Here we asked whether oestrogen would effectively promote healing in our LPS bacterial infection model. Intriguingly, co-treatment with 17β-estradiol was able to promote re-epithelialisation, dampen inflammation and induce collagen deposition in our LPS-delayed healing model. Collectively, these studies validate K. pneumoniae-derived LPS treatment as a simple yet effective model of bacterial wound infection, while providing the first indication that oestrogen could promote cutaneous healing in the presence of infection, further strengthening the case for its therapeutic use.

With advantages of high cation exchange capacity, swelling-shrinking property and large specific surface area, monmtorillonite is chosen as a carrier and modified with Fe3+ saturation for estrogen decontamination. 17β-Estradiol (βE2) has highest estrogenic activity among estrogens and is selected as representative compound. Rapid βE2 transformation in the presence of Fe3+ - saturated montmorillonite in aqueous system was observed and βE2 oligomers were the major βE2 transformation products. About 98% of βE2 were transformed into oligomers which are >107 times less water-soluble than βE2 and therefore are much less bioavailable and mobile. Fe3+ -saturated montmorillonite catalysis achieved highest βE2 removal efficiency at neutral solution pH and higher temperature. Common cations did not have impact on the reaction efficiency. Dissolved organic matter slightly reduced βE2 removal efficiency. Regardless of wastewater source, ~40% βE2 removal efficiency was achieved for wastewater effluents when they were exposed to same dosage of Fe3+ -saturated montmorillonite as that for simple water systems which achieved ~83% removal efficiency. For real wastewater that contained higher organic matter, higher dosage of Fe3+ -saturated montmorillonite would be needed to create available reaction sites for βE2. This thesis also reports that Fe3+ -saturated montmorillonite effectively deactivate wastewater microorganisms. Microbial deactivation rate was 92±0.6% when secondary wastewater effluent was mixed with Fe3+ -saturated montmorillonite at 35 mg/mL for 30 min, and further increased to 97±0.6% after 4-h exposure. Freeze-drying Fe3+ -saturated montmorillonite iii after each usage resulted in 82±0.5% microbial deactivation efficiency even after fourth consecutive use. For convenient application, Fe3+ -saturated montmorillonite was further impregnated into filter paper through wet-end addition and formed uniformly impregnated paper. Scanning electron microscopy (SEM) imaging showed Fe3+ -saturated montmorillonite was evenly dispersed over cellulose fiber surface. When filtering 50 mL and 200 mL water spiked with live Escherichia coli (E. coli) cells at 3.67×108 CFU/mL, Fe3+ -saturated montmorillonite impregnated paper with 50% mineral weight loading deactivated E. coli with 99% and 77%, respectively. Dielectrophoresis and impedance analysis of filtrate confirmed that the deactivated E. coli passing through Fe3+ -saturated montmorillonite paper did not have trapping response due to higher membrane permeability and conductivity. The results demonstrate feasibility of using Fe3+ -saturated montmorillonite impregnated paper for convenient point-of-use drinking water disinfection. / Ph. D. / In this thesis, Fe³⁺-saturated montmorillonite was produced in an eco-friendly way to serve as cost-effective material for both efficient estrogen removal and microbial deactivation from wastewater. 17β-Estradiol (βE2), a common estrogen compound, was quickly removed by Fe³⁺- saturated montmorillonite and the transformation products could be easily settled down from wastewater and became less bioavailable. Fe³⁺-saturated montmorillonite also demonstrated durability over different environmental conditions in wastewater and still achieved satisfied βE2 removal efficiency. Moreover, Fe³⁺-saturated montmorillonite could rapidly deactivate the microbes in wastewater effluent and can be promising wastewater disinfection method in the future. Fe³⁺ -saturated montmorillonite immobilized filer paper was also produced and has great potential to be used as a cost-effective filtration purifier for safe drinking water.