A Síndrome dos Ovários Policísticos (PCOS) é a endocrinopatia mais frequente em mulheres na idade reprodutiva, além de ser a causa mais comum de hiperandrogenismo e anovulação crônica. Diferentes fenótipos da PCOS foram identificados, e um melhor entendimento dessas diferentes apresentações clínicas se faz necessário para o reconhecimento de riscos, medidas preventivas e terapêuticas específicas para cada fenótipo. Associações entre polimorfismos de substituição de um único nucleotídeo (SNPs) em genes que codificam enzimas responsáveis pelo metabolismo androgênico e PCOS foram descritos. O anticoncepcional oral (ACO) é utilizado para o tratamento de mulheres com PCOS por seu efeito supressivo na secreção de androgênios ovarianos e melhora do hirsutismo. Entretanto, os dados na literatura são conflitantes quanto aos efeitos do ACO nos parâmetros metabólicos de mulheres com PCOS. Além disso, não está bem estabelecido se a presença de alelos polimórficos está associada com diferenças nos fenótipos da PCOS e se pode influenciar na resposta ao tratamento com ACO. Os objetivos desta tese foram investigar a influência dos SNPs -71 AG no gene AKR1C3 e SNP50 no gene CYP19 gene (substituição de G por A) na resposta de mulheres com PCOS ao tratamento com ACO e verificar se o SNP50 está associado com fenótipos da PCOS. Cento e sessenta e duas mulheres com PCOS foram estratificadas em PCOS clássicas (hiperandrogenismo e disfunção ovulatória, c-PCOS) e PCOS ovulatórias (hiperandrogenismo, ciclos ovulatórios, aparência policística dos ovários, ov-PCOS) e uma subamostra de 51 mulheres (que não apresentavam resistência insulínica evidente) completaram 6 meses de tratamento com ACO (20 ug etinilestradiol e 75 ug gestodeno, 21/28 dias por ciclo). A presença ou ausência dos alelos polimórficos foram consideradas para expressar os resultados que avaliaram os SNPs -71 AG e SNP50. O escore de hirsutismo foi similar em c-PCOS e ov-PCOS, e as diferenças nos parâmetros hormonais e metabólicos observadas foram independentes da presença do alelo A do SNP50. Após os 6 meses de tratamento com ACO, como era esperado, os níveis de testosterona total e o escore clínico de hirsutismo diminuíram, enquanto os níveis da globulina carreadora de hormônios sexuais aumentaram. Houve uma pequena redução da pressão arterial sistólica e do hormônio luteinizante. As medidas de insulina e do índice HOMA permaneceram inalteradas após o tratamento. Houve um aumento dos níveis de lipídios, mas os valores permaneceram dentro dos limites da normalidade. Nenhuma das alterações observadas esteve associada com a presença dos alelos polimórficos dos SNPs -71 AG ou SNP50. As conclusões são de que o ACO é uma alternativa eficaz para o tratamento dos sintomas do hiperandrogenismo, sem comprometimento dos parâmetros metabólicos, pelo menos naquelas mulheres sem resistência insulínica prévia ao tratamento. Os SNPs -71AG no gene AKR1C3 e SNP50 no gene CYP19 não contribuem para as melhoras observadas com o uso do ACO. Além disso, o SNP50 parece não estar associado com as diferenças existentes entre os fenótipos clássico e ovulatório da PCOS. / Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women at reproductive age, and also the most common cause of hyperandrogenism and chronic anovulation. Different phenotypes of PCOS have been identified, and a better knowledge of these clinical symptoms is necessary to recognize risks, prevention, and treatment strategies for each phenotype. Associations between single nucleotide polymorphisms (SNPs) in genes that codify enzymes responsible for the androgenic metabolism and PCOS have been described. The oral contraceptive pill (OCP) is used to treat women with PCOS due to the suppressive effect on ovarian androgen secretion, with consequent amelioration of hirsutism. However, data are conflicting in literature regarding the effects of OCP on metabolic variables in PCOS. Besides that, it is not well established whether the presence of polymorphic alleles is associated with PCOS phenotypes and whether can influence on the response to OCP treatment. The aims of this thesis were to investigate the influence of the SNPs -71 AG at AKR1C3 gene and SNP50 of CYP19 gene (G to A substitution) on the response of PCOS to treatment with oral contraceptive pills and to assess whether the SNP50 is associated with PCOS phenotypes. A hundred sixty two hirsute women were stratified into a classic PCOS group (hyperandrogenism and ovulatory dysfunction, c-PCOS) and an ovulatory PCOS group (hyperandrogenism, ovulatory cycles, polycystic ovaries, ov-PCOS), and a subsample of 51 women (without evidences of insulin resistance) completed a 6-month OCP trial (20 ug ethinylestradiol plus 75 ug gestodene, 21/28 days per cycle). We considered the presence or absence of the polymorphic alleles to express results and to perform the comparisons regarding the SNPs -71 AG and SNP50. Hirsutism score was similar in c-PCOS and ov-PCOS, and the differences in hormone and metabolic variables between phenotypes were independent of the presence of allele A for SNP50. After 6 months of OCP treatment, as expected, total testosterone and hirsutism score declined, while sex hormone binding globulin increased. There was a slight reduction in systolic blood pressure and luteinizing hormone levels. Insulin and homeostasis model assessment remained unchanged after treatment. There was an increase in lipids, but the values remained at the normal range. None of these changes were associated with the presence of polymorphic alleles for -71 AG or SNP50 polymorphisms. We conclude that OCP is an alternative to ameliorate androgenic symptoms without compromising metabolic parameters, at least in women without insulin resistance before treatment. The -71AG SNP of AKR1C3 gene and the SNP50 of CYP19 gene did not contribute to the improvements observed. Besides that, SNP50 may not be associated to the existing differences between classic and ovulatory PCOS phenotypes.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

The disposal of waste from agricultural activities has been recognised as a source of environmental contamination by endocrine disrupting chemicals (EDCs). The New Zealand dairy industry produces a large volume of dairy farm effluent, which contains EDCs in the form of estrogens. Most of this dairy farm effluent is applied onto the land for disposal. Groundwater and soil contamination by estrogens following waste application on the land have been reported overseas, but our understanding of the processes and factors governing the fate of estrogens in the soil is poor. Therefore the main goal of the present study was to better understand the fate and transport of estrogens, in particular 17β-estradiol (E2) and estrone (E1) in soil. In order to quantify E1 and E2 in drainage water and soil samples, chemical analysis by gas-chromatography mass-spectrometry (GC-MS) was carried out. This included sample extraction, sample clean-up through silica gel and gel permeation chromatography, and sample extract derivatisation prior to analysis. In order to develop a reliable method to extract estrogens from soil, research was conducted to optimise E1 and E2 extraction conditions by adjusting the number of sonication and shaking events, as well as the volume and type of solvent. Among five solvents and solvent mixtures tested, the best recovery on spiked and aged soil was obtained using an isopropanol/water (1:1) mix. A microcosm experiment was carried out to determine the dissipation rates of E2 and E1, at 8°C and at field capacity, in the Templeton soil sampled at two different depths (5-10 cm and 30-35 cm). The dissipation rates decreased with time and half-life values of 0.6-0.8 d for E1 and 0.3-0.4 d for E2 were found for the two depths studied. A field transport experiment was also carried out in winter, over three months, by applying dairy farm effluent spiked with estrogens onto undisturbed Templeton soil lysimeters (50 cm in diameter and 70 cm deep). The hormones were applied in dairy farm effluent at 120 mg m⁻² for E2 and 137 mg m⁻² for E1. The results of the transport experiment showed that in the presence of preferential/macropore flow pathways 0.3-0.7% of E2 and 8-13% of E1 was recovered in the leachate at the bottom of the lysimeters after 3 months, and 1-7% of the recovered E2 and 3-54% of the recovered E1 was leached within 2 days of application. These results suggest that leaching of estrogens via preferential/macropore flow pathways is the greatest concern for groundwater contamination. In the absence of preferential/macropore flow pathways, a significant amount (> 99.94%) of both hormones dissipated in the top 70 cm of soil, due to sorption and rapid biodegradation. Surprisingly, in all cases, estrogen breakthrough occurred before that of an inert tracer (bromide). This could not be explained by the advection-dispersion transport of estrogens, nor by their presence as antecedent concentrations in the soil. It was therefore suggested that colloidal enhanced transport of estrogens was responsible for the earlier breakthrough of estrogens and caused the leaching of a fraction of the applied estrogens to a soil depth of 70 cm. A two-phase model, adapted from a state-space mixing cell model, was built to describe the observed estrogen transport processes under transient flow. The model takes into account 3 transport processes namely, advection-dispersion, preferential/macropore flow and colloidal enhanced transport. This model was able to successfully describe the estrogen transport observed from the lysimeters.

Indiana University-Purdue University Indianapolis (IUPUI) / 17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH.