Phyto-estrogens are known to bind to estrogen receptors of the uterus and they caninitiate the early events of estrogen stimulation including water imbibition and synthesis of induced protein as well as late events such as uterine growth. There is indirect evidence that these compounds affect the functioning of the hypothalamus and pituitary with respect to the release of gonadotropins. The purpose of this study was to determine if the phytoestrogen compounds, genistein and coumestrol, could interact with the estrogen receptor molecules in the cytoplasm of pituitary and hypothalamus tissue from ewes. Estrogen binding characteristics were also examined. Cytosol preparations from the various experimental, tissues were incubated fifteen minutes at thirty degrees C. with ³H-estradiol; separation of bound from free label was carried out on Sephadex LH-20 columns. Estrogen binding parameters were determined by double reciprocal plots. Competitions with ³H-estradiol in the presence of either coumestrol or genistein were carried out in a similar manner. Apparent inhibition constants (K[sub I]) were determined from Dixon plots. The.apparent dissociation constants (K[sub D]) for estradiol in ewe pituitary cytosol was determined to be 0.26±0.12 nM. The apparent for K[sub I] coumestrol in the ewe pituitary cytosol was determined to be 59-61 nM and the apparent K[sub I] for genistein was determined to be 130-210 nM. These compounds were shown to displace estradiol from receptors in ewe hypothalamus and amygdala cytosols. Results of preliminary binding experiments with ewe pineal and uterus cytosols are also presented. These results suggest that phyto-estrogens can interfere with normal estrogen feedback mechanisms with respect to gonadotropin release in the ewe. / Land and Food Systems, Faculty of / Graduate

Estrogenic endocrine disrupting chemicals (EEDCs) are defined as chemicals that bind to estrogen receptors (ERs) and augment estrogenic functions, either through promoting or blocking estrogen receptor signaling. Recent reports highlight the growing concern surrounding environmental exposure to EEDCs and immune system modulation. A commonly prescribed EEDC, 17α-ethinyl estradiol, is a synthetic analog of 17β-estradiol (E2), and is also found in many environmental reservoirs of human and animal exposure. Little is known regarding the immunomodulatory effects of this EEDC. Autoimmune diseases, such as systemic lupus erythematosus (SLE), are characterized by a dysregulated immune system that has lost tolerance to self-antigens. The pathogenesis of SLE is still poorly understood. However, it is likely that genetics, epigenetics, hormones, and environmental factors, such as EEDC exposure, contribute to the pathogenesis and severity of SLE. The work presented in this dissertation focused on investigating the immunomodulatory effects of exogenous estrogens in mouse models of SLE. Chapter 1 describes an overview of environmental endocrine disruptors and autoimmune disease, with a particular emphasis on estrogens. Chapter 2 represents a review of the current and pertinent literature surrounding the contributions of sex differences, hormones, and EDCs to the induction of autoantibodies and development of autoimmunity, as well as the contributions of anti- microbial responses to SLE. We explored the contribution of dietary components to SLE disease severity. Mice fed a diet devoid of exogenous phytoestrogens developed significantly reduced glomerulonephritis and glomerular immune complex deposition compared to mice fed a diet containing soy isoflavones. Diet also influenced cytokine production and epigenetics of LPS-stimulated splenic leukocytes. We identified similar effects of E2 and EE implantation with regards to innate immunity, and distinct cellular subset, cytokine production profiles, gene expression, and epigenetic responses between E2 and EE treated NZB/WF1 mice. Oral exposure to a very low human relevant dose of EE promoted glomerulonephritis and augmented responses to viral and bacterial mimics in MRL/lpr mice. Overall, our findings suggest that chronic exposure to environmental EEDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals and have greatly expanded the current body of knowledge surrounding 17α-ethinyl estradiol. / Doctor of Philosophy / Chemicals that can disrupt the normal effects of hormones are termed endocrine disrupting chemicals (EDCs). Estrogenic EDCs promote or suppress the ability of estrogen receptors to carry out normal functions within the body. Normal immune system functions require a fine balance of inflammatory and anti-inflammatory cellular responses. This delicate balance is a prime target for dysregulation by EDC exposure. Autoimmune diseases, such as systemic lupus erythematosus, are characterized by a loss of immune tolerance to ones’ own cells and tissues. There is a lack of knowledge surrounding the immunomodulatory effects of a commonly prescribed EDC, 17α-ethinyl estradiol, especially as it pertains to autoimmune disease patients. The aim of this dissertation work is to investigate the immunomodulatory effects of exogenous EDC exposure in mouse models of SLE. We found that MRL/lpr mice fed a diet devoid of phytoestrogens had reduced kidney disease and immune-complex deposition and had augmented cytokine response and epigenetics in LPS-stimulated splenic leukocytes compared to mice fed a diet high in isoflavones. We next compared the immunomodulatory effects of chronic pharmacologic dose exposure to 17β-estradiol or EE, and found that while both estrogens have similar effects on innate immune cellular responses, EE has distinct effects on T cell population subsets, cytokine production, gene response and epigenetic alterations in female NZB/WF1 mice. Finally, chronic low-dose oral exposure to EE exacerbated clinical signs of kidney disease and suppressed the normal response of toll-like receptor 9 in MRL/lpr mice. Overall, we have found that chronic exposure to environmental estrogenic EDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals.

Two studies were conducted to assess factors affecting estrogen excretion in dairy heifers. The objective of the first study was to quantify estrogenic activity in feces and urine during the estrous cycle. Ten non-pregnant Holstein heifers were fed the same diet for 28 d. Plasma, feces, and urine samples were collected daily. Plasma 17-β estradiol (17-β E2) was quantified with RIA and used to confirm day of estrous. Feces and urine samples from days -12, -6, -2, -1, 0, 1, 2, 6, 12 of the estrous cycle were analyzed with RIA and Yeast Estrogen Screen (YES) bioassay. Plasma 17-β E2 concentrations peaked on day of estrus, with feces and urine estrogenic excretion peaking a day after. The objective of the second study was to quantify variation in estrogenic activity in feces and urine due to increased dietary phytoestrogen content. Ten Holstein heifers were randomly assigned to treatment sequence in a two-period crossover design. Dietary treatments consisted of grass or red clover hay, and necessary supplements. Feces and urine samples were collected and pooled for analysis. Estrogenic activities of pooled samples were quantified using the YES bioassay. Estrogenic excretion in feces and urine was higher for heifers fed red clover hay. Fecal and urine samples from five heifers were analyzed using LC/MS/MS to quantify excretion of phytoestrogenic compounds. Heifers fed red clover hay excreted more equol than heifers fed grass. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation. / Master of Science

Obesity-related comorbidities, including type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) have become a major public health concerns. These complications primarily arise in response to cellular changes in white adipose tissue (WAT). In particular, when a majority of fat cells become hypertrophic it promotes a metabolically unhealthy phenotype, which is characterized by chronic low-grade inflammation, insulin resistance, and ectopic lipid accumulation. Research has implicated synthetic (i.e., Bisdehydrodoisynolic acid, BDDA) and natural (i.e., genistein and daidzein) xenoestrogens in the protection against obesity-related pathologies. Bisdehydrodoisynolic acid (BDDA) reduced weight gain and adiposity, as well as improved lipid homeostasis in obese rodents. Alternatively, phytoestrogens, such as genistein and daidzein were reported to induce adipocyte differentiation through potential interactions with PPARγs, canonical WNT proteins, and estrogen receptors (ER) signaling. The current investigation was conducted to test the effects of synthetic and natural estrogenic compounds (BDDA, daidzein, and genistein) for their effects on the induction of adipogenic signaling, which may potentially improve WAT morphology by reducing adipocyte hypertrophy.

Phytoestrogens are naturally occurring chemicals of plant origin that have the ability to cause estrogenic and/or anti-estrogenic effects due to their structural similarities to the human hormone oestradiol. It has been proposed that phytoestrogens protect against a wide range of ailments, including breast and prostate cancers, cardiovascular disease, osteoporosis, and menopausal symptoms. Daidzein, biochanin A and especially genistein which has been reported to be the most biologically active dietary phytoestrogen attract great deal of interest in today&rsquo / s researches. In this study, twenty different food items, including legumes, fruits, nuts and herbs, (haricot beans, chickpeas, green lentils, red lentils, soybeans, licorice root, yarrow, dried chestnuts, prunes, raisins, currants, black cumin, dried apricots, dried parsley, dried dates, dried figs, sage (from Aegean region), sage (from Mediterranean region), grapevine leaves, gilaburu) were selected. Following an extraction procedure employing acid hydrolysis and heating / they were analysed for their daidzein, genistein and biochanin A contents using a reversed-phase C18 column with linear gradient elution on a high-performance liquid chromatography (HPLC) coupled with diode-array detector (DAD). Soybeans were found to contain high amounts of daidzein (91.36 mg/100 g) and genistein (85.57 mg/100 g). Chickpeas were found to contain much less amount of genistein (0.89 mg/100 g) compared with that of soybeans and also biochanin A (0.95 mg/100 g) which was not detected in soybeans. None of daidzein, genistein and biochanin A was detected in the remaining eighteen food items.

Regular soy consumption has been shown to reduce cardiovascular (CV) risk through plasma cholesterol reduction. According to the current health claim, this benefit is attributed to soy protein (SP). Dietary intervention trials indicate that isoflavones (ISO), weak phytoestrogens in soy, may also contribute by offering additional vascular and metabolic protection. Equol, a metabolite of the ISO daidzein (DAZ) with greater estrogenic potency, may be an important mediator of such effects. This thesis examines effects of soy, in particular, ISO consumption on CV risk factors and the potential for ISOs to enhance cognition, possibly through improvements of circulatory function. Two crossover design intervention trials were undertaken: a food-based intervention, investigating differential effects of SP and ISO on plasma lipids and other risk factors for CVD, and an ISO supplementation trial, examining effects on cognition and vascular function. Both addressed whether benefits were dependent on equol production. In the first trial, 91 subjects with untreated mild hypercholesterolemia were randomised to consume each of the following three diets in random order for sequential 6 week periods: (S) soy foods containing 24 g of SP and 75-90 mg ISO per day, (SD) soy/dairy foods containing 12 g SP, 12 g dairy protein (DP) and 75-90 mg ISO per day or (D) dairy foods containing 24 g DP only per day. At the end of each diet period, blood lipids, flow-mediated dilatation (FMD) of the brachial artery, blood pressure, arterial compliance and anthropometric measures were assessed. Compared with the control diet (D), there was a small but significant reduction in total cholesterol on the S diet only (2.8 + 1.1%, P<0.05), which could be accounted for by a decrease in saturated fat intake. FMD was found to be significantly improved when SD and S diet data were nested (P=0.03). Plasma triglycerides (TG) improved on both the SD and S diets compared with D (P<0.01). Other lipid, metabolic and vascular parameters did not differ between diets. There were no differences in outcomes between equol (n=30) and non equol producers (n=61). In a subsequent 12 week double-blind supplementation trial, 34 healthy males were randomised to take 4 capsules providing 120mg ISO per day or a matching placebo for 6 weeks, after which they crossed over to the alternate supplement. FMD and cognitive assessments relating to measures of memory and executive function were performed at the beginning and end of each treatment phase. Spatial working memory, a test in which females consistently perform better than males, was significantly improved by ISO supplementation (P<0.02). However, other measures of cognition and FMD were unaffected and there were no differences between equol (n=8) and non-equol producers (n=26). These interventions indicate that ISOs offer specific health benefits, independent of equol production. ISO supplementation can enhance specific cognitive processes which appear dependent on estrogen activation. Additionally, soy foods containing ISOs improved FMD and TG but were unable to improve LDL cholesterol, even in equol producers. Thus dietary ISOs may reduce CV risk but the validity of the current health claim for SP is questioned. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345614 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

A wealth of research has reported possible risk and predictive factors for dementia, the variance across populations and the possible reasons for this variance. This thesis attempts to describe demographic and modifiable risk factors for dementia, with the emphasis on the association between (phyto) estrogens and cognitive function.

Breast cancer is the second leading cause of death in women in the U.S. The CDC estimates that between 2010 and 2020, there will be an increase in breast cancer in women in the United States by 21%, or greater 900,000 new cases per year. (CDC) Women are at increased risk for breast cancer due to the fact that some breast cancers have cells which are responsive to the hormone estrogen. Further, the risk of developing cancer increases as women age. Women who experience menopause after age 55 have an increased risk of developing breast cancer. (Surakasula et al., 2014) Phytoestrogens, which include isoflavones, are compounds found in plants and processed foods, which structurally and functionally mimic the hormone estrogen. Soy is frequently found in processed foods regularly consumed in the average American diet and is a source of isoflavones, including genistein, daidzein, and daidzein’s metabolite, equol. Phytoestrogens can bind to the estrogen receptor and change the expression of genes that respond to estrogen, such as the oncogene c-Fos, a protein in breast cancer cells which can suppress breast cancer cell growth when it is mutated (Lu et al., 2005). Phytoestrogens have been shown to reduce the severity and frequency of some of the symptoms of menopause, including a reduction in hot flashes and other estrogen deficiency-related symptoms of menopause. (Chen et al., 2015). Phytoestrogens have been used therapeutically in menopausal women, as they can compensate for lower levels of estrogen in the body. There is, however, limited research regarding the consumption of soy and increased risk for cancer in postmenopausal women. The objective of this study was to determine whether increasing levels of urinary phytoestrogen are correlated with a decreased risk of postmenopausal breast cancer. The analysis observes 2,439 postmenopausal female subjects equal to or greater than 45 years of age who had urinary phytoestrogen and reproductive health data in the 1999-2010 National Health and Nutrition Examination Survey database. The dietary data was obtained from the U.S. Department of Agriculture’s Agricultural Research Service database. Logistic regression models constructed using SAS were used to assess the change in the relative risk for cancer across low, medium, and high levels of urinary daidzein, genistein, and equol. Variables of interest, including demographic, body measures, dietary patterns, and lab measures, were compared to a base model, which adjusts for age only. It was found that the relative risk for postmenopausal breast cancer was not significantly different in the tertiles and quintiles of urinary phytoestrogens. The greatest increase in the likelihood of postmenopausal breast cancer was found to be 1.53 times in the second tertile of daidzein, increasing the risk by 53%. There was no significant protective effect of increasing levels of urinary phytoestrogens against postmenopausal breast cancer.

L’érythropoïèse est un processus extrêmement prolifératif, et qui doit donc être très étroitement régulé. L’érythropoïétine (EPO) est l’un des facteurs absolument nécessaires à l’érythropoïèse. Cependant, dans la moelle osseuse, la quantité d'EPO circulante est sous-optimale et la capacité des érythroblastes à survivre dépend donc de leur sensibilité à l'EPO. Les facteurs modulant la réponse à l'EPO au cours de l'érythropoïèse sont encore largement inconnus.Nous avons donc voulu explorer plusieurs facteurs pouvant potentiellement être impliqués dans la régulation de l’érythropoïèse et plus précisément dans la réponse à l’EPO : tout d’abord, la transferrine ainsi que ses récepteurs (TfR), la transferrine et le TfR1 étant également essentiels à l’érythropoïèse, ainsi qu’un phytoestrogène provenant d’une plante nommée Curcuma comosa, les oestrogènes étant eux aussi connus pour favoriser l’érythropoïèse.Concernant la transferrine, nous avons voulu principalement explorer son rôle sur la signalisation, ayant récemment montré au laboratoire que le TfR1, essentiellement connu pour son rôle dans l’endocytose du fer, est également capable d’entraîner une signalisation.Nous avons montré que la transferrine potentialise la stimulation induite par l’EPO des voies ERK, AKT et STAT5. Cet effet est conservé même en l’absence d’endocytose du TfR1. Aucune coopération n’a été trouvée entre la transferrine et le stem cell factor (SCF).Nous avons également observé qu’en l’absence du TfR2, il existe une augmentation de l’expression de l’EPO-R et de la signalisation induite par l’EPO, sans impact de la transferrine dans ce contexte. Par ailleurs, nous avons montré que le Curcuma comosa améliore la prolifération et la différenciation des progéniteurs érythroïdes précoces, par un mécanisme de potentialisation de la signalisation induite par l’EPO impliquant le récepteur aux oestrogènes ER-α.En conclusion, la transferrine et ses récepteurs, ainsi qu’un phytoestrogène et l’ER-α, sont impliqués dans la régulation de l’érythropoïèse via leur action sur la signalisation induite par l’EPO. L’approfondissement de ces données pourrait ouvrir de nouvelles pistes thérapeutiques dans le traitement de l’anémie. / Erythropoiesis is an extremely proliferative process and must be very closely regulated. Erythropoietin (EPO) is one of the major factors necessary for erythropoiesis. However, in the bone marrow, the amount of circulating EPO is suboptimal and the ability of erythroblasts to survive therefore depends on their sensitivity to EPO. The factors modulating the response to EPO during erythropoiesis are still largely unknown. We therefore wanted to explore several factors that could potentially be involved in the regulation of erythropoiesis and more specifically in the response to EPO: first, transferrin and its receptors (TfR), transferrin and TfR1 being also essential for erythropoiesis, as well as a phytoestrogen from a plant called Curcuma comosa, as estrogens are also known to promote erythropoiesis. Regarding transferrin, we mainly wanted to explore its role on signaling, having recently shown in the laboratory that TfR1, essentially known for its role in iron endocytosis, is a signaling-competent receptor. We have shown that transferrin potentiates EPO-induced stimulation of the ERK, AKT and STAT5 pathways. This effect is maintained even in the absence of TfR1 endocytosis. No cooperation was found between transferrin and stem cell factor (SCF). We also observed that in the absence of TfR2, there is an increase in EPO-R expression and EPO-induced signaling, without any impact of transferrin in this context.In addition, we have shown that Curcuma comosa improves the proliferation and differentiation of early erythroid progenitors through a mechanism involving the ER-α estrogen receptor, able to potentiate EPO-induced signaling. In conclusion, transferrin and its receptors, as well as a phytoestrogen and ER-α, are involved in the regulation of erythropoiesis through their action on EPO-induced signaling. Further investigation of these data could provide new therapeutic strategies in the treatment of anemia.

According to the American Cancer Society, breast cancer is the most commonly diagnosed cancer and is the second leading cause of cancer death in American women. Breast cancer screenings and improvement in treatments have resulted in the rising number of survivors in the recent decade. This urged the need for post-diagnosis lifestyle changes to improve breast cancer patients' quality of life. Many studies found soy food, the primary dietary source of phytoestrogens, has a protective effect against breast cancer recurrence and mortality. Dietary phytoestrogens can be classified into two groups: isoflavones and lignans. Daidzein and genistein were identified as the most common isoflavones. Due to their structural similarities to 17- beta estradiol, isoflavones exert agonistic and antagonistic effects. However, limited studies have evaluated the effect of phytoestrogen on health-related quality of life (HRQOL) among cancer survivors. This thesis examined the association between genistein levels and HRQOL and whether these associations are altered by menopausal status using data collected by a research team in South Korea. Women aged 21 to 81 years old, diagnosed with stage I – III primary breast cancer, and received breast cancer surgery at least six months prior were enrolled from five hospitals. Plasma genistein level was measured by orbitrap liquid chromatography-mass spectrometry at the University of Hawaii Cancer Center, Honolulu, Hawaii, U.S.A. HRQOL was self-reported using a validated Korean version of the SF-36 questionnaire. SF-36 has eight domain scores. The scores are summarized into physical (PCS) and mental (MCS) component summary scores, with a higher score indicating better HRQOL. The association between plasma genistein and HRQOL was assessed using a multiple linear regression model adjusting for potential confounders. Stratified analysis was conducted to examine whether menopause status modified this association. 407 women (mean age: 52.1 ± 8.3) with all available data were included in the analysis. The mean blood genistein concentration was 144.7 nM (range: 1.1- 2073.0 nM), and the PCS and MCS scores were 49.5 ± 7.1 and 49.2 ± 9.8, respectively. Analysis for the entire sample showed no significant associations between genistein level and HRQOL score across all domains. Further subgroup analysis by menopausal status revealed that only pre- menopausal women in the highest tertile level of genistein had a higher social function domain score (48.9 vs. 47.2) than those in the lowest tertile with a borderline statistical significance (p = 0.083). These findings suggest beneficial effects of genistein may depend on women's menopausal status, but further investigation is necessary.