<p>Consumption of soy foods has been shown to provide beneficial health outcomes such as reduction of menopause symptoms, reduced risk of breast cancer and prostate cancer, improved cardiovascular health, and improved bone health. The mechanism hypothesized to be driving these outcomes is the conversion of the soy isoflavone daidzein into the metabolite equol by bacteria in the gut microbiome. Equol is an exclusively microbially produced metabolite with a high binding affinity to mammalian estrogen receptors. Not all humans harbor equol-producing microbes in their gut, and less than half of the population can be classified as equol producers. To date, soy feeding research published suffers from confounding factors that make assessing the causal impact of equol production in health difficult due to: (i) large interpersonal variation of the human microbiome and human genomes and that (ii) all lab-raised rodent models harboring natural microbiomes are highly efficient equol producers. In this study, we sought to establish a gnotobiotic mouse model harboring synthetic bacterial communities with divergent equol-producing capacities by designing two communities: the Equol(-) community and Equol(+) community. The Equol(-) community was designed to include ten bacterial strains commonly found within a human microbiome without equol-producing capacity<em>.</em> To create the Equol(+) community, the equol-producing bacteria <em>Adlercreutzia equolifaciens</em> was added to the Equol(-) community<em>.</em> Female and male germ-free C57BL/6 mice were colonized with either the Equol(-) or Equol(+) community for 4 weeks. Daidzein was administered by dietary supplementation (1.5% wt/wt daidzein) in a semi-purified diet containing fermentable fiber starting two weeks prior to bacterial colonization. As expected, equol was detected in the serum of mice colonized with the Equol(+) community, but not detectable in those colonized with the Equol(-) community. There were no sex differences detected in equol production. 16S rRNA gene sequencing of mouse cecal content revealed that ~50-80% of the strains from each community colonized within the mice at detectable levels. Strain-specific qPCR improved the detection of strains not observed consistently through 16S rRNA gene sequencing. Our results demonstrated that this model is reliable in producing the expected equol producing and non-equol producing phenotypes when colonized with the Equol(+) and Equol(-) communities, respectively. This model system can be utilized in a broad range of future studies to conclusively determine the causal impact of endogenous equol production in many areas, such as cardiometabolic health and bone health.</p>

Orientador: Mary Rosa Rodrigues de Marchi / Banca: Anderson Rodrigo Moraes de Oliveira / Banca: Marcia Cristina Bisinoti / Resumo: É consenso entre cientistas do mundo inteiro que existem moléculas com o poder de alterar o funcionamento do sistema endócrino humano e de diversas espécies de animais, conhecidas atualmente como alteradores endócrinos. Dentro deste grupo de substâncias, merece destaque as isoflavonas presentes em maior abundância nos grãos de soja, principalmente a genisteína (GEN) e a daidzeína (DAID), apontadas como responsáveis por desencadear inúmeros efeitos endócrinos em espécies aquáticas. Portanto, o objetivo do presente trabalho foi otimizar e validar um novo método para determinação de GEN e DAID em efluente de ETE, água subterrânea, potável e de rio; aplicando o método já validado nestas matrizes na cidade de Araraquara e avaliar o comportamento sazonal destes alteradores endócrinos. O método apresentado é baseado na pré-concentração e simplificação de amostra proporcionado pelo uso de SPE aplicado a 2 litros de amostras de água de rio e subterrânea e ao volume de 1,5 litro de efluente de ETE, analisados por HPLC-UV utilizando um sistema MeOH/Água como alternativa ao uso de acetonitrila. A exatidão mostrou-se na faixa de 75 a 110%, com precisão entre 0,5 e 14 %. O LQ alcançado com o referido método foi de 6,3 e 188 ng L-1 para daidzeína e 2,17 e 187 ng L-1 para a genisteína nas matrizes de água subterrânea, superficial e efluente de ETE, respectivamente. A aplicação do método nas amostras de água subterrânea e do Ribeirão das Cruzes demostrou que as concentrações de DAID e GEN situaramse abaixo dos LD. Já para as amostras de efluente de ETE, estas moléculas foram identificados em todas as amostras coletadas, com concentrações variando entre 60-306 ng L-1 para GEN e de 76 a 288 ng L-1 para DAID. Comparando-se os dados de temperatura e pluviosidade com as concentrações destes fitoestrógenos no período amostrado, percebe-se que a temperatura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is a common thought among scientists worldwide that there are molecules with the capacity of altering the functioning of the endocrine system of humans and several species of animals, currently known as endocrine modifiers. Inside this group of substances, the highlighted ones are the isoflavones more abundantly present in soybean grains, especially genistein (GEN) and daidzein (DAID), pointed as those responsible for triggering a number of endocrine effects in aquatic species. Therefore, the aim of the present work was optimize and validate a new method for the determination of GEN and DAID in an effluent of a Sewage Treatment Unit (STU), as well as subterranean, drinking and river waters, applying the method already validated in these matrixes in the city of Araraquara, and then evaluate the seasonal behavior of these endocrine modifiers. The method presented is based on the sample pre-concentration and simplification enabled by the use of SPE applied to 2 liters of samples from subterranean and river waters, and to the volume of 1.5 liter of STU effluent, analyzed through HPLC-UV using a system of MeOH/Water as an alternative to the use of acetonitrile. The accuracy was shown to be in the range of 75 to 110%, with a precision between 0.5 and 14 %. The LQ reached with the method referred was of 6.3 and 188 ng L-1 for daidzein, and 2.17 and 187 ng L-1 for genistein in the matrixes of subterranean and superficial waters, as well as STU effluent, respectively. The application of the method in the samples of subterranean water and also in that of the Cruzes stream demonstrated that the DAID and GEN are below the LD. For the samples of STU effluent, such molecules were identified in all samples collected, with concentrations ranging between 60-306 ng L-1 for GEN and 76-288 ng L-1 for DAID. Comparing the temperature and rainfall data with the concentrations of these... (Complete abstract click electronic access below) / Mestre

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.

Made available in DSpace on 2014-06-11T19:29:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:18:55Z : No. of bitstreams: 1 cavassani_tb_me_araiq.pdf: 1899529 bytes, checksum: c90c7f090f98ce6f94ef871d0d70f937 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / É consenso entre cientistas do mundo inteiro que existem moléculas com o poder de alterar o funcionamento do sistema endócrino humano e de diversas espécies de animais, conhecidas atualmente como alteradores endócrinos. Dentro deste grupo de substâncias, merece destaque as isoflavonas presentes em maior abundância nos grãos de soja, principalmente a genisteína (GEN) e a daidzeína (DAID), apontadas como responsáveis por desencadear inúmeros efeitos endócrinos em espécies aquáticas. Portanto, o objetivo do presente trabalho foi otimizar e validar um novo método para determinação de GEN e DAID em efluente de ETE, água subterrânea, potável e de rio; aplicando o método já validado nestas matrizes na cidade de Araraquara e avaliar o comportamento sazonal destes alteradores endócrinos. O método apresentado é baseado na pré-concentração e simplificação de amostra proporcionado pelo uso de SPE aplicado a 2 litros de amostras de água de rio e subterrânea e ao volume de 1,5 litro de efluente de ETE, analisados por HPLC-UV utilizando um sistema MeOH/Água como alternativa ao uso de acetonitrila. A exatidão mostrou-se na faixa de 75 a 110%, com precisão entre 0,5 e 14 %. O LQ alcançado com o referido método foi de 6,3 e 188 ng L-1 para daidzeína e 2,17 e 187 ng L-1 para a genisteína nas matrizes de água subterrânea, superficial e efluente de ETE, respectivamente. A aplicação do método nas amostras de água subterrânea e do Ribeirão das Cruzes demostrou que as concentrações de DAID e GEN situaramse abaixo dos LD. Já para as amostras de efluente de ETE, estas moléculas foram identificados em todas as amostras coletadas, com concentrações variando entre 60-306 ng L-1 para GEN e de 76 a 288 ng L-1 para DAID. Comparando-se os dados de temperatura e pluviosidade com as concentrações destes fitoestrógenos no período amostrado, percebe-se que a temperatura... / It is a common thought among scientists worldwide that there are molecules with the capacity of altering the functioning of the endocrine system of humans and several species of animals, currently known as endocrine modifiers. Inside this group of substances, the highlighted ones are the isoflavones more abundantly present in soybean grains, especially genistein (GEN) and daidzein (DAID), pointed as those responsible for triggering a number of endocrine effects in aquatic species. Therefore, the aim of the present work was optimize and validate a new method for the determination of GEN and DAID in an effluent of a Sewage Treatment Unit (STU), as well as subterranean, drinking and river waters, applying the method already validated in these matrixes in the city of Araraquara, and then evaluate the seasonal behavior of these endocrine modifiers. The method presented is based on the sample pre-concentration and simplification enabled by the use of SPE applied to 2 liters of samples from subterranean and river waters, and to the volume of 1.5 liter of STU effluent, analyzed through HPLC-UV using a system of MeOH/Water as an alternative to the use of acetonitrile. The accuracy was shown to be in the range of 75 to 110%, with a precision between 0.5 and 14 %. The LQ reached with the method referred was of 6.3 and 188 ng L-1 for daidzein, and 2.17 and 187 ng L-1 for genistein in the matrixes of subterranean and superficial waters, as well as STU effluent, respectively. The application of the method in the samples of subterranean water and also in that of the Cruzes stream demonstrated that the DAID and GEN are below the LD. For the samples of STU effluent, such molecules were identified in all samples collected, with concentrations ranging between 60-306 ng L-1 for GEN and 76-288 ng L-1 for DAID. Comparing the temperature and rainfall data with the concentrations of these... (Complete abstract click electronic access below)

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.

Soybeans and products thereof are considered as a very contradictory legume across layman and expert opinions. Some see it almost as a “superfood” while others avoid it due to its biologically active content with an unclear effect on the organism. As a lot of research on soybeans was already performed the content varies considerably and it is not easy to grasp the issue correctly. The objective of the research part of the thesis is a structuralized study of already published scientific knowledge to clarify the current state of the art. Experimental part of the thesis focuses on characterisation of soy and its products in terms of active ingredients content and determination of antioxidants and antimicrobial effect. After that, the cytotoxic effect on human heterogeneous colorectal adenocarcinoma epithelial cell line and mouse melanoma cell line is determined by in vitro testing. All obtained information and results of experimental part could possibly serve as a starting point for further study of soy and soy products, both in the dissertation and in the submission of a grant project.

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This protection could be linked to isoflavone-induced alterations in immune function. However, recent in vitro and animal studies suggest that soy isoflavones may either enhance or suppress immunocompetence, depending upon the isoflavone concentration, target tissue, and a number of other factors. To date, no study has investigated the effect of dietary soy isoflavone supplementation on immune parameters in humans. Therefore, the purpose of this double-blind, placebo-controlled, 4 wk intervention trial was to investigate whether supplementation with soy isoflavones alters indices of immune function in postmenopausal women. Twenty healthy women (50-69 yr), who were not on hormone replacement therapy, were randomly divided into 2 treatment groups. The supplemented group (n=10) consumed soy isoflavone tablets (100 mg/d) for 4 wk, while the control group (n=10) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess specific immune parameters. In addition, plasma concentrations of genistein and daidzein were quantified at baseline and at the end of the intervention period. Despite high individual variability among subjects, there was a significant increase (p<0.005) in plasma isoflavone concentration in the supplemented group. However, all assessed immune parameters remained unchanged after supplementation and did not differ between the 2 treatment groups. In conclusion, this study suggests that short-term soy isoflavone supplementation at physiologically attainable concentrations does not alter the aforementioned immune parameters in healthy postmenopausal women. Due to the conflicting data concerning the effect of dietary soy isoflavones on immune function, further research in this area is warranted. / Master of Science

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This defense could be linked to isoflavone-induced alterations in immune function. However, to date, no study has examined the effect of soy isoflavone supplementation on human immunity in vivo. Establishing whether isoflavones affect immunity in aging adults is particularly relevant since compromised immune function has been observed in this population. Therefore, the purpose of this double-blind, placebo-controlled, 4-wk intervention trial was to investigate whether supplementation with soy isoflavones influenced the distribution and/or function of specific lymphocytes in postmenopausal women. Healthy postmenopausal women (50-69 y), who were not using hormone replacement therapy, were randomly divided into 2 treatment groups. The experimental group (n=9) consumed two-50 mg soy isoflavone tablets/d for 4 wk, while the control group (n=9) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess distribution of T-helper cells (CD3+CD4+), T-cytotoxic cells (CD3+CD8+), total T lymphocytes (CD3+), B lymphocytes (CD19+) and natural killer (NK) cells (CD16+CD56+) via flow cytometry. Cytotoxicity of NK cells was quantified based on lactate dehydrogenase release of lysed K562 cancer cells following co-culture with NK cells from subjects. Analysis of plasma isoflavone concentrations by HPLC demonstrated a significant increase (p<0.005) in plasma genistein concentration in the experimental group after 4 wk of supplementation. However, there was no alteration in lymphocyte distribution or NK cell activity in response to isoflavone supplementation, suggesting that short-term soy isoflavone supplementation does not alter these parameters of immunity in healthy postmenopausal women. / Master of Science

Die Osteoporose ist eine systemische Skeletterkrankung, die durch eine niedrige Knochenmasse und eine Verschlechterung der Mikroarchitektur des Knochengewebes mit nachfolgend erhöhter Knochenbrüchigkeit charakterisiert ist. Hauptverantwortlich für den Verlust der Knochenmasse ist vor allem der Östrogenmangel, der die mit Abstand häufigste Form, die postmenopausale Osteoporose, verursacht. Jahrelang ist diese durch die Substitution von Östrogenen therapiert worden. Die Hormonersatztherapie, die unter vielen Substanzen nachweislich das höchste antiosteoporotische Potenzial besitzt, scheidet jedoch aufgrund diverser unerwünschter Nebenwirkungen als Therapieoption aus. Daher gilt es, Therapiealternativen zur Prävention und Behandlung der Osteoporose zu finden. Als potenzielle Therapiealternativen werden in dieser Arbeit ein Phytohormon namens Daidzein und ein endokriner Disruptor namens 4-MBC an der Lendenwirbelsäule ovariektomierter und konsekutiv an Osteoporose erkrankter Sprague-Dawley-Ratten über eine Therapiedauer von 35 (5 Wochen), respektive 70 Tagen (10 Wochen), untersucht. Als Kontrolle über die Wirksamkeit der getesteten Substanzen dienten eine sojafrei behandelte sowie eine mit Östrogenen behandelte Kohorte. Phytoöstrogene sind hauptsächlich Bestandteil von Sojabohnen, Klee, Alfalfasprossen und Leinsamen. Bevorzugt binden sie an Östrogenrezeptor β, wenn auch mit einer geringeren Affinität. Eines dieser Phytoöstrogene ist Daidzein, dessen osteoprotektive Wirkung zuvor bereits vielfach nachgewiesen wurde. 4-MBC als zweite Substanz ist ein sogenannter endokriner Disruptor, der in Europa zur Herstellung von Kosmetika zugelassen ist. Bevorzugt bindet 4-MBC an Östrogenrezeptor β und seine osteoprotektive Wirkung an ovariektomierten Ratten ist ebenfalls bereits nachgewiesen. In der vorliegenden Arbeit wurden 3 Monate alte ovariektomierte Sprague-Dawley-Ratten über einen Zeitraum von 8 Wochen mit einer sojafreien Nahrung gefüttert. In dieser Zeit entwickelt die Ratte eine schwere Osteoporose. Anschließend wurde bilateral eine Tibiaosteotomie, die osteosynthetisch versorgt wurde, durchgeführt. Die Ratten wurden in 4 Gruppen eingeteilt und die sojafreie Basisnahrung um die zu testenden Substanzen 4- MBC (200 mg), Daidzein (50mg) und Östradiol (0,4mg) pro Kilogramm Körpergewicht ergänzt. Nach 5, respektive 10 Wochen wurden 12 Ratten je Futtergruppe per Dekapitation getötet und jeweils an den Lendenwirbelkörpern wurden verschiedene Untersuchungen durchgeführt. So wurde der zweite Lendenwirbelkörper mikroradiographisch analysiert, der dritte Lendenwirbelkörper wurde verascht und der vierte Lendenwirbelkörper biomechanisch getestet. Auf den erhobenen Daten basierend konnte nachgewiesen werden, dass sowohl Daidzein als auch 4-MBC einen positiven Einfluss auf das Knochengewebe während der gesamten Therapiedauer besitzen. Östradiol verzeichnet wie erwartet den größten Effekt, gefolgt von 4-MBC und Daidzein. Bezogen auf die Therapiedauer zeigte sich, dass die größere Wirkungsentfaltung innerhalb der Kurzzeittherapie zu verzeichnen ist. Nach einer Langzeittherapie zeigt die sojafrei ernährte Kontrollgruppe ebenfalls verbesserte Knochenparameter, möglicherweise zurückzuführen auf Adaptationsmechanismen der Osteoporose. 4-MBC ist von seinem osteoprotektiven Wirkungspotenzial dem der Östrogene am ähnlichsten. Eine Therapie mit 4-MBC verbessert sowohl spongiöse, als auch kortikale Knochenparameter und könnte somit als potenzielle Therapiealternative der Osteoporose dienen. Allerdings sind die Wirkungsmechanismen des endokrinen Disruptors zum gegenwärtigen Zeitpunkt noch nicht verstanden und es bedarf diesbezüglich weiterer intensiver Forschung. Ein Aspekt, der einen Gebrauch von 4-MBC als antiosteoporotisches Medikament verhindern könnte, ist das noch nicht endgültig erforschte Nebenwirkungsspektrum. Auch hier sind weitere Untersuchungen erforderlich. Daidzein zeigt insgesamt im Vergleich zu Östradiol und 4-MBC den geringsten osteoprotektiven Effekt. Als potenzielle Therapiealternative ist es 4-MBC und Östradiol unterlegen. Möglicherweise können Patienten mit osteoporosebedingten Frakturen jedoch von einer Behandlung mit Daidzein profitieren. Komrakova et al. haben 2011 an Ratten, bei denen die Tibia osteotomiert und die anschließend osteosynthetisch versorgten wurde, nachgewiesen, dass nach einer 5 wöchigen Therapie mit Daidzein die größte Kallusdichte zu verzeichnen war, während bei 4-MBC und Östradiol eine Kallusdichte aufgewiesen wurde, die der sojafreien Kontrollgruppe ähnlich war. Ob sich Daidzein als Therapieoption zur Unterstützung der Frakturheilung eignet, ist zum aktuellen Zeitpunkt noch nicht geklärt und in weiteren Untersuchungen zu eruieren. Des Weiteren gilt es, auch für Daidzein den genauen Wirkungsmechanismus zu klären und sein Nebenwirkungsspektrum zu untersuchen.

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