Orientador: Sonia Nair Bao / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T07:53:58Z (GMT). No. of bitstreams: 1 Cardoso_JulioRoquete_D.pdf: 2342812 bytes, checksum: 608836a3972af8e207e90ef5ae664a4a (MD5) Previous issue date: 2007 / Resumo: Este estudo foi proposto para avaliar se a exposição perinatal (gestacional e lactacional) ou crônica a isoflavonas em dieta contendo soja ou na forma de concentrados de isoflavonas pode comprometer a saúde reprodutiva de coelhos machos. No primeiro experimento, fêmeas foram alimentadas com dieta contendo soja ou dieta isenta de soja e alfafa, suplementada com 10 ou 20 mg/kg/dia de isoflavonas ao longo da gestação e lactação. O grupo controle foi mantido somente com a dieta isenta de soja e alfafa. Na desmama, foram avaliados o peso e a morfologia dos órgãos do aparelho reprodutor e os níveis séricos de testosterona de parte dos filhotes machos. O restante deles foi submetido à dieta controle desde a desmama até a fase adulta. Após a puberdade, os animais foram avaliados quanto ao comportamento sexual, qualidade do sêmen e morfologia dos órgãos reprodutivos. No segundo experimento, fêmeas foram alimentadas com as mesmas dietas empregadas no primeiro experimento, porém a suplementação com isoflavonas foi realizada com doses variando de 2,5 a 20 mg/kg/dia. As doses de isoflavonas foram selecionadas com base em estimativas da ingestão de isoflavonas a partir do consumo de alimentos derivados da soja. Após a desmama, os filhotes machos receberam a mesma dieta fornecida para suas respectivas mães até o fim do experimento. Foi avaliado nestes animais a idade à puberdade, qualidade do sêmen e o comportamento sexual, e, na 33ª semana de vida o peso e a morfologia dos órgãos reprodutivos. Os resultados deste estudo foram baseados em dados obtidos da avaliação de 100 machos num período de 3 anos. O número de espermatozóides esteve de acordo com os valores da literatura para coelhos da raça Nova Zelândia e não variou significativamente em relação ao grupo controle, embora o volume de sêmen tenha sido menor em coelhos expostos à alta dose de isoflavonas (20 mg/kg/dia). O peso dos órgãos reprodutivos não diferiu estatisticamente do grupo controle e não houve evidência de malformações genitais, alterações metaplásicas, ou qualquer outra alteração histopatológica correlacionada com os tratamentos. Nos jovens, a análise histológica dos testículos não revelou diferenças no desenvolvimento gonadal. Coelhos suplementados de forma crônica com 20 mg/kg/dia de isoflavonas apresentaram menor ingestão de alimentos e peso corporal na fase adulta. Este achado é economicamente importante na produção animal; todavia os animais alimentados com a dieta contendo soja apresentaram na 33ª semana de idade consumo de alimento e peso corporal maiores em 6 e 4% respectivamente do que os animais do grupo controle (P < 0,05). Apesar dos recentes alertas, os resultados deste estudo não suportam a hipótese de que a exposição à isoflavonas em doses compatíveis com o consumo de alimentos à base de soja possa comprometer a saúde reprodutiva masculina / Abstract: This study was proposed to determine if perinatal (that is gestation and lactation) or chronic exposure to isoflavones trough consumption of soy containing diet or semipurified soy isoflavones may disrupt male reproductive health of rabbits. In the first experiment, groups of dams were fed either soy containing diet or soy and alfafa free diet supplemented with soy isoflavones at levels of 10 and 20 mg/kg/day throughout gestation and lactation. The control group was kept on soy and alfafa free diet only. Reproductive organs weight and morphology and serum levels of testosterone of part of the male offspring were evaluated at weaning. Remaining males were subjected to the control diet from weaning to adulthood. Sexual behavior, semen quality and reproductive organs morphology were evaluated after puberty. In the second experiment, groups of dams were fed same diets employed in the experiment 1, but supplementation with isoflavones were performed with doses ranging from 2,5 to 20 mg/kg/day. Dose levels of isoflavones were selected on the basis of the reported estimative of isoflavones intake from the consumption of soy-based foods. After weaning, male offspring received the same diet, which was given to the respective mother. The age that males reached puberty, semen quality and sexual behavior were evaluated in these animals and at 33 weeks of age reproductive organs weight and morphology were analyzed. Results of this study were sustained by data from the evaluation of 100 males in a period of 3 years. Sperm counts was within literature values for New Zealand rabbits and did not vary significantly in relation to control group, although semen volume has been lesser in rabbits exposed to high levels of isoflavones (20 mg/kg/dia). Reproductive organs weight did not differ statistically from the control, and there was no gross evidence of genital malformations, metaplastic changes, or any histopathologic alteration that was correlated with the treatments. In the young rabbits, histological analysis of the testes did not revel differences in gonadal development. Rabbits chronically supplemented with 20 mg/kg/day of soy isoflavones showed lesser food intake and body weight at adulthood. This find is economically important in animal production; however the animals fed soy containing diet showed food consumption and body weight 6 and 4%, respectively higher than animals of the control group at 33 weeks of age (P <0.05). In conclusion, despite recent alerts, results of this study did not support the hypothesis that isoflavones consumption at dietary levels may impair male reproductive health / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Soy based products are growing in popularity in food supplementation, and a larger population of the world is consuming soy on a regular basis. Soy contains phytoestrogens, plant based mimics of the hormone estrogen. Estrogen has many functions in humans, but one relatively unexplored function is its ability to regulate the levels of the hormone oxytocin (OT) and its receptor (OTR) in the brain. OT is a hormone traditionally known for its role in birth, but recently has been as a key regulator in many different behaviors. These behaviors that OT may affect include increased maternal behaviors, increased sexual behaviors, increased social interactions, increased trust, decreased anxiety, and increased potential for pair bonding. Key phytoestrogens found in soy are of the isoflavone family, and genistein and diadzein are the main two isoflavones that have been shown to exert physiologic effects when ingested by binding to estrogen receptors in the brain. The isoflavones can be estrogen agonists or estrogen antagonists, based on the preexisting, endogenous levels of estrogen in the individual. For men and postmenopausal women, it is believed that ingesting soy can cause an increase in production of OT and OTR, resulting in an increased in OT driven behaviors. For premenopausal women, there is a high endogenous level of estrogen present, so the ingested soy can cause a decrease in production of OT and OTR in the brain, resulting in a decrease in OT driven behaviors. While there is strong evidence to suggest that this may in fact occur in humans, more human based studies, rather than animal models, must be conducted to further verify and validate this hypothesis. An important area yet unexplored is the onset and duration of these OT driven behaviors. It is unclear if these are transient, or more long lasting effects, and future studies must be done to answer this question. This area of research is certainly more relevant as soy based diets are becoming more common; moving forward additional research is needed to determine the extent of oxytocin's ability to alter behaviors in individuals in a significant way.

Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells.

After menopause, a woman's production of 17-estradiol, the predominant female sex hormone, declines. This change is associated with increased risk of osteoporosis/osteopenia and atraumatic bone fracture, cardiovascular disease, and breast and ovarian cancers. Phytoestrogens are non-steroidal compounds isolated from plants that have antagonistic, weak agonistic, or super-agonistic estrogenic effects in mammalian tissues; they have emerged as a potential therapeutic to alleviate post-menopausal symptoms. While some epidemiological evidence indicates that dietary consumption of phytoestrogens can alleviate post-menopausal health risks, other research suggests that phytoestrogens may not be completely safe. The research presented in this thesis indicates that a high concentration and sustained dose of phytoestrogens may be necessary to achieve antiestrogenic effects. MCF-7 cells, an estrogen-sensitive breast adenocarcinoma cell line, were used as a model system, and proliferating cell nuclear antigen (PCNA) was used as a marker of cell proliferation. Immunoblotting shows that genistein, a commercially purified phytoestrogen, promotes cell proliferation when administered for 24 hours, but may reduce proliferation when cells were treated for 48 hours. Genistein and estrogen have an additive effect on cells that were treated simultaneously with both hormones for 24 hours. In contrast, Promensilâ„¢, an over-the-counter phytoestrogen dietary supplement, was able to abolish expression of PCNA after 48 hours, and at high concentrations prevented estrogen-induced upregulation of PCNA after 48 hours. The clinical significance of these findings is that phytoestrogens may reduce the risk of breast cancer, but only after sustained high doses, which may be difficult if patient non-compliance is at issue. Additionally, because cell proliferation and not cell survival was investigated, we cannot say whether phytoestrogens are cytotoxic to breast cancer cells, only that they reduce proliferation.

Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic acitivity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006. / The phytoestrogenic activity of Cyclopia, used to prepare honeybush tea, was evaluated and compared with that of the endogenous estrogen, 17-β-estradiol (E2) and the known phytoestrogen, genistein. Phytoestrogens are plant polyphenols much in demand in the nutraceutical market as they mediate an estrogenic effect through binding to estrogen receptor (ER) subtypes, ERα and ERβ.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Phytoestrogens are perceived as a safer alternative to conventional hormone replacement therapy (HRT) for the alleviation of menopausal symptoms as they present a decreased side-effect profile. The Cyclopia subternata (honeybush) methanol extract, SM6Met, displays estrogenic attributes desirable for the development of an phytoestrogenic nutraceutical, namely, estrogen receptor (ER) α antagonism, ERβ agonism, and antagonism of 17β-estradiol (E2)-induced breast cancer cell proliferation. Activity-guided fractionation was employed in an attempt to isolate and identify the compounds inducing the specific estrogenic profile of SM6Met. Fractions were evaluated for estrogenic attributes and major polyphenols present. Initial liquid-liquid fractionation of SM6Met yielded a polar fraction (PF) and a non-polar fraction (NPF), with the estrogenic attributes of interest retained and concentrated in NPF. Subsequent high performance counter-current chromatography (HPCCC) fractionation of NPF yielded three fractions (F1-F3). Interestingly, the fractions revealed separation of the previously demonstrated positive estrogenic attributes of NPF into separate fractions, with F1 and F2 acting as ERα antagonists, only F2 inducing antagonism of E2-induced breast cancer cell proliferation and only F3 retaining ERβ agonist activity. Although ERβ agonism displayed by F3 was robust and significantly higher than that of 10-11 M E2, it also displayed weak ERα agonism. Fractionation also for the first time in the study revealed ERβ antagonism, as induced by F1. In terms of major polyphenols HPCCC fractionation resulted in a divergence with F1 emerging as the dihydrochalcone-rich fraction and F2 as the flavanone and benzophenone-rich fraction, while the xanthones, flavones and phenolic acids were retained in F3. In addition, a preliminary absorption study was conducted using the ex vivo flow-through diffusion assay whereby the permeability of porcine small and large intestine for polyphenols in SM6Met was evaluated. The major compounds present in SM6Met were not able to penetrate the large intestinal mucosa, but small intestinal permeation of all major compounds in SM6Met ensued, with apparent permeability coefficient (Papp) values ranging from 1.91-3.74 x 10-6 cm.s-1, indicative of good intestinal absorption. Open source programs used for theoretical prediction of absorption gave conflicting results, emphasising the need to confirm predictions experimentally. ACD/Labs predicted poor intestinal absorption of SM6Met compounds based on physicochemical profiling, while OSIRIS and ChemAxon anticipated good absorption. In conclusion, activity-guided fractionation results suggest that retention of all the positive estrogenic attributes of the original SM6Met in one fraction is not an attainable goal. This suggests that several of the polyphenols present in SM6Met or NPF, through antagonistic, synergistic, or additive effects, may together be conferring these desired estrogenic traits. Thus production or isolation of a mixture of compounds, i.e. an “intelligent” mixture, should serve as a superior strategy in designing a nutraceutical product tailored to user demand of estrogenic activity. / AFRIKAANSE OPSOMMING: Fitoestrogene word beskou as ‘n veiliger alternatief vir konvensionele hormoon-vervangingsterapie (HVT) vir die verligting van simptome geassosiaeer met menopause aangesien dit ‘n verminderde newe-effek profiel vertoon. Die metanol ekstrak van Cyclopia subternata (heuningbos), SM6Met, vertoon estrogeniese eienskappe wat wenslik is vir die ontwikkeling van ‘n fitoestrogeen nutraseutiese middel, naamlik, estrogeen reseptor (ER) α antagonisme en ERβ agonisme, asook antagonisme van 17β-estradiol (E2) geïnduseerde proliferasie van borskankerselle. Aktiwiteit-begeleide fraksionering (ABF) is gebruik om die verbindings wat die spesifieke estrogeniese profiel aan SM6Met verleen te probeer isoleer en identifiseer. Fraksies is ge-evalueer vir estrogeniese eienskappe, asook vir die hoof polifenole teenwoordig. Aanvankilike vloeistof-vloeistof fraksionering van SM6Met het ‘n polêre fraksie (PF) en ‘n nie-polêre fraksie (NPF) opgelewer met behoud en konsentrering van die wenslike estrogeniese eienskappe in NPF. Daaropeenvolgende hoë werkverrigting teen-vloei chromatografie (HPCCC) van NPF het drie fraksies (F1-F3) opgelewer. Interessant genoeg het hierdie fraksies ‘n verdeling van die wenslike estrogeniese eienskappe van NPF in die individuele fraksies teweeggebring, deurdat F1 en F2 ERα antagonisme getoon het, F2 E2-geïnduseerde proliferasie van borskankerselle antagoniseer het, en net F3 ERβ agonis-aktiwiteit behou het. Alhoewel die ERβ agonis-aktiwiteit van F3 betekenisvol hoër was in vergelyking met die aktiwiteit van 10-11 M E2, het dit ook swak ERα agonisme getoon. Verder het fraksionering, vir die eerste keer in hierdie studie, ERβ antagonisme meegebring soos getoon deur F1. HPCCC het ook ‘n skeiding van die hoof polifenole veroorsaak, waarvolgens F1 as die dihidrogalkoon-ryke fraksie, F2 as die bensofenoon-ryke en flavanoon-ryke fraksie, en F3 as die xantoon-, flavoon- en fenoliese suur-ryke fraksie tevoorskyn gekom het. ‘n Voorlopige absorpsie studie, wat gebruik gemaak het van die ex vivo deurvloei diffusie toetssisteem, is uitgevoer om die deurlaatbaarheid van vark dik- en dunderm vir SM6Met polifenole te evalueer. Die hoof verbindings van SM6Met kon nie die dikderm mukosa penetreer nie, maar die deurlaatbaarheid van die dunderm vir alle SM6Met hoof polifenole is aangetoon, met skynbare deurlaatbaarheidskoëffisiënt (Papp) waardes wat strek vanaf 1.91 tot 3.74 x 10-6 cm.s-1, ooreenstemmend met goeie intestinale absorpsie. Oopbron programme, wat gebruik is vir die teoretiese voorspelling van absorpsie deur gebruik te maak van fisiese-chemiese profilering van verbindings, het teenstellende resultate opgelewer wat daarop dui dat hierdie voorspellings eksperimenteel bevestig moet word. ACD/Labs, wat van die fisiese-chemiese eienskappe van die verbinding gebruik maak, het swak intestinale absorpsie van die polifenole voorspel, terwyl OSIRIS en ChemAxon goeie absorpsie voorspel het. Ten slotte, resultate van aktiwiteit-begeleide fraksionering het getoon dat behoud van al die gewensde estrogeniese eienskappe van SM6Met in een fraksie nie ‘n haalbare doelwit is nie. Hierdie bevinding dui daarop dat verskeie van die polifenole teenwoordig in SM6Met of NPF saam, deur middel van antagonistiese, sinergistiese of additiewe effekte, die wenslike estrogeniese eieskappe verleen. Die ontwikkeling of isolering van ‘n mengsel van verbindings, met ander woorde ‘n ―intelligente‖ mengsel is dus ‘n beter strategie vir die ontwerp van ‘n estrogeniese nutraseutiese produk om die verbruikers-aanvraag van estrogeniese aktiwiteit te voorsien.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Le cancer de la prostate est le plus fréquent et représente la troisième cause de mortalité par cancer chez l’homme en France. Outre la théorie de la génétique dans le développement des cancers, l’implication des modifications épigénétiques au cours de la carcinogenèse prostatique ne fait plus aucun doute. L’épigénétique est définie comme l’étude des modifications de l’expression des gènes qui sont transmissibles lors de la mitose et/ou la méiose, mais ne découlent pas de modifications dans la séquence de l’ADN (Berger et al., 2009). Autrement dit, les altérations épigénétiques sont capables de moduler le niveau d’expression des gènes en agissant sur la chromatine. Depuis quelques années, l’attention des chercheurs porte de plus en plus sur l’implication des modifications épigénétiques dans la carcinogenèse prostatique. Une activité anormale d’un ou de plusieurs acteurs épigénétiques entraîne des modifications de profil d’expression des gènes pouvant être associées à la carcinogenèse. Dans le cancer de la prostate, on retrouve ainsi une forte activité des ADN méthyltransférases (DNMT1) et une dérégulation des modificateurs d’histones, notamment de l’histone méthyle transférase EZH2, associée à la répression de la transcription de certains gènes (Gillio-Tos et al., 2012; Gravina et al., 2013; Koh et al., 2011; Yu et al., 2007). En plus de ces facteurs épigénétiques, des facteurs environnementaux et plus particulièrement la nutrition intervient dans les processus de carcinogenèse. La thématique du laboratoire a été longtemps axée sur la nutrition et le développement des cancers. En effet, des études sur les effets préventifs des phyto-oestrogènes du soja dans la carcinogenèse prostatique ont largement été abordées (Adjakly et al., 2011). Les phyto-oestrogènes du soja ont montré leur effet déméthylant sur les promoteurs des gènes suppresseurs de tumeurs. L’hypothèse de ces travaux a été établie du fait de la faible incidence du cancer de la prostate retrouvée dans les pays asiatiques où une forte consommation de soja a été remarquée. Les micronutriments contenus dans le soja auraient ainsi la capacité de moduler les modifications épigénétiques dans le cancer de la prostate (Vardi et al., 2010). Dans le cadre de cette thèse, nous nous sommes focalisés sur les modifications des histones notamment la méthylation des histones qui reste peu étudiée dans le cancer de la prostate. Premièrement, nous avons étudié le rôle de la triméthylation de la lysine 27 de l’histone H3 (H3K27me3) dans l’implication et la progression du cancer de la prostate. La deuxième partie a consisté en l’identification de nouveaux marqueurs pronostiques et épigénétiques liés à la méthylation des histones afin d’établir un profil épigénétiques des tumeurs prostatiques. Nous avons également évoqué l’effet des phyto-oestrogènes du soja sur la modulation des modifications épigénétiques dans le cancer de la prostate et leurs rôles protecteurs dans le développement tumoral. / Prostate cancer is the most common and represents the third leading cause of cancer death in men in France. In addition to the theory of genetics in the development of cancers, the involvement of epigenetic changes during prostate carcinogenesis is no longer in doubt. Epigenetics is defined as the study of changes in the expression of genes that are transmissible during mitosis and / or meiosis, but do not result from modifications in the DNA sequence. In other words, epigenetic alterations are able to modulate the level of gene expression by acting on chromatin. In recent years, the attention of researchers has increasingly focused on the involvement of epigenetic modifications in prostatic carcinogenesis. Abnormal activity of one or more epigenetic actors leads to changes in gene expression patterns that may be associated with carcinogenesis. In prostate cancer, there is thus a strong activity of DNA methyltransferases (DNMT1) and a deregulation of histone modifiers, especially histone methyl transferase EZH2, associated with the repression of the transcription of certain genes (Gillio- Tos et al., 2012, Gravina et al., 2013, Koh et al., 2011, Yu et al., 2007). In addition to these epigenetic factors, environmental factors and more particularly nutrition is involved in the processes of carcinogenesis. The work of the laboratory has long focused on nutrition and cancer development. Indeed, studies on the preventive effects of soy phytoestrogens in prostate carcinogenesis have been widely discussed (Adjakly et al., 2011). The soy phytoestrogens showed their demethylating effect on the promoters of tumor suppressor genes. The hypothesis of this work was established because of the low incidence of prostate cancer found in Asian countries where high consumption of soybeans was noticed. The micronutrients contained in soy thus have the ability to modulate epigenetic modifications in prostate cancer (Vardi et al., 2010). In this thesis, we focused on histone modifications including histone methylation, which remains poorly studied in prostate cancer. First, we investigated the role of trimethylation of histone H3 lysine 27 (H3K27me3) in the involvement and progression of prostate cancer. The second part consisted of the identification of new prognostic and epigenetic markers related to histone methylation in order to establish an epigenetic profile of prostate tumors. We also discussed the effect of soy phytoestrogens on the modulation of epigenetic changes in prostate cancer and their protective roles in tumor development.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 256-292). Also available in print.