La graine de soja contient de grandes quantités d'isoflavones (génistéine, daidzéine et glycitéine). En raison de leurs propriétés phytoestrogéniques, ces composés peuvent avoir des effets bénéfiques sur la santé humaine, mais ils peuvent aussi être perçus comme perturbateurs endocriniens, en particulier dans les laits pour nourrissons. La teneur et la composition en isoflavones de la graine diffèrent selon la fraction considérée. Les cotylédons contiennent de la génistéine et de la daidzéine, tandis que les germes, avec une teneur quatre à dix fois supérieure, contiennent majoritairement de la daidzéine et de la glycitéine. Le génotype influence fortement la teneur en isoflavones totales. Le déterminisme du pourcentage des isoflavones est essentiellement génétique. Ce travail porte sur l'étude du déterminisme génétique à l'origine des variations de teneurs et de compositions en isoflavones dans les germes et les cotylédons de la graine, en tenant compte également du net décalage de l'accumulation entre ces deux compartiments, au cours du développement de la graine. Dans un premier temps, les gènes des isoflavone synthases (IFS) de variétés très différenciées pour leurs teneurs et profils d'isoflavones ont été séquencés, puis les expressions des gènes clefs de la biosynthèse (neuf chalcone synthases (CHS), une chalcone réductase (CHR), quatre chalcone isomérases (CHI) et les deux isoflavone synthases (IFS) ont été suivies par RT-PCR quantitative dans les cotylédons et dans les germes, à trois stades critiques du développement de la graine (25, 40 et 60 jours après floraison). La seconde partie de ce travail a été consacrée à l'étude de l'expression de différents gènes candidats de la flavonoïde 6-hydroxylase (F6H) catalysant la première étape de la synthèse de la glycitéine. Le polymorphisme des séquences génomiques IFS1 et IFS2 des isoflavone synthases n'a pas montré de lien avec les différences de teneurs en isoflavones entre les variétés. L'activité transcriptionnelle des gènes de biosynthèse des isoflavones souligne l'existence d'une régulation bien distincte de cette synthèse dans ces deux compartiments. Les taux d'expression des gènes cibles ne sont pas toujours reliés avec les différences de teneurs ou de profils dans les germes et les cotylédons, suggérant ainsi l'effet prépondérant des régulations post-traductionnelles, notamment dans la formation du complexe multienzymatique de biosynthèse de ces composés. Nous avons aussi mis en évidence une forte expression du gène CHS9 codant pour la chalcone synthase 9, avec un profil correspondant plus à l'accumulation des isoflavones dans le germe que dans les cotylédons. Les gènes CHS7 et CHS8 codant pour les chalcone synthases 7 et 8, déjà signalés comme fortement corrélés à la synthèse des isoflavones, sont plus liés à l'accumulation dans les cotylédons que dans les germes. Ces travaux montrent aussi que le gène F6H signalé dans la littérature ne s'exprime pas dans les germes. En revanche, deux candidats dont la séquence est similaire à 79% ont été étudiés. Le gène F6H3 est le seul à s'exprimer dans la graine, uniquement dans le germe. Son expression n'a pas été détectée dans les germes d'une lignée mutante qui ne produit pas de glycitéine. Ce gène est donc un candidat potentiel clef pour la synthèse de la glycitéine dans le germe. La structure particulière de l'enzyme correspondante pourrait indiquer une forte implication de l'architecture du complexe enzymatique et des contraintes qui en découlent dans l'utilisation préférentielle d'une voie ou d'une autre dans ce schéma de biosynthèse. / The soybean seed contains large amounts of isoflavones (genistein, daidzein, and glycitein). Owing to their phytoestrogenic properties, these compounds can have beneficial effects on human health, but they can also be considered as endocrine disruptors, for example in infant formulas. The isoflavone content and composition in the seed depend on the considered fraction. The cotyledons contain only genistein and daidzein, while the hypocotyls are four to ten times more concentrated and contain three isoflavones, mostly daidzein and glycitein. The genotype has a strong influence on total isoflavone content, and even more on the percentage of individual isoflavones in cotyledons and hypocotyls. The objective of this work is to investigate the genetic determinism that underlies such contrasted contents and compositions between the two seed fractions, and the relation between main biosynthetic steps and genotypic differences. First, the genes of isoflavone synthases (IFS) were sequenced in varieties with highly contrasted content and composition. The expression of different keys genes of the biosynthesis (nine chalcone synthases (CHS), a chalcone reductase (CHR), four chalcone isomerases (CHI) and the two isoflavone synthases (IFS) have then been followed by quantitative RT - PCR in the cotyledons and hypocotyls, at three critical stages of seed development (25, 40 and 60 days after flowering). Second, the expression of different candidate genes for the flavonoid 6-hydroxylase (F6H) which catalyzes the first step in the synthesis of the glycitein has been investigated. The polymorphism of the genomic sequences IFS1 and IFS2 of isoflavone synthases was not correlated with differences in isoflavone contents. The transcriptional activity of key genes of the biosynthesis of isoflavones pointed out the existence of a distinct regulation of isoflavone biosynthesis between the two seed fractions. The expression levels of target genes were not always related to differences in isoflavone content or compositions in the hypocotyls and cotyledons. This suggests the overriding effect of post-translational regulation, especially in the formation of multienzyme complex of biosynthesis of these compounds. The chalcone synthase gene CHS9 was highly expressed, with a profile similar to the accumulation of isoflavones in hypocotyls. The chalcone synthase genes CHS7 and CHS8 expressions, already reported as highly correlated to the biosynthesis of isoflavones were more related to accumulation in the cotyledons than in hypocotyls. This work has also shown that the F6H gene, reported in the literature was not expressed in the hypocotyls. However, two candidates with as highly similar coding sequence (79%) have been studied. The F6H3 gene is the only one expressed in the seed, more precisely in the hypocotyls but it was not expressed in the cotyledons. Moreover, it was not expressed in a mutant line which did not accumulate glycitein. This gene is therefore a key potential candidate for the synthesis of the glycitein in hypocotyls. The particular structure of the corresponding enzyme may indicate a strong involvement of the architecture of the multienzyme complex of isoflavones biosynthesis and the constraints arising in the preferential use of a track or another in this scheme of biosynthesis.

Chan, Ming Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-106). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Aromatase --- p.1 / Chapter 1.2 --- Tissue Specific Promoter for Aromatase Expression --- p.4 / Chapter 1.3 --- Signaling Pathway --- p.7 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.9 / Chapter 2.1 --- Chemicals And Materials --- p.9 / Chapter 2.2 --- Mammalian Cell Culture --- p.9 / Chapter 2.2.1 --- Maintenance of Cells --- p.10 / Chapter 2.2.2 --- Preparation of Cells Stock --- p.10 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.11 / Chapter 2.3 --- Tritiated Water Release Assay --- p.11 / Chapter 2.3.1 --- Aromatase Activity in Intact Cell --- p.11 / Chapter 2.3.2 --- Aromatase Assay on Recombinant Supersomes --- p.12 / Chapter 2.4 --- RNA Isolation and cDNA Synthesis --- p.13 / Chapter 2.5 --- Semi-Quantitative PCR Reaction --- p.13 / Chapter 2.6 --- Quantitative Real Time PCR Using Taqman Probe --- p.15 / Chapter 2.7 --- Western Blotting --- p.17 / Chapter 2.8 --- Measurement of Promoter Activity --- p.18 / Chapter 2.8.1 --- Plasmid Preparation --- p.18 / Chapter 2.8.2 --- Transient Transfection and Dual Luciferase Assay --- p.18 / Chapter 2.9 --- Statistical Methods --- p.19 / Chapter CHAPTER 3 --- Genistein up-regulate aromatase in Estrogen receptor alpha-transfected HepG2 cells --- p.21 / Chapter 3.1 --- Introduction --- p.21 / Chapter 3.1.1 --- Cardiovascular Disease (CVD) --- p.21 / Chapter 3.1.2 --- Phytoestrogen --- p.21 / Chapter 3.1.3 --- Estrogen Receptor --- p.24 / Chapter 3.1.4 --- Protective Mechanism Against CVD Protection --- p.25 / Chapter 3.1.5 --- Effects of genistein on LDL Receptor and Apolipoprotein A-I --- p.26 / Chapter 3.1.6 --- Effects of estradiol on LDL Receptor and Apolipoprotein A-I　 --- p.26 / Chapter 3.1.7 --- Aim of study and hypothesis --- p.27 / Chapter 3.2 --- Result --- p.29 / Chapter 3.2.1 --- ERa increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.2 --- Genistein increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.3 --- Differential Effect of MAP kinase Inhibitors --- p.35 / Chapter 3.2.4 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in ERa-transfected HepG2 cells" --- p.35 / Chapter 3.2.5 --- Genistein Increased Aromatase Protein Expression in ERa-transfected HepG2 cells --- p.38 / Chapter 3.2.6 --- Genistein Induced Aromatase mRNA Expression Attributed to Induction of Exon ̐ơ.1 Expression --- p.40 / Chapter 3.2.7 --- Genistein Induced Promoter 1.1 Transcriptional Activity in ERa- transfected HepG2 cells --- p.44 / Chapter 3.2.8 --- Genistein Increased ERE and AP-1 Reporter Activity Through Interaction with ERa --- p.47 / Chapter 3.3 --- Discussion --- p.51 / Chapter CHAPTER 4 --- "Effect of 2,3,7,8-tetrachlorodibenzo- para-TCDD (TCDD) on aromatase in MCF-7 cells" --- p.54 / Chapter 4.1 --- Introduction --- p.54 / Chapter 4.1.1 --- Breast Cancer --- p.54 / Chapter 4.1.2 --- TCDD --- p.54 / Chapter 4.1.3 --- CYP Enzymes --- p.55 / Chapter 4.1.4 --- TCDD and Breast Cancer --- p.56 / Chapter 4.1.5 --- Aim of Study --- p.56 / Chapter 4.2 --- Result --- p.57 / Chapter 4.2.1 --- Effect of TCDD on Aromatase Activity in Different Cell Lines --- p.57 / Chapter 4.2.2 --- TCDD Increased Aromatase Activity in MCF-7 Cells --- p.62 / Chapter 4.2.3 --- Effect of TCDD on Human CYP 19 Recombinant Supersomes® and MCF-7aro Cells --- p.66 / Chapter 4.2.4 --- TCDD Increased Aromatase Protein Expression in MCF-7 Cells --- p.66 / Chapter 4.2.5 --- Effect of TCDD in Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.6 --- Effect of TCDD in CYP 19 Promoter and AP-1 Promoter Activity in MCF-7 Cells --- p.70 / Chapter 4.2.7 --- Effect of TCDD in CYP 19 mRNA Half-life --- p.75 / Chapter 4.2.8 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in MCF-7 Cells" --- p.78 / Chapter 4.2.9 --- TCDD induced ERK1/2 Activation --- p.78 / Chapter 4.2.10 --- Induction of aromatase activity in MCF-7erk cells --- p.78 / Chapter 4.3 --- Discussion --- p.87 / Chapter CHAPTER 5 --- Summary --- p.90 / BIBLIOGRAPHY --- p.92

Previous studies showed that early exposure to soy isoflavones resulted in improved bone mineral density (BMD) and bone quality that resulted in stronger bones in CD-1 mice. This study investigated whether the benefits to bone health are transferred to second generation (F2) females and if there are any adverse effects on reproductive health. First generation (F1) female CD-1 mice received subcutaneous injections of the isoflavones daidzein (DAI) and genistein (GEN) or corn oil from postnatal day (PND) 1 to 10 or 21. F1 and F2 treated-females experienced earlier pubertal onset and lengthened anogenital distance but only F1 had reduced fertility, histological abnormalities in the uterus and ovaries, and altered estrous cycling. F2 had higher BMD and stronger bones at 4 months of age. In conclusion, early life exposure to soy isoflavones compromise reproductive function but confer a transgenerational benefit to bone development in CD-1 mice.

Previous studies showed that early exposure to soy isoflavones resulted in improved bone mineral density (BMD) and bone quality that resulted in stronger bones in CD-1 mice. This study investigated whether the benefits to bone health are transferred to second generation (F2) females and if there are any adverse effects on reproductive health. First generation (F1) female CD-1 mice received subcutaneous injections of the isoflavones daidzein (DAI) and genistein (GEN) or corn oil from postnatal day (PND) 1 to 10 or 21. F1 and F2 treated-females experienced earlier pubertal onset and lengthened anogenital distance but only F1 had reduced fertility, histological abnormalities in the uterus and ovaries, and altered estrous cycling. F2 had higher BMD and stronger bones at 4 months of age. In conclusion, early life exposure to soy isoflavones compromise reproductive function but confer a transgenerational benefit to bone development in CD-1 mice.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 126-148). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 126-148). Also available on the Internet.

Endophyte-infected tall fescue produces many ergot alkaloids, which have been shown to be vasoconstrictive in various vessel types of bovine. On the other hand, substantial evidence has been reported on the vasodilative effects of formononetin and biochanin A in different vessel types in humans and rats. So, a study was conducted using mesenteric vasculature collected from heifers shortly after slaughter. After 2-h incubation with formononetin (F), biochanin A (B), or an ergovaline-containing tall fescue seed extract (EXT) and their combinations, vessels were mounted in a multi-myograph to determine their ergotamine-induced contractility. Results indicated that F and B at 1 × 10-6 M and their combination did not impact the contractile response to ergotamine in mesenteric vasculature. The pre-myograph incubation of mesenteric vasculature with EXT altered the contractile response manner to ergotamine. However, at higher concentration, F and B may alleviate the reduction of vasoconstriction caused by prior exposure to EXT. To our knowledge, this study was the first to investigate the interaction of ergot alkaloids and isoflavones on in vitro bovine mesenteric vasculature. However, further investigations are necessary to understand the mechanism behind the interaction of ergot alkaloids and isoflavones on vasoactivity.

This work presents an investigation of the potential development of phenoxodiol, a novel isoflavone, for the treatment of prostate cancer. The literature concerning isoflavone epidemiology, pharmacology, clinical use and their effect on prostate cancer is reviewed. Isoflavone impact on signal transduction pathways is also reviewed with a particular focus on the Akt / mTOR signal transduction pathway, a key signaling pathway in prostate cancer cells. In vitro experiments and xenograft nude mice studies show that phenoxodiol inhibits the growth of DU145 and PC3 prostate cancer cells, when used alone and in combination with cisplatin or carboplatin. Phenoxodiol in combination with cisplatin is highly synergistic when inhibiting the growth of DU145 cells in particular. We show for the first time, that phenoxodiol and cisplatin or carboplatin, inhibits phosphorylation of Akt and mTOR in DU145 and PC3 cells. A first-in-human study of single dose phenoxodiol investigating its pharmacokinetic properties in cancer patient volunteers was performed. Extensive conjugation of phenoxodiol and a short half-life was noted in this study. A Phase I study of intravenous phenoxodiol administered weekly to patients with advanced cancer was then performed to assess toxicity of a repeated dose schedule, as well as the maximum tolerated dose. This schedule was well tolerated in patients, with lymphocytopenia noted as the main toxicity. Given the short half-life of phenoxodiol and our preclinical studies suggesting that combination with cisplatin and carboplatin synergistically inhibited prostate cancer cell growth, we also undertook a Phase I dose escalation study of oral phenoxodiol administered in combination with cisplatin or carboplatin to patients with advanced cancer. The main adverse events include hyperglycemia, hypocalcemia, mild transaminase rises, as well as nausea, constipation, infusion site reactions and lethargy. Three patients with ovarian cancer responded to treatment according to CA125 criteria, and there was a 45% reduction in prostate specific antigen level in one man with androgen independent prostate cancer. The bioavailability of the oral formulation of phenoxodiol was calculated to be 17.5%. Phenoxodiol shows promise for further development in the potential treatment of prostate cancer.

This work presents an investigation of the potential development of phenoxodiol, a novel isoflavone, for the treatment of prostate cancer. The literature concerning isoflavone epidemiology, pharmacology, clinical use and their effect on prostate cancer is reviewed. Isoflavone impact on signal transduction pathways is also reviewed with a particular focus on the Akt / mTOR signal transduction pathway, a key signaling pathway in prostate cancer cells. In vitro experiments and xenograft nude mice studies show that phenoxodiol inhibits the growth of DU145 and PC3 prostate cancer cells, when used alone and in combination with cisplatin or carboplatin. Phenoxodiol in combination with cisplatin is highly synergistic when inhibiting the growth of DU145 cells in particular. We show for the first time, that phenoxodiol and cisplatin or carboplatin, inhibits phosphorylation of Akt and mTOR in DU145 and PC3 cells. A first-in-human study of single dose phenoxodiol investigating its pharmacokinetic properties in cancer patient volunteers was performed. Extensive conjugation of phenoxodiol and a short half-life was noted in this study. A Phase I study of intravenous phenoxodiol administered weekly to patients with advanced cancer was then performed to assess toxicity of a repeated dose schedule, as well as the maximum tolerated dose. This schedule was well tolerated in patients, with lymphocytopenia noted as the main toxicity. Given the short half-life of phenoxodiol and our preclinical studies suggesting that combination with cisplatin and carboplatin synergistically inhibited prostate cancer cell growth, we also undertook a Phase I dose escalation study of oral phenoxodiol administered in combination with cisplatin or carboplatin to patients with advanced cancer. The main adverse events include hyperglycemia, hypocalcemia, mild transaminase rises, as well as nausea, constipation, infusion site reactions and lethargy. Three patients with ovarian cancer responded to treatment according to CA125 criteria, and there was a 45% reduction in prostate specific antigen level in one man with androgen independent prostate cancer. The bioavailability of the oral formulation of phenoxodiol was calculated to be 17.5%. Phenoxodiol shows promise for further development in the potential treatment of prostate cancer.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: leaf 279-310.