Genistein, daidzein, and resveratrol are polyphenolic plant compounds called phytoestrogens (PEs) because they are capable of binding and activating estrogen receptor (ER) isoforms due to structural similarity to estrogen (E2). The central hypothesis governing these investigations is that PEs can bind to plasmamembrane ERs, cross-talk with epidermal growth factor receptor (EGFR), and signal downstream to an array of molecular pathways including those involved in cell motility. This is relevant to breast cancer metastasis as PEs have been shown to have both preventative and promotional effects on breast cancer cells. The data herein investigates the role of E2, genistein, daidzein, and resveratrol in cell migration, actin cytoskeleton organization, focal adhesion assembly, as well as EGFR, focal adhesion kinase (FAK), and Rho family GTPase activation in ER (+/-) human breast cancer cells. We report that E2 and EGF increase cell migration, induce Rac-dependent lamellipodia formation, increase focal adhesion assembly, and increase EGFR, FAK, and Rac activity in ER[beta] (+) cells. We report that genistein and daidzein also increase cell migration, Rac-dependent lamellipodia formation, focal adhesion assembly, and FAK activity in ER[beta] (+) cells. Resveratrol demonstrates a biphasic concentration-dependent effect on the same signaling pathways. Resveratrol at 5 [micromolar] increases cell migration, lamellipodia formation, and increases FAK and Rac activity in ER[beta] (+) cells similar to E2. Conversely, resveratrol at 50 [micromolar] inhibits cell migration/invasion, blocks E2/EGFinduced migration, induces sustained and unpolarized filopodia, decreases focal adhesion assembly, increases EGFR activity, and decreases FAK, Cdc42, and Rac activity in ER[beta] (+) cells. The induction of filopodia by 50 [micromolar] resveratrol is partially Rho GTPaseindependent and can be observed in serum and on extracellular matrices. The induction by 50 [micromolar] resveratrol of a global, sustained extension of filopodia in conjunction with inhibition of focal adhesion assembly, as well as FAK, Cdc42, and Rac activity is hypothesized to negatively affect breast cancer cell motility. Ultimately, the elucidation of these cell structures and signaling mechanisms in response to PEs will help to determine a preventive or promotional role for plant compound-based therapies in breast cancer metastasis. / text

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

No description available.

The activity of plant estrogens, genistein (GEN) and enterolactone (ENL), on mammalian cells is not well understood. Previously, they have been shown to exhibit a biphasic effect, being stimulatory at low doses and inhibitory at high doses. Therefore, consumption of plant estrogens by women with breast cancer has become controversial. Simplified flow cytometry applications to quantify the degree of proliferation and apoptosis were used to measure differentiation and growth inhibitory properties of GEN and/or ENL on two estrogen receptor-positive, MCF-7 and T47D, and two estrogen receptor-negative, MDA-MB-231 and MDA-MB-468, breast cancer cell lines in the presence and absence of I7β-estradiol (E2). Activity in the presence of 4-hydroxytamoxifen (OHT) was also evaluated. A live cell assay using propidium iodide (PI) and fluorescein diacetate was used for evaluation of live, apoptotic and necrotic populations. The biological activity of ENL and GEN in tissue culture was confirmed by MTT assay. An Annexin V/PI live cell assay confirmed the presence and degree of apoptosis. No effect was seen at lower doses, ie. less than 10µM and 5011M in estrogen receptor-positive, ER (+), cells and decreased viability was seen at ENL 10 µM in one estrogen receptor-negative, ER (-), cell line. A significant reduction in viability was seen with treatments of 100 µM of ENL and GEN in both ER (+) cell lines. The effect of combining E2 with treatments of GEN 50 and 100 µM was a reduction in viability in one ER (+) cell line. The effect of combining treatments of low doses of ENL and GEN (10 MM) resulted in a reduction in viability in one ER (+) and one ER (-) cell line. Treatment of ER (+) breast cancer cell lines with low dose GEN (10 µM) and high dose ENL and GEN (50 MM) combined with low dose OHT (10 µM ) reduced viability in excess OHT 10 µM alone. High dose OHT (50 µM ) significantly reduced viability in all cell lines. However, when high dose OHT was combined with low dose ENL and GEN, there was an effect of rescuing ER (-) cells from OHT-induced apoptosis and necrosis. Although the use of high dose OHT is not typically used to treat ER (-) breast cancer, these results suggest the action of plant estrogens is more varied than previously thought. This work supports the notion that there may be dissimilarity in the actions between ENL and GEN which may warrant separate recommendations regarding dietary consumption, especially in women with breast cancer.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Equol is known to be a selective androgen modulator and has the ability to bind and inhibit 5-alpha dihydrotestosterone (5α-DHT). Equol is also a selective estrogen receptor modulator and is able to bind beta estrogen receptors with high affinity. As estrogen receptors are found in the hypothalamus, pituitary, and gonads, prenatally administered equol could affect the morphological and reproductive development of offspring. To test this hypothesis, during gestational days 14 to 20, forty-two pregnant Long-Evans rats were given one of six treatments: 1) no treatment, 2) injection with dimethyl sulfoxide (DMSO), 3) injection with 10 mg/kg equol, 4) injection with 21.0 mg/kg equol, 5) injection with 63.0 mg/kg equol, or 6) injection with 90.0 mg/kg flutamide. At birth the pups were weighed, anogenital distance measured, and sex was determined. Some of the animals were sacrificed and trunk blood collected from both the mothers and pups. Serum levels of phytoestrogens, estradiol, testosterone, and 5α-DHT levels were determined. Some pups were allowed to grow up to day 29 and were tested on the forced-swim test with the parameters of time mobile, time immobile, swim distance, and average speed measured. The flutamide treated pups had the lowest anogenital distance. The low equol dose animals had the largest anogenital distance. There were no significant differences in 5α-DHT serum levels in the male offspring among the treatments. However in non-injected control female offspring displayed significantly lower 5α-DHT levels than all the other groups. Mothers treated prenatally with equol displayed significantly higher circulating equol levels compared to controls values. Rats injected with 63.0 mg/kg of equol gained the least weight during pregnancy. Their offspring also had the lowest body weights at birth. Male and female offspring displayed similar behaviors in the Porsolt forced-swim test among the treatment groups. The low and high equol groups displayed the least depressive-like behaviors. The offspring from mothers treated with the medium and high equol doses both gained the most weight from birth to day 29. Treating pregnant rats with equol during the last week of gestation does not appear to have any affect on morphological genital development of the offspring.

Yuen Yee Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 108-125). / Abstracts in English and Chinese. / TITLE PAGE --- p.1 / ACKNOWLEGDEMENTS --- p.2 / ABSTRACT --- p.3 / 摘要 --- p.5 / table of contents --- p.7 / list of figures and tables --- p.13 / CHAPTER 1 GENERAL INTRODUCTION --- p.16 / Chapter 1.1 --- role of PHYTOESTROGENS in soy and red WINE the PREVENTION OF CARDIOVASCULAR DISEASES (CVD) --- p.17 / Chapter 1.1.1 --- INTRoduction and Classification of Phytoestrogens --- p.17 / Chapter 1.1.2 --- estrogenic1ty of phytoestrogens and theIr abundancesin Plasma --- p.18 / Chapter 1.1.3 --- phytoestrogens as one of the active components In cvd Protection --- p.21 / Chapter 1.1.4 --- effects of Phytoestrogens on LDL Receptor and Apolipoprotein A-1 --- p.22 / Chapter 1.2 --- role of estrogen receptors (ers) in gene regulation --- p.24 / Chapter 1.2.1 --- "structure, Classification and tissue distribution of ERS" --- p.24 / Chapter 1.2.2 --- ligands for ERS --- p.25 / Chapter 1.2.3 --- mechaniSMS OF LIgands-ERS complex in GENE regulation --- p.27 / Chapter 1.2.4 --- ligand-independent ER activation --- p.28 / Chapter 1.3 --- aims and scopes of investigation --- p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- chemicals and materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Plasmids --- p.30 / Chapter 2.2 --- mammalian cell culture maintainence --- p.30 / Chapter 2.2.1 --- Maintenance of Cells --- p.31 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.31 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.31 / Chapter 2.3 --- manipulation of dna --- p.31 / Chapter 2.3.1 --- isolation of HEPG2 cells genonmic DNA --- p.31 / Chapter 2.3.2 --- separation and purification of dna from agarose gel --- p.31 / Chapter 2.3.3 --- Restriction digestionof DNA --- p.32 / Chapter 2.3.4 --- Ligation of DNA Fragments --- p.32 / Chapter 2.3.5 --- Transformation of --- p.32 / Chapter 2.3.6 --- Small Scale Plasmids Purification from DH5a --- p.32 / Chapter 2.4 --- construction of expression and reporter plasmids --- p.33 / Chapter 2.4.1 --- Construction of Estrogen Receptorα (Erα) Expression Vectors --- p.33 / Chapter 2.4.2 --- construction of reporter vectors of LDLR promoter and the Respective Mutants --- p.33 / Chapter 2.4.3 --- Construction of Reporter Vectors of APOAI Promoter and the Respective Mutants --- p.33 / Chapter 2.5 --- determination of promoter transcrtiption activities --- p.34 / Chapter 2.5.1 --- Transient Transfection of Cell with ERa Expression Vector and Promoter Reporter using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.34 / Chapter 2.6 --- semi-quantitative and quantitative rt-pcr assay --- p.34 / Chapter 2.6.1 --- Transient transfection of Cell with ERa Expression Vector Using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.6.2 --- "Isolation of RNA using TRIzol® Reagent (Life Technology, USA)" --- p.35 / Chapter 2.6.3 --- Quantitation of RNA --- p.35 / Chapter 2.6.4 --- First Strand cDNA Synthesis --- p.35 / Chapter 2.6.5 --- Sem卜Quantitative PCR Reactions --- p.35 / Chapter 2.6.6 --- Quantitative PCR Reactions --- p.36 / Chapter 2.7 --- western blotting analysis --- p.36 / Chapter 2.8 --- statistical methods --- p.36 / Chapter CHAPTER 3 --- REGULATION BY PHYSIOLOGICAL LEVEL OF 17B-ESTRADIOL ON APOLIPOPROTEIN A-I AND LOW-DENSITY- LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.37 / Chapter 3.1 --- introduction --- p.37 / Chapter 3.2 --- results --- p.39 / Chapter 3.2.1 --- Determination of transient transfection functionality of estrogen receptors in hepg2 cells --- p.39 / Chapter 3.2.2 --- Effect of 17β-Estradiolon LDLR promoter transcription activity --- p.39 / Chapter 3.2.3 --- Effect of 17β-Estradiol on apoai promoter transcription activity --- p.40 / Chapter 3.2 --- discussion --- p.47 / Chapter CHAPTER 4 --- SOY ISOFLAVONES AND RESVERATROL DISPLAY DIFFERENT MECHANISM IN THE UP-REGULATION OF LOVV-DENSITY-LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.49 / Chapter 4.1 --- introduction --- p.49 / Chapter 4.2 --- results --- p.54 / Chapter 4.2.1 --- Association of ERα and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.2 --- Association of ERβ and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol induced LDLR promoter transcription" --- p.55 / Chapter 4.2.4 --- Identification of promoter regions responsible for induction of LDLR transcription by isoflavones in the presence OF ERα --- p.55 / Chapter 4.2.5 --- Identification of promoter regions responsible for induction of LDLR TRANSCRIPTION BY resveratrol IN THE ABSENCE OF ERα --- p.56 / Chapter 4.3 --- DISCUSSION --- p.75 / Chapter CHAPTER 5 --- SOY ISOFLAVONES AND RESVERATROL UP-REGULATE APOLIPOPROTEIN A-I SIMILAR TO 17B-ESTRADIOL IN HEPG2 CELLS --- p.80 / Chapter 5.1 --- INTRODUCTION --- p.80 / Chapter 5.2 --- RESULTS --- p.84 / Chapter 5.2.1 --- Association of ERα phytoestrogens on APCAI gene expression --- p.84 / Chapter 5.2.2 --- Association of ERβ and isoflavones or resveratrol on APOAI promoter transcription activity --- p.85 / Chapter 5.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol in APOAI promoter transcription in the presence of ERα" --- p.85 / Chapter 5.2.4 --- Identification of promoter regions responsible for induction of APOAI transcription by isoflavones and resveratrol in the presence of ERα --- p.85 / Chapter 5.3 --- DISCUSSION --- p.100 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.103 / Chapter CHAPTER 7 --- SUMMARY --- p.106 / BIBLIOGRAPHY --- p.108 / APPENDIX 1 ABBREVIATIONS --- p.126 / APPENDIX 2 MATERIALS AND METHODS --- p.129 / APPENDIX 3 PRIMER LISTS --- p.145 / APPENDIX 4 REAGENTS AND BUFFERS --- p.147

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study investigated the “Effect of Phytogenic Feed Additives on Gonadal Development in Mozambique tilapia (Oreochromis mossambicus). Aquaculture remains the fastest growing animal food-producing sector and it is set to overtake capture fisheries as a source of food fish, and also to outpace population growth. This rapid growth can be attributed to the increasing demand for aquaculture products, and an urgent need for a sustainable food resource and the safe production of food. Globally, fish provides over 3.5 billion people with up to 15 percent of their average per capita intake of animal protein. The introduction of aquaculture to sub-Saharan African (SSA) took place during the 1940s and 1950s, and the main objectives were to improve nutrition in rural areas, allow for the diversification of activities to reduce the risk of crop failures, to generate additional income, and to create employment opportunities. Aquaculture was seen as a viable option for rural development in SSA, and substantial resources were invested to support its development. Poor results were however, recorded in terms of production and sustainability. Tilapia is the most widely cultured of all fish species farmed, and the second most important group of freshwater and brackish water fish after carps. Tilapia has all the necessary traits that makes it an excellent species to culture, but costefficient production is hampered by the animals attaining sexual maturity at a an early age, which then result in precocious breeding in aquaculture systems. The production of single-sex populations (i.e. all male) is a potential means to address the problem of precocious breeding, but the technologies used to establish single-sex populations are not readily available to resource-poor communities that farm with tilapia for food purposes. Phytochemicals, also known as phytoestrogens, are plant-derived compounds that structurally or functionally mimic mammalian estrogens that affect the sexual differentiation of fish. Phytochemicals occur in plants like Pawpaw (Carica papaya) and Moringa (Moringa oleifera). The study thus had a threefold objective. Firstly, the study investigated the potential of Pawpaw seed meal (P) and Moringa seed meal (M), as part of a commercial tilapia diet, to be used as endocrine disrupting compounds (EDC’s) to control the reproduction of sexually mature Mozambique tilapia (20-45g). Secondly, assess the potential of P and M to inhibit the attainment of sexual maturity in immature tilapia (2-8g). Finally, P and M to determine its influence on sexual differentiation of tilapia fry (9-12 days posthatch) to produce all-male populations. The study indicated that both P and M seeds contain bio-active chemicals that are capable of disrupting the gonad function, differentiation and sexual maturation of Mozambique tilapia. Sperm production was affected, evident in the degeneration of the testicular tissue samples. Egg production, ovulation and spawning were all affected, as evident in the difference in colour of the degrading eggs, as well as the absence of spawning. Ovo-testes were observed in cases where diets containing 10.0g P and 10.0g M /kg basal diet were fed. Eggs were observed in the ovaries of sexually immature fish, but spawning did not occur. The study also presents the first report on the isolation of Oleanolic acid in Moringa seeds. The evident of antifertility properties of both Pawpaw and Moringa seeds can be exploited to control or prevent reproduction of Mozambique tilapia in SSA aquaculture systems. This could be of particular importance to aquaculture development in rural areas of Sub-Saharan African countries, given the abundant year round availability of these compounds. Further studies are required to optimise the preparation of the experimental compounds; as well as determining the optimal inclusion level of the phytogenic compounds, as well as how their efficacy to manipulate the reproductive potential and ability of Mozambique tilapia are influenced by environmental factors such as water temperature. / AFRIKAANSE OPSOMMING: Akwakultuur is die vinnigste groeiende dierlike voedsel-sektor. Daar word verwag dat dit visserye sal oortref as ʼn bron van voedsel en dat groei in dié bedryf selfs die bevolkingsgroei sal verbysteek. Die vinnige groei in die sektor kan toegeskryf word aan die toenemende vraag na akwakultuur produkte en 'n dringende behoefte vir 'n volhoubare voedsel hulpbron, wat ook die veilige produksie van voedsel sal verseker. Wêreldwyd voed vis meer as 3.5 miljard mense en dra tot 15% van die gemiddelde hoeveelheid dierlike proteïen per kapita ingeneem, by. Die bekendstelling van akwakultuur in sub-Sahara Afrika (SSA) het gedurende die 1940's en 1950's plaasgevind, met die belangrikste doelwitte om voeding in landelike gebiede te verbeter, geleenthede vir diversifisering te skep wat die risiko van misoeste verminder, om bykomende inkomste te genereer en werksgeleenthede te skep. Akwakultuur is gesien as 'n lewensvatbare opsie vir die ontwikkeling van die landelike gebiede in SSA en aansienlike hulpbronne is belê om die ontwikkeling daarvan te ondersteun. Swak resultate is egter in terme van produksie en volhoubaarheid behaal. Tilapia is die mees algemene spesies wat gekweek word en is die tweede mees belangrike groep van varswater en brak water vis soesies, na Karp. Tilapia beskik oor al die nodige eienskappe wat dit ʼn uitstekende spesie vir voedselproduksie maak, maar koste-doeltreffende produksie daarvan word gekortwiek deur die feit dat die spesie seksuele volwassenheid op 'n vroeë ouderdom bereik, wat dan lei tot vroeg-rype teling en die gevolglike oorbevolking en swak groei van tilapia in ʼn akwakultuur sisteem. Die produksie van enkel-geslag bevolkings (d.i. slegs manlike vis) is ʼn potensiële oplossing vir dié probleme, maar die tegnologie wat gebruik word om enkel-geslag bevolkings te produseer is nie geredelik toeganklik vir hulpbron-arme gemeenskappe wat met Tilapia vir kosdoeleindes boer nie. Fitochemikalieë, anders ook bekend as fito-estrogene, is verbindings wat in plante voorkom en wat struktureel of funksioneel die werking van die natuurlike soogdier estrogene/androgene naboots, met die fitochemikalieë wat die seksuele differensiasie van vis beïnvloed. Fitochemikalieë kom in plante soos papaja (Carica papaya) en Moringa (Moringa oleifera) voor. Die studie het dus gepoog om die potensiaal van papaja saad meel (P) en Moringa saad meel (M), as deel van 'n kommersiële tilapia dieet, om as endokriene ontwrigters (EDC's) gebruik te word om die reproduksie van seksueel volwasse Mosambiek tilapia (20-45g) te beheer, om te voorkom dat onvolwasse tilapia (2-8g) geslagsrypheid te vroeg bereik en ook om die geslagsdifferensiasie van tilapia vingerlinge (9-12 dae na uitkom) te manipuleer om enkel-geslag (manlike) produksiegroepe te produseer. Die studie het aangedui dat beide papaja en Moringa sade bio-aktiewe chemikalieë wat die werking van die gonadotrofien hormone, geslagsdifferensiasie die stadium waarop Mosambiek tilapia geslagsrypheid bereik, kan beïnvloed. Spermproduksie is negatief beïnvloed, soos waargeneem in die degenerasie van die testisweefsel. Eierproduksie, ovulasie en die vrystelling van eiers is almal negatief beïnvloed, soos duidelik waargeneem in die kleurverskil (van normale eiers) van eiers wat ʼn mate van reabsorbsie aandui en die feit dat geen eiers vrygestel is nie. Ovo-testes is waargeneem in gevalle waar diëte met 10.0g papaja en / of Moringa / kg basale dieet gevoer is. Eiers is waargeneem in die eierstokke van seksueel onvolwasse vis, maar vrystelling het nie plaasgevind nie. Die studie is die eerste verslag oor die isolasie van Oleanoliese suur in Moringa sade. Die duidelike reproduksie-beperkende (anti-vrugbaarheid) eienskappe van beide papaja en Moringa sade kan benut kan word om reproduksie in Mosambiek tilapia te voorkom of te manipuleer in SSA akwakultuur stelsels. Dit is veral van besondere belang vir akwakultuur ontwikkeling in die landelike gebiede van SSA lande, gegewe dat beide sade regdeur die jaar geredelik beskikbaar is. Verdere studies word benodig om protokolle vir die voorbereiding van die eksperimentele verbindings te optimaliseer, sowel as die bepaling van die optimale insluitingsvlakke van die fitogeniese verbindings, asook hoe die doeltreffendheid van hierdie verbindings deur omgewingsfaktore soos water temperatuur beïnvloed word. / The University for Development Studies (UDS), Ghana, particularly Prof S.K. Nokoe, and Ghana Education Trust Fund (GETFund), for the initial financial support that enabled me to enrol for the programme. The Division of Aquaculture, together with the Postgraduate and International Office (Postgraduate Funding Section), Stellenbosch University (SU) for financial support during my studies.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, may serve as a source of new estrogen analogues. It would be of great benefit if these new analogues would not only prevent the development and progression of breast cancer which, globally, is responsible for the highest number of cancer associated deaths among females, but also have a reduced side-effect profile when compared to current treatments and, in addition, also alleviate menopause associated symptoms. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for their potential to modulate molecular targets involved in prevention and treatment of breast cancer. We show that the phytoestrogenic extracts of Cyclopia antagonise estrogen-induced cell proliferation both in vitro as well as in vivo. Furthermore, our study presents various molecular mechanisms whereby the Cyclopia extracts may be eliciting this effect. Importantly, we show, for the first time, that the Cyclopia extracts behave as ERα antagonists and ERβ agonists which, with respect to the known role of the ER subtypes in breast cancer, where the ERα subtype is associated with the stimulation of cell proliferation and the occurrence of breast cancer, while ERβ ameliorates the action of ERα in breast cancer and could act as an inhibitor of breast cancer development, may be beneficial for the prevention or treatment of breast cancer. In addition, we also show that the extracts of Cyclopia behave as selective estrogen receptor degraders by down-regulating ERα protein levels while stabilising ERβ protein levels, which not only provides a possible molecular explanation for the observed ERα antagonism and ERβ agonism, but, in addition, may be beneficial as higher ERα levels are associated with malignant breast cancer tumours, while higher ERβ levels are associated with benign tumours. Furthermore, we show that the Cyclopia extracts affect the nuclear localization and distribution of both ER subtypes in a manner that provides an additional molecular explanation for the observed ERα antagonism and ERβ agonism. Investigation of the molecular processes involved in the promotion and progression of breast cancer, such as the distribution of cells between the phases of the cell cycle, cancer cell invasion, and the regulation of genes governing these processes provides evidence that the Cyclopia extracts are not as proliferative as estrogen. In addition, Cyclopia extracts display anti-inflammatory properties, which may be beneficial as inflammation is an enabling characteristic in cancer development and progression. Furthermore, this study, for the first time, shows that the phytoestrogenic extracts of Cyclopia are absorbed, are not toxic, and display biological ERα antagonist activity in vivo by retarding uterine growth. Thus, we propose that the Cyclopia extracts act as selective estrogen receptor subtype modulators with potential to be developed as a nutraceutical for the treatment or prevention of breast cancer. / AFRIKAANSE OPSOMMING: Fitoëstrogeen-bevattende ekstrakte van Cyclopia, ‘n inheemse Suid Afrikaanse fynbosplant wat gebruik word vir die voorbereiding van heuningbostee, mag as ‘n bron van nuwe estrogeen-analoë dien. Dit sal baie voordelig wees indien hierdie nuwe analoë nie net die ontwikkeling en progressie van borskanker sal voorkom nie, aangesien borskanker wêreldwyd verantwoordelik is vir die grootste getal kankerverwante sterftes onder vroue, maar ook ‘n verminderde newe-effek profiel vertoon in vergelyking met huidige behandelings en ook, boonop, simptome wat met menopouse geassosieer word, sal verlig. In hierdie studie is drie ekstrakte, P104, SM6Met, en cup-of-tea, vanaf twee spesies van Cyclopia, C. genistoides en C. subternata, geëvalueer vir hul potensiaal om die molekulêre teikens betrokke by die voorkoming en behandeling van borskanker te moduleer. Ons wys dat die fitoëstrogeniese ekstrakte van Cyclopia antagoniseer estrogeen-geïnduseerde selproliferasie beide in vitro as ook in vivo. Verder bied ons studie ook verkskeie molekulêre meganismes aan oor hoe die Cyclopia ekstrakte hierdie effek mag ontlok. ‘n Belangrike bevinding is dat ons vir die eerste keer wys dat die Cyclopia ekstrakte hulself as ERα -antagoniste en ERβ-agoniste gedra wat, met betrekking tot die erkende rol van die ER-subtipes in borskanker, waar die ERα-subtipe geassosieer word met die stimulasie van selproliferasie en die gebeurtenis van borskanker, terwyl ERβ die aksie van ERα onderdruk en as ‘n inhibeerder van borskankerontwikkeling kan dien, voordelig mag wees vir die voorkoming of behandeling van borskanker. Ons wys boonop ook dat die ekstrakte van Cyclopia hulself soos selektiewe estrogeen- reseptor-degradeerders gedra deurdat hul ERα-proteïnvlakke verlaag terwyl hul ERβ-proteïnvlakke stabiliseer. Dit verksaf nie net ‘n moontlike molekulêre verduideliking vir die waargeneemde ERα-antagonisme en ERβ-agonisme nie, maar mag ook voordelig wees in borskanker aangesien hoër ERα-vlakke geasosieer word met kwaadaardige borskankertumors en hoër ERβ-vlakke met nie-kwaadaardige tumors. Verder wys ons dat die Cyclopia ekstrakte die lokalisering en verspreiding van beide ER-subtipes in die selkern op so ‘n wyse beïnvloed dat dit ‘n addisionele molekulêre verduideliking bied vir die ERα-antagonisme en ERβ-agonisme wat waargeneem is. Verdere ondersoek van die molekulêre prosesse betrokke by die promosie en progressie van borskanker, soos die verspreiding van selle tussen die fases van die selsiklus, die beweging van kankerselle na omliggende weefsels, en die regulering van gene wat hierdie prosesse beheer, verskaf bewyse dat die Cyclopia-ekstrakte nie so proliferatief is soos estrogeen nie. Die ekstrakte van Cyclopia vertoon boonop ook anti-inflamatoriese eienskappe, wat voordelig mag wees aangesien inflammasie ‘n bydraende eienskap in kankerontwikkeling en -progressie is. Verder wys hierdie studie vir die eerste keer dat die fitoëstrogeniese ekstrakte van Cyclopia geabsorbeer word, nie toksies is nie, en dat hulle biologiese ERα-antagonis aktiwiteit vertoon deurdat hulle uterus-groei vertraag in vivo. Dus stel ons voor dat die Cyclopia-ekstrakte optree soos selektiewe-estrogeen-reseptor-subtipe-moduleerders met die potensiaal om ontwikkel te word as ‘n nutraseutiese middel vir die behandeling of voorkoming van borskanker.

Metabolomics is an emerging field that entails the detailed characterization of the ensemble of metabolites produced by living organisms; subfields include drug metabolism and natural environmental toxin production. The first part of the dissertation pursued metabolism of glyceollins, i.e., isoflavones produced by soybeans, that are potential cancer therapy agents. In vivo glyceollin metabolites produced in rats were investigated by on-line Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry. An odd-electron fragment ion at m/z 148, formed in violation of the even-electron rule, and diagnostic of the glyceollin backbone, was discovered. Based on this finding, a negative mode precursor ion scanning method was developed to screen for glyceollins and their metabolites from biological samples. Products of both Phase I and Phase II metabolism were identified, none of which have been previously reported. Sulfated metabolites were confirmed by accurate mass measurement, while glucuronide conjugation was confirmed by enzyme-assisted glucuronidation by rat liver microsomes. Intact GSH-glyceollin conjugates were not observed, but breakdown products of the GSH pathway, i.e., cysteinylglyceine, cysteine, and acetylated cysteine, were identified as conjugates of oxygenated glyceollins. The identification of GSH by-product conjugates was confirmed in product ion spectra acquired in the negative mode (where peptide anions, and glyceollin-bearing cleaved peptide portions were observed), as well as in the positive mode (where intact oxygenated glyceollin fragments appeared without the initially-present peptide portion). Mass spectral evidence strongly supports a metabolic pathway involving initial epoxidation of glyceollins followed by GSH addition at the epoxidation site. The second part of the dissertation undertook the investigation of secondary metabolites called microbial volatile organic compounds (MVOCs) produced by fungi (mold) that have been reported to have adverse human health effects. MVOCs were collected onto different sorbent materials and analyzed by Thermal Desorption Analysis coupled with on-line Gas Chromatography-Mass Spectrometry. Fungal MVOCs were characterized from various simulated flooding conditions (brackish, freshwater, and saltwater) and different substrates (nutrient rich vs. low nutrient) to determine diagnostic MVOCs. Ten fungi from simulated environments were identified by genetic sequencing. Cladosporium sp. and Chaetomium sp. were cultivated and their emitted MVOCs, 3-furaldehyde and 3-(4-hydroxy-3-methoxyphenyl)-2-propenal, were proposed as diagnostic indicators of these fungi.