Bradyrhizobium japonicum ist ein Bodenbakterium, welches in der Lage ist mit der bedeutenden Agrarpflanze Sojabohne zu interagieren und deren Wachstum zu fördern. In der vorliegenden Arbeit wurde das Genistein-Stimulon und verschiedenen Stressantworten von B. japonicum mittels Mikroarrayanalyse bestimmt. 101 Gene werden nach Genisteinzugabe induziert. NodW ist der Hauptaktivator der Genistein-induzierbaren Gene, welche zum Großteil kein nod-Box-Motiv in der Promotorregion aufweisen. Einzig acht Gene, wovon sieben für Transportersysteme kodieren, zeigen in der NodW-Mutante 613 nach Genisteinzugabe weiterhin ein erhöhtes Expressionsniveau. In weiterführenden Arbeiten konnte für zumindest ein Transportersystem (Bll4319/Bll4320/Bll4321) neben der Genistein-Abhängigkeit auch eine Regulation durch den Genistein-abhängigen TetR-Regulator FrrA nachgewiesen werden [Günther 2007; Bhandari 2008]. Dies zeigt, dass NodW in weiterführende Regulationskaskaden eingebunden sein muss, wobei diese in der vorliegenden Arbeit nicht näher charakterisiert wurden. Die Mikroarraydaten geben lediglich Hinweise auf mögliche Regulationskaskaden. Anhand der Mikroarraydaten liegt die Vermutung nahe, dass die Transkriptionsaktivierung des lateralen Flagellenclusters ebenfalls einer indirekten NodW-abhängigen Regulation unterliegt. Durch Pflanzentests war bekannt, dass es B. japonicum 901 möglich ist, den NodW-Defekt bei der Nodulation der Wirtspflanzen zu überwinden [Grob et al. 1993]. Dies beruht auf die Überexpression des 2-Komponentenregulators NwsB. So können Symbiose-relevante Gene wie die nod-Box-assoziierten Gene ähnlich dem Wildtyp nach Genistein-Zugabe induziert werden. NwsB ist aber nicht in der Lage, das Flagellarcluster der lateralen Flagellen (bis auf blr6846) nach Genistein-Zugabe zu induzieren. Dies bedeutet, dass trotz der hohen Ähnlichkeit zwischen den 2-Komponentenregulatoren NodW und NwsB, die Bindestellen in der Promotorregion der Genistein-induzierbaren Genen nicht identisch sind. Die NodD1-Mutante 1267 ist in der Lage, weiterhin Knöllchen mit allen Wirtspflanzen von B. japonicum zu bilden [Göttfert et al. 1992], was wahrscheinlich auf ein funktionelles NodW als Hauptaktivator der nodYABC-Operons zurückzuführen ist. Anhand der Mikroarraydaten ist festzustellen, dass kein weiteres nod-Box-assoziiertes Operon in der NodD1-Mutante 1267 verstärkt exprimiert vorliegt. Dies könnte bedeuten, dass NodW die Transkription von nodD1 und des nodYABC-Operons startet und später durch NodD1 in der Aktivierung der nod-Box-assoziierten Gene unterstützt wird. In der vorliegenden Arbeit wurde die transkriptionelle Stressantwort von B. japonicum hinsichtlich pH 4, pH 8, 80 mM NaCl, Hitzeschock und Temperaturstress analysiert. Dabei konnten sowohl Aussagen über Gene, welche in die allgemeine als auch in die spezifische Stressantwort eingebunden sind, getroffen werden. Die transkriptionelle Antwort auf pH 8 war mit 1636 differenziell exprimierten Genen die umfangreichste Stressantwort der vorliegenden Arbeit. Hierbei konnte gezeigt werden, dass B. japonicum bei pH-Stress besonders Gene der pHi-Homöostase aktiviert. Dies umfasst sowohl Transportergene als auch enzymatische Gene. Interessant waren die differenziell exprimierten Gene, welche bei pH 8 verstärkt und bei pH 4 verringert exprimiert vorlagen. Diese Gene besitzen im Promotorbereich eine RegR-Box und sind in der transkriptionellen Aktivierung von RegR abhängig [Lindemann et al. 2007]. Aufgrund der Homologie des RegSR-Systems von B. japonicum mit dem pH-abhängigen ActSR-System von S. meliloti besteht die Möglichkeit, dass RegSR ebenfalls in die pH-abhängige Regulation dieser Gene eingebunden ist. Neben solch spezifischen Stressantworten konnte auch eine allgemeine Stressantwort ermittelt werden. So weisen fünf Gene ein gleiches Expressionsmuster in den untersuchten Stressanalysen dieser Arbeit auf. blr5264 kodiert hierbei für einen 2-Komponenten-Hybridsensor und -Regulator und könnte als Regulator in die allgemeine Stressantwort von B. japonicum eingebunden sein. Aus diesem Grund wurde Blr5264 in GscR (general stress control regulator) umbenannt. Mittels Mutagenese von gscR wurde B. japonicum D826 erzeugt, dessen Transkriptom bezüglich Salzstress und Hitzeschock verifiziert wurde. Es wurden 87 Gene identifiziert, welche bei Stresseinfluss von GscR abhängig sind. Hierunter sind sieben verringert exprimierte Gene, welche bei Stress im Wildtyp verstärkt exprimiert vorliegen. Diese scheinen demzufolge bei Stress durch GscR transkriptionell aktiviert zu werden. Das interessanteste Gen war hierbei blr7881, welches für einen ArsR-Typ-Regulator kodiert und einen weiteren Schritt in der allgemeinen Stressantwort von B. japonicum sein könnte.

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the United States. It is also well recognized that the incidence of CVD is substantially increased in postmenopausal women due to the loss of estrogen. Experimental and clinical data support vascular protective effects of estrogen by various mechanisms. However, administration of estrogen is also associated with an increased incidence of heart disease which limits its therapeutic potential. Given the demonstrated risks of conventional estrogen therapy, a search for novel, cost-effective, alternative vasoactive agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity. Genistein, a major soy isoflavone, may be one of those alternative agents because of its selective affinity to estrogen receptor-beta and various beneficial effects on CVD. However, the mechanism of the cardioprotective effects of genistein is still unclear. The objectives of this study were (1) to investigate the effect of genistein on the expression of endothelial nitric oxide synthase (eNOS) both in vitro and in vivo; (2) to define the mechanism by which genistein regulates eNOS expression; and, (3) to examine whether genistein protects against tumor necrosis factor-alpha (TNF-α)-induced apoptosis in human aortic endothelial cells (HAECs). The results demonstrated that genistein, at physiologically achievable concentrations (1-10 μM) in individuals consuming soy products, enhanced the expression of eNOS protein and subsequently elevated nitric oxie (NO) synthesis in both HAECs and human umbilical vein endothelial cells, concomitant with the increased eNOS mRNA expression (2.6-fold of control) and eNOS promoter activity, suggesting that genistein activates eNOS transcription. Furthermore, dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt kinase, protein kinase C or inhibition of typrosine kinases, but possibly through activating the cAMP/protein kinase A/cAMP responsive elemant binding protein pathway. These data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increase in eNOS expression and NO synthesis, thereby improving vascular homeostasis. We also found that genistein (5-10 μM) significantly inhibited TNF-α-induced apoptosis in HAECs as determined by caspase-3 activation, apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of anti-apoptotic Bcl-2 protein and its promoter activity that was ablated by TNF-α. Moreover, this anti-apoptotic effect of genistein was not mediated by extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptor. However, inhibition of p38 mitogen-activated protein kinase (p38) by SB203580 completely abolished the cytoprotective effect of genistein, suggesting that genistein acted through the p38-dependent pathway. Accordingly, stimulation of HAECs with genistein resulted in rapid and dose-dependent activation of p38. Unlike TNF-α which specifically activated p38α, genistein selectively induced phosphorylation of p38β, suggesting that p38β, but not p38α, is essential for the cytoprotective effect of genistein. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Taken together, the resuls of the present study suggest that genistein can act directly on vascular ECs, improves endothelium homeostasis by promoting eNOS expression and endothelial-derived NO synthesis through activating the cAMP/PKA/CREB cascade, and protects against TNF-α-induced apoptosis via activation of p38 β. These data potentially provide a basic mechanism underlying the physiological effects of genistein in the vasculature. / Ph. D.

Stressors in the aquaculture environment can lead to negative impacts on growth and immune health, resulting in susceptibility to infectious diseases. These stressors are expected to increase as the growth of aquaculture continues to rise to meet demands for quality fish protein. Isoflavones, as a crude extract or as a pure isolate, may be effective in modulating the stress response, promoting growth and immunity. The objective of these studies was to examine the effect of various pure isoflavone isolates and crude isoflavone extracts on stress, growth, and immunity. Nile tilapia (Oreochromis niloticus) were stressed by adding hydrocortisone to the feed. In a 7-week study, pure isoflavone isolates of genistein and puerarin were evaluated to determine their respective effects on stress, growth, and immunity. A separate 10-day physiological and 6-week growth study focused on crude isoflavone extracts from kudzu (Pueraria lobata), red clover (Trifolium pratense), and soybean (Glycine max) was performed to determine their respective effects on stress, growth, and immunity. Numerous physiological parameters of the fish were measured (serum cortisol concentration, blood glucose concentration, hematocrit, hepatosomatic index, plasma protein concentration, lysozyme activity, and spleensomatic<br>index) to determine the effects of these pure isoflavone and crude isoflavone extracts on the modulation of stress and immunity. Many growth parameters were examined (length, weight, condition factor, weight gain, specific growth rate, feed intake, feed conversion ratio, and protein efficiency ratio) as well to determine the effects of these pure isoflavones and isoflavone extracts on growth. The addition of isoflavone and crude isoflavone extracts to the diet of Nile tilapia ameliorated some of the negative consequences of stress. Compared to stressed fish fed commercial feed, genistein and puerarin added to the diet appeared to improve serum cortisol concentrations, which resulted in increased plasma protein, albeit at different durations of stress. Puerarin, as well as all three crude isoflavone extracts, significantly increased spleen-somatic index compared to non-supplemented stressed fish, although the crude isoflavone extracts did not appear to improve serum cortisol concentrations. Crude isoflavone extracts also showed overall increases in lysozyme activity compared to non-supplemented stressed fish, although this was not significant. Genistein, puerarin, and red clover showed increased growth rates, feed conversion ratio, and protein efficiency. Overall, pure isolates of isoflavone appear to be more effective in modulating stress, immunity, and growth than the crude isoflavone extracts, although red clover extract showed promises in the ability to modulate the stress response and improve growth and immunity. There are likely substantial interactions between the isoflavones in the crude extracts that cannot be fully understood by measuring the effects of single isoflavones. Regardless, isoflavone supplementation (pure or crude) appeared to generally have an overall positive impact on stressed Nile tilapia, requiring more research to better understand the effects and mechanisms behind these isoflavones.

This study determined the impact of vitamin C dose on genistein-induced apoptosis in LNCaP cancer cells at various treatment regimens in vitro. Although the linear regression of viability assay (MTT) indicated a p-value = 0.11; NBT assay reveal a declining SOD activity during cell death. Apoptosis induction was the main mode of treatment induced cell death. The overall data showed the trend of treatment efficacy as;(Gen 10uM + Vit C 40uM) > (Gen 30uM + Vit C 40uM) > (Gen 70uM + Vit C 40uM) > 10uM genistein > 70uM genistein. The chi-square test for comparing necrosis, apoptosis and life cells showed that Vitamin C could impact genistein-induced apoptosis in LNCaP cells (p = 0.0003). This study forms the basis for in vivo studies of the impact of vitamin C on genistein-induced apoptosis in LNCaP prostate cancer cells. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

An estimated 220,800 new prostate cancer cases and 27,540 deaths are expected to occur in US men by the end of 2015. Despite the increased treatment modes for prostate cancer, there is still no definite cure, and prognosis remains, at best, cautiously optimistic. The explicit amalgamation of two or more cancer therapeutic modalities such as surgery, radiation, and chemotherapy, has been one of the main interests of clinical investigation for several decades. Genistein (GN) and Beta-lapachone (BL) are two of the most promising anticancer phytochemical compounds. However, the anticancer activities of BL have been correlated with the enzyme activity of NQO1. The aim of this study was to investigate the enhancing effects of VLDR derived from a portable pyroelectric crystal generator on the chemopreventive and/or chemotherapeutic effects of GN and BL in NQO1+ PC3 and NQO1± (deficient) LNCaP prostate cancer cells (PCa) in vitro. The combination treat ment-induced cytotoxicity was investigated via MTT and Trypan blue exclusion assays. Dicoumarol (an NQO1 inhibitor) was co-administered to assess the effect of VLDR on NQO1 modulation. Nitro-blue tetrazolium assay was used to assess the intracellular ROS levels. Fluorescence microscopy was also used to assess the mode of cell death. In this study, a novel quantitative modeling approach was employed to comparably assess the cytotoxic effects of specific drugs used alone or in combinations with VLDR and to predict the potential synergistic therapeutic combinations. The data suggests that VLDR induced a rise in ROS levels, followed by upregulation in NQO1 levels. Pharmacodynamic indices were developed to quantify and characterize the combination treatment as synergistic, additive or antagonistic per dose or time-interval. Synergism was found to be dose and time-interval dependent. The major mode of cell death by this combination therapeutic regimen was found to be via apoptosis . In conclusion, our results confirm that VLDR enhanced cytotoxicity effects of both drugs dose- and time-dependently. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Made available in DSpace on 2017-07-21T19:59:54Z (GMT). No. of bitstreams: 1 Camila Juraszeck.pdf: 2382188 bytes, checksum: de4167e36249ed23da96ba41c11fe677 (MD5) Previous issue date: 2011-04-19 / Genistein has estrogenic activity and can bind to estrogen receptors (ER), so it is considered a phytoestrogen. ER have been reported in macrophages and in that sense, the estrogens modulate immune responses. Despite the sexual dimorphism of the immune responses in females and males is well established, there are few studies that elucidate the role of bioactive compounds such as genistein among the genders. We investigated the effects of genistein on mice macrophage functionality, benchmarking males and females, which are divided into three groups according to sex and stage of the estrous cycle. First we checked the cytotoxicity,employing the technique of MTT reduction, in the following compounds: genistein, quercetin and 17-estradiol in the presence or absence of lipopolysaccharide (LPS). And none of these altered cell viability. To test the functionality of macrophages, the cells were treated for 24 h with two concentrations of genistein and quercetin (5M and 10M) as well as two concentrations of 17-estradiol (0,01 and 10 M), besides the control of the vehicle, the dimethylsulfoxide (DMSO) at 0,5%. The macrophages’ funtion standarts determined by spectrophotometry were the NO production in both basal and LPS induced, the production of H2O2 induced by LPS and by light microscopy was evaluated the phagocytic ability of macrophages, which were challenged to phagocytize Zymosan particles opsonized or not, after two hours of treatments. The results show a inhibition percentage of basal NO production in macrophages treated with genistein 10M, 34% and 40% for males and females in diestrus, respectively. In the macrophages obtained from females in proestrus quercetin treatment alone (in lower and higher concentration) showed a decrease in basal NO production significantly compared to control, with inhibition of 24% and 34% respectively. As NO production induced by LPS, macrophages from males showed significant inhibition of production with all treatments (except quercetin 5 μM), while in females in diestrus, treatments genistein (5 and 10 μM) and quercetin (5 and 10 μM ), this parameter significantly decreased functional macrophages, with values corresponding respectively to 25%, 30%, 17% and 31%. Once in females in proestrus, basal NO production induced by LPS and was inhibited only by quercetin (10M). All treatments of macrophages collected from the male reduced the production of H2O2 and genistein inhibition percentage of 10M of 27%. In females in diestrus was observed that treatment with genistein 5 e 10 M and quercetin 10M inhibited respectively 29%, 32% and 37% the production of H2O2. The H2O2 production by macrophages from females in proestrus was significantly inhibited by all treatments (except estradiol 0,01 ηM and quercetin 10M), and the percentage inhibition of genistein 5 e 10M was 22%. The phagocytic activity of macrophages was not affected by treatment with genistein for either group. Our results suggest that NO production, macrophages premodulated by exposure to 17-estradiol, the effect of genistein is less pronounced, may possible be explained by downregulation of ER in ex vivo treatments. In conclusion we can infer that, the use of genistein should account for sex, and therefore variations in serum hormone concentrations of 17-estradiol (sexual cycle) in females. / A genisteína têm atividades estrogênicas, e pode se ligar aos receptores de estrogênio (RE), por isso é considerada um fitoestrógeno. RE têm sido relatados em macrófagos e nesse sentido, os estrogênios modulam as respostas imunes. Apesar do dimorfismo sexual das respostas imunes de fêmeas e machos estar bem estabelecido, existem poucos estudos que elucidem o papel de compostos bioativos como a genisteína entre os sexos. Neste estudo, investigamos os efeitos da genisteína na funcionalidade de macrófagos de ratos, avaliando comparativamente machos e fêmeas, sendo estes divididos em três grupos de acordo com o sexo e a fase do ciclo estral. Primeiramente verificamos a citotoxicidade, empregando a técnica de redução do MTT, dos seguintes compostos: genisteína, quercetina e 17-estradiol, na presença ou ausência de lipopolissacarídeo (LPS). E nenhum destes alterou a viabilidade celular. Para o ensaio da funcionalidade dos macrófagos, as células foram tratadas por 24 horas com duas concentrações de genisteína e quercetina (5M e 10M), como também duas concentrações de 17-estradiol (0,01 e 10 M), além do controle do veículo, o dimetilsulfoxido (DMSO) na concentração de 0,5%. Dos parâmetros de funcionalidade dos macrófagos, foram determinadas por espectrofotometria a produção de NO tanto basal quanto a induzida por LPS, bem como a produção de H2O2 induzida por LPS, e por microscopia de luz realizou-se a avaliação da capacidade fagocítica dos macrófagos, que foram desafiados a fagocitar partículas de Zimosan opsonizadas ou não, após tratamentos de 2 horas. Os resultados encontrados revelam uma porcentagem de inibição da produção de NO basal de macrófagos tratados com genisteína 10 M de 34% e 40%, para machos e fêmeas em diestro, respectivamente. Nos macrófagos obtidos das fêmeas em proestro apenas o tratamento quercetina (em menor e maior concentração) apresentou diminuição na produção de NO basal significativa em relação ao controle, com inibição de 24% e 34% respectivamente. Quanto a produção de NO induzida por LPS, macrófagos de machos apresentaram inibição significativa da produção com todos os tratamentos (exceto quercetina 5M), enquanto nas fêmeas em diestro, os tratamentos genisteína (5 e 10 M) e quercetina (5 e 10 M), diminuíram significativamente esse parâmetro funcional de macrófagos, com os valores correspondentes respectivamente a 25%, 30%, 17% e 31%. Já nas fêmeas em proestro, a produção de NO basal e induzida por LPS foi apenas inibida pela quercetina (10M). Todos os tratamentos dos macrófagos obtidos de machos reduziram a produção de H2O2, sendo porcentagem de inibição da genisteína 10 μM de 27%. Nas fêmeas em diestro observa-se que os tratamentos com genisteína 5 e 10M e quercetina 10 M inibiram respectivamente em 29%, 32% e 37% a produção de H2O2. A produção de H2O2 pelos macrófagos das fêmeas em proestro foi inibida significativamente por todos os tratamentos (exceto estradiol 0,01 M e quercetina 10 M), sendo que a porcentagem de inibição da genisteína 5 e 10 M foi de 22%. A atividade fagocítica dos macrófagos não foi influenciada pelo tratamento com a genisteína para nenhum dos grupos. Nossos resultados são sugestivos de que a produção de NO nos macrófagos prémodulados pela exposição ao hormônio estradiol o efeito da genisteína é menos acentuado, podendo ser explicado por possível downregulation dos RE em tratamentos ex vivo. Em conclusão podemos inferir que para o emprego da genisteína deve se levar em consideração o sexo, e por conseguinte, a variação da concentração hormonal sérica de 17-estradiol (ciclo sexual) em fêmeas.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2014-09-09T11:45:33Z No. of bitstreams: 2 Dissertacao_Lorena_Maione_Silva_Ciencias_Farmaceuticas.pdf: 516603 bytes, checksum: ec25498f249629c65bc31a3ef2bd8588 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-09T11:45:33Z (GMT). No. of bitstreams: 2 Dissertacao_Lorena_Maione_Silva_Ciencias_Farmaceuticas.pdf: 516603 bytes, checksum: ec25498f249629c65bc31a3ef2bd8588 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Genistein (GEN), isoflavone contained in soybeans, shows activity against a large number of cancers, including skin cancer. However, to be used topically it is essential the association of GEN in an appropriate formulation. The aim of this study was the development and characterization of solid lipid nanoparticles (SLN) contends genistein for topical application. A bioanalytical method was developed and validated for GEN quantification in skin layers with High Performance Liquid Chromatographer (HPLC) with UV detection. The SLN was obtained with glyceryl behenate, polysorbate 80, sorbitan trioleate and different amounts of cetylpyridinium chloride (CPC) (0.5 - 0.05%). The characteristics in terms of particle size, size distribution, zeta potential, entrapment efficiency and drug recovery were evaluated. In vitro release, passive and iontophoretic, skin permeation studies were performed. Cytotoxicity studies were carried out in melanoma cells (B16F10) with free drug and SLN with and without GEN. The analytical method was linear in the concentration range of 0.1 to 60 mg/mL. Limit of quantification was 100 ng/mL for both skin layers. Recovery of the drug ranged from 95.57 to 97.57%. The method was able to analyze the GEN without suffering interference from endogenous skin components. SLN loaded with GEN showed positive surface (+ 23 mV) and average size of 343 nm. Particles were obtained with high entrapment efficiency (93%) and drug load was 5.45%. In vitro release studies demonstrated that the release of GEN from SLN occurs in two stages with a large amount of drug released within the first six hours, followed by a slow release of the remaining drug in the lipid matrix of SLN in the following hours. When administered in the skin, during in vitro passive permeation studies, the SLN increased retention of GEN in stratum corneum and remaining skin compared with free GEN. After iontophoresis application in formulation of SLN containing GEN thirteen times more GEN was retained on stratum corneum and three times more drug was retained on remaining skin. In cellular cytotoxicity studies SLN favored the increase of interaction of drug with the cells and the cytotoxicity was concentration-dependent. Thus, GEN loaded SLN increased drug skin permeation and retention and shows to be a potential formulation for topical application. / A genisteína (GEN), isoflavona contida nos grãos da soja, apresenta atividade contra um grande número de tipos de câncer, incluindo o câncer de pele. No entanto, para ser usada topicamente é fundamental que a GEN esteja associada a uma formulação adequada. O objetivo deste trabalho foi desenvolver e caracterizar nanopartículas lipídicas sólidas (NLS) contendo genisteína para aplicação tópica. Para a quantificação da genisteína nas diferentes camadas da pele foi desenvolvida e validada uma metodologia bioanalítica em cromatografia líquida de alta eficiência (CLAE) com detecção no UV. As NLS foram obtidas com behenato de glicerila, polissorbato 80, trioleato de sorbitano e tensoativo catiônico cloreto de cetilpiridínio (CPC) em diferentes quantidades (0,5 – 0,05%). As NLS foram caracterizadas quanto ao tamanho, PdI, potencial zeta, eficiência de encapsulação e recuperação. Foram realizados estudos in vitro de liberação e permeação cutânea passiva e iontoforética. Estudos de citotoxicidade foram realizados em linhagem de melanoma (B16F10) com fármaco livre e NLS com e sem GEN. O método de quantificação do fármaco mostrou-se linear na faixa de concentração de 0,1 a 60 μg/mL. O limite de quantificação foi de 100 ng/mL para ambas as camadas da pele. A recuperação do fármaco variou entre 95,57 a 97,57%. Ainda, o método foi capaz de analisar a GEN sem sofrer interferência dos componentes endógenos da pele. As NLS carregadas com fármaco apresentaram carga superficial positiva (+ 23 mV) e tamanho médio de 343 nm. Também foram obtidas partículas com alta eficiência de encapsulação (93%) e carga de fármaco de 5,45%. Os estudos de liberação in vitro demonstraram que a liberação da GEN a partir das NLS ocorre em duas fases, com uma grande quantidade de fármaco liberada nas seis primeiras horas, seguida por uma liberação lenta do restante do fármaco da matriz lipídica das NLS nas horas seguintes. Quando administrada na pele, nos estudos de permeação passiva in vitro, as NLS aumentaram a retenção da GEN tanto no estrato córneo quanto na pele remanescente em comparação à administração da GEN livre. Após a aplicação da iontoforese na formulação de NLS contendo GEN, treze vezes mais GEN ficou retida no EC e três vezes mais fármaco ficou retido na pele remanescente. Nos estudos de citotoxicidade, as NLS favoreceram o aumento da interação do fármaco com as células e a citotoxicidade foi concentração-dependente. Deste modo, a encapsulação da GEN em NLS aumentou a permeação e retenção do fármaco na pele, demonstrando assim, potencial para aplicação tópica.

Die Behandlung menopausaler und postmenopausaler Beschwerden ist mit einem erhöhten Risiko verbunden, an Mamma- und Endometriumskarzinomen zu erkranken. Darüber hinaus zeigen epidemiologische Studien, dass in ostasiatischen Ländern postmenopausale Beschwerden, osteoporotische Frakturen und Herz-Kreislauferkrankungen seltener auftreten als in westlichen Ländern. Vor diesem Hintergrund war es Ziel der Untersuchungen dieser Arbeit, in dem Tiermodell der ovarektomierten Ratte die mögliche Bedeutung von estrogenrezeptorsubtypspezifischen Einflüssen für hormonell bedingte Erkrankungen und Beschwerden zu untersuchen. Hierbei sollten gewebespezifische Wirkungen estrogenrezeptorsubtypspezifischer Liganden untersucht und explizit die Bedeutung der Estrogenrezeptorsubtypen ERα und ERβ bei der Gewebehomöostase in Knochen und Darm analysiert werden. Darüber hinaus sollten vor dem Hintergrund estrogenrezeptorsubtypspezifischer Wirkungsweisen gewebespezifische Einflüsse des Phytoestrogens Genistein (Gen) näher charakterisiert werden. Es konnte gezeigt werden, dass nach einer subkutanen Applikation der knochenprotektive Einfluss von Gen mit dem von Estradiol (E2) vergleichbar ist, durch die Kombination mit Bewegung verstärkt wird und über den ERα vermittelt zu sein scheint. Es zeigte sich außerdem, dass der stimulierende Einfluss von E2 auf den motorischen Antrieb ERα-vermittelt ist und ERβ-spezifische Liganden ebenso wie Gen diesen Effekt antagonisieren. Des Weiteren wurde deutlich, dass E2, nicht aber Gen über den ERα Einfluss auf die Körperfettverteilung nimmt. Mit einer phytoestrogenreichen Diät konnten in adulten Ratten physiologisch relevante Gen/Dai-Plasmaspiegel erreicht werden, allerdings blieben hierbei die nach einer subkutanen Applikation beobachteten knochenprotektiven Effekte dieser Phytoestrogene aus. Bei der näheren Betrachtung der Gewebehomöostase im Dünndarm zeigte sich, dass über den im Darm verstärkt exprimierten ERβ antiproliferative und proapoptotische Effekte vermittelt werden und Gen in diesem Gewebe wie ein ERβ-spezifischer Agonist wirkt. Bezogen auf eine hormonell bedingte Osteoporose, wie sie bei einem Großteil postmenopausaler Frauen auftritt, scheint das Phytoestrogen Genistein eine mögliche Alternative zur Hormonersatztherapie darzustellen. Außerdem zeigt sich, dass Genistein gewebe- und estrogenrezeptorsubtypspezifische antagonistische und agonistische Einflüsse hat und somit die Charakterisierung als „Phyto-SERMs“ (pflanzlicher selektiver Estrogenrezeptormodulator) zutreffend ist. Sollten sich in weiterführenden Studien die beobachteten Effekte im Dünndarm auch für die Gewebehomöostase im Kolon beschreiben lassen, können vor diesem Hintergrund Genistein und ERβ-spezifische Liganden für die Darmkrebsprävention diskutiert werden…

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