Phytoestrogens Inhibiting Androgen Receptor Signal and Prostate Cancer Cell Proliferation

The androgen receptor(AR) signaling activated by dihydrotestosterone(DHT) plays critical roles in prostate cancer development and progression. Phytoestrogens, which are diphenolic compounds with estrogen and anti-estrogen effects, can bind to estrogen receptors. However, their function on AR signaling has not been fully elucidated. In this study, dual-luciferase reporter assay, immunobloting, docking system test, MTT assay, immunofluorescence and chromatin immunoprecipitation(ChIP) assays were employed to examine the potential effects of three phytoestrogens(genistein, daidzein, flavone) on DHT-activated prostate specific antigen(PSA) activation, cell proliferation and AR transactivation in lymph node carcinoma of prostate(LNCaP) cells. Phytoestrogens were detected to down-regulate DHT-activated AR-mediated PSA promoter transactivation by dual-luciferase reporter system. Furthermore, three phytoestrogens, especially genistein, were demonstrated to significantly decrease AR-activated PSA protein expression by Western blotting analysis. MTT experiment proves that phytoestrogens, especially genistein, remarkably inhibits the DHT-induced cell proliferation in LNCaP cells. To provide reasonable explanations for experimental phenomena mentioned above, we did docking system test and detected phytoestrogens to share the same AR-binding site with DHT. To further prove the competition between phytoestrogen and DHT on AR binding, we examined the effects of phytoestrogens on DHT-activated AR nuclear translocation and immunofluorescence analysis which confirms that phytoestrogens, especially genistein, inhibit DHT-activated androgen receptor nuclear translocation. Results from ChIP show that phytoestrogens down-regulate DHT-induces AR binding to the androgen response elements(AREs, including AREI, AREII, and AREIII) in PSA promoter. Genistein remarkably down-regulates AR, binding to the AREI located in -250― -39 bp and AREIII in -4170― -3978 bp in the presence of DHT. In general, three phytoestrogens were identified to inhibit DHT-AR binding by competitively binding to AR and inhibit AR-mediated transactivation. And genistein shows the strongest effects among three phytoestrogens. Our findings confirm that phytoestrogens are AR antagonist in the regulation of AR-related PSA activation and cell proliferation, which provides valuable insights into the treatment of prostate cancer.     

Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry

This investigation describes a reliable and sensitive method for simultaneously determining bisphenol A (BPA) and two major phytoestrogens, daidzein and genistein, in powdered milks and infant formulas by gas chromatography-mass spectrometric analysis after trimethylsilylation. To reduce the matrix interference associated with the constituents of the formulas, the dissolved formula solutions were firstly ultra-centrifuged and the analytes in the supernatant were then extracted using a C18 solid-phase extraction column. The accuracy and precision of the method were determined and the technique was successfully employed to measure trace concentrations of BPA, daidzein and genistein in powdered formulas. The results show that BPA, daidzein and genistein were detected in all the testing samples (n = 6) at concentrations from 45 to 113 ng/g (except one infant formula), 20 to 2050 ng/g and 21 to 6510 ng/g, respectively. The highest concentrations of daidzein and genistein (i.e., 2050 and 6510 ng/g) were detected in a soy-based powdered infant formula. The quantitation limits were 1.0 ng/g for BPA, and 10 ng/g for daidzein and genistein using 0.5 g powdered milk samples.     

Simultaneous monitoring of seven phenolic metabolites of endocrine disrupting compounds (EDC) in human urine using gas chromatography with tandem mass spectrometry

A gas chromatographic-tandem mass spectrometric (GC-MS/MS) method for the simultaneous determination of the three well-known endocrine disruptors, bisphenol A, daidzein and genistein, as well as of four human pesticide metabolites which are supposed to have proper endocrine activity or which are metabolites of endocrine-disrupting compounds, viz., 1- and 2-naphthol, 2-isopropoxyphenol and 3,5,6-trichloropyridinol, has been developed and validated. The method involves enzymatic cleavage of the conjugates using β-glucuronidase/arylsulfatase followed by solid-phase extraction and derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Isotopically labelled internal standards were used for all analytes, to achieve best analytical error correction. The method proved to be both sensitive and reliable in human urine with detection limits ranging from 0.1 to 0.6 μg/L for all analytes. Precision and repeatability was determined to range from 1 to 15 %. Compared with other published analytical procedures, the present method enables the simultaneous determination of a couple of phenolic agents with competitive or improved analytical reliability. Thus, the present method is suitable for a combined monitoring of the exposure to prominent xenobiotics with effects on the human endocrine system (bisphenol A, carbaryl, chlorpyrifos, chlorpyrifos-methyl, naphthalene, propoxur, triclopyr) and phytoestrogens (daidzein, genistein) in population studies.     

Soy Isoflavones Inhibit Both GPIb-IX Signaling and αIIbβ3 Outside-In Signaling via 14-3-3ζ in Platelet

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s⁻¹) and high (1800 s⁻¹) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbβ3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s⁻¹). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbβ3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbβ3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.     

Determination of some phytoestrogens in soybeans and their processed products with HPLC and coulometric electrode array detection

A reliable and sensitive method for quantification of daidzein and genistein by HPLC with coulometric electrode array detection is presented using bisphenol A as internal standard. Acid hydrolysis during extraction of foods allows the quantitative determination of total phytoestrogens as aglycones. The substances are separated on a reversed phase column (Hypersil^® Elite C-18), eluted with methanol/acetonitrile/50 mM sodium acetate pH 4.8 (40/ 20/40, v/v/v) and detected in a coulometric electrode array detector using an oxidative detection mode (+390 to +810 mV in increments of 60 mV, vs palladium reference electrode). Phytoestrogen levels from several food items were determined. High levels of daidzein (819 mg/kg) and genistein (960 mg/kg) were found in soy products whereas biochanin A could not be detected in any of these food samples. The recovery depends on the kind of food and was found to be between 72 and 94% for daidzein and genistein.     

Determination of some phytoestrogens in soybeans and their processed products with HPLC and coulometric electrode array detection

A reliable and sensitive method for quantification of daidzein and genistein by HPLC with coulometric electrode array detection is presented using bisphenol A as internal standard. Acid hydrolysis during extraction of foods allows the quantitative determination of total phytoestrogens as aglycones. The substances are separated on a reversed phase column (Hypersil^? Elite C-18), eluted with methanol/acetonitrile/50 mM sodium acetate pH 4.8 (40/ 20/40, v/v/v) and detected in a coulometric electrode array detector using an oxidative detection mode (+390 to +810 mV in increments of 60 mV, vs palladium reference electrode). Phytoestrogen levels from several food items were determined. High levels of daidzein (819 mg/kg) and genistein (960 mg/kg) were found in soy products whereas biochanin A could not be detected in any of these food samples. The recovery depends on the kind of food and was found to be between 72 and 94% for daidzein and genistein. Received: 29 September 1998 / Revised: 30 November 1998 / Accepted: 4 December 1998     

Biomolecules and Nutritional Quality of Soymilk Fermented with Probiotic Yeast and Bacteria

Soymilk was fermented with five isolates of probiotic lactic acid bacteria and in combination with probiotic yeast Saccharomyces boulardii. Nutritional profile like fat, protein, ash, pH, acidity, polyphenol, and protein hydrolysis were analyzed. Polyphenol content decreased from 265.88 to 119 microg/ml with different cultures. Protein hydrolysis ranged from 2.46 to 2.83 mmol l(-1) with different cultures. The antioxidant activity was assessed using different methods like 1, 1-diphenyl-2-picrylhydrazyl free radical-scavenging assay, inhibition of ascorbate autoxidation, and measurement of reducing activity. The activities varied with the starters used but, nevertheless, were significantly higher than those found in unfermented soymilk. Bioconversion of the isoflavone glucosides (daidzin + genistin) into their corresponding bioactive aglycones (daidzein + genistein) was observed during soymilk fermentation. Total glucosides in soyamilk were 26.35 mg/100 ml. In contrast, aglycones genistein and daidzein were quantitatively lesser accounting 2.91 mg/100 ml (genistein 1.17 mg/100 ml and daidzein 1.19 mg/100 ml). Soymilk fermented with probiotic cultures resulted in the reduction of glycosides ranging from 0.40 mg to 1.36 mg/100 ml and increase in aglycones ranging from 6.32 mg to 13.66 mg/100 ml.     

Interactions of genistein and related isoflavones with lipid micelles

Genistein (5,7,4'-trihydroxyisoflavone) modulates the function of several transmembrane ion-channel proteins by mechanisms that are unrelated to phosphorylation events. Daidzein (7,4'-dihydroxy-isoflavone) typically exhibits modest effects, whereas genistin (7-O-glucosyl-genistein) usually exhibits no effect on ion-channel activities. Genistein appears to modulate gramicidin A ion channels by alteration of bilayer mechanical properties, but the associated molecular interactions have not been defined. The incorporation of daidzein into phosphatidylcholine liposomes promotes aggregation and precipitate formation which is problematic for structural studies based on NMR spectroscopy. In the present study, daidzein was incorporated into sodium dodecyl sulfate (SDS) micelles to provide a stable system with no evidence of micelle aggregation. For this reason genistein, daidzein, genistin, and osajin (a diprenyl-genistein derivative) were incorporated into SDS micelles (in D(2)O) to evaluate differences in position and orientation within micelle structures. The (1)H NMR line widths, as a function of Mn(2+) concentration, indicate that genistein is quite mobile and buried within the hydrophobic micelle core. Daidzein and genistin also are mobile but exhibit average positions near the micelle/aqueous interface, with polar groups oriented toward the aqueous compartment. These results demonstrate that daidzein, with only two hydroxyl substituents, has a greater affinity for a polar environment than genistein with three hydroxyl substituents. The 5-hydroxyl group of genistein forms an intramolecular hydrogen bond with the 4-carbonyl group, which diminishes the molecular affinity for a polar matrix. These results suggest an explanation for the relative abilities of these compounds to increase gramicidin channel lifetimes and modulate other ion-channel types.     

Gas chromatographic–mass spectrometric fragmentation study of phytoestrogens as their trimethylsilyl derivatives: Identification in soy milk and wastewater samples

An analytical method for the identification of eight plant phytoestrogens (biochanin A, coumestrol, daidzein, equol, formononetin, glycitein, genistein and prunetin) in soy products and wastewater samples was developed using gas chromatography coupled with ion trap mass spectrometry (GC/MS–MS). The phytoestrogens were derivatized as their trimethylsilyl ethers with trimethylchlorosilane (TMCS) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The phytoestrogens were isolated from all samples with liquid–liquid extraction using ethyl acetate. Daidzein-d₄ and genistein-d₄ labeled standards were used as internal standards before extraction and derivatization. The fragmentation patterns of the phytoestrogens were investigated by isolating and fragmenting the precursor ions in the ion-trap and a typical fragmentation involved the loss of a methyl and a carbonyl group. Two characteristic fragment ions for each analyte were chosen for identification and confirmation. The developed methodology was applied to the identification and confirmation of phytoestrogens in soy milk, in wastewater effluent from a soy-milk processing plant, and in wastewater (influent and effluent) from a treatment plant. Detected concentrations of genistein ranged from 50,000 μg/L and 2000 μg/L in soy milk and in wastewater from a soy-plant, respectively, to 20 μg/L and <1 μg/L for influent and effluent from a wastewater treatment plant, respectively.     

Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.     

Identification of phytoestrogens in bovine milk using liquid chromatography/electrospray tandem mass spectrometry

In an international context of promoting scientific research on food safety, the interest in molecules having potential hormonal disrupting effects is growing. While industrial endocrine disruptors (phthalates, alkylphenols, PCBs, etc.) have been studied for several years, natural compounds like phytoestrogens remain less investigated. Accordingly, a research project was initiated with its main objectives to develop efficient analytical methods for a wide range of phytoestrogens in various food matrices, and to evaluate their occurrence in food products. Electrospray ionization with tandem mass spectrometric (MS/MS) analysis of isoflavones (genistein, daidzein, equol, formononetin, biochanin A), lignans (enterolactone, enterodiol), and coumestans (coumestrol) was investigated. This study revealed the formation of a large number of fragment ions in both positive and negative modes, corresponding to specific cleavages of the hydroxyl, carbonyl, and/or methoxy groups, and to Retro-Diels-Alder reactions. An LC/ESI-MS/MS method was developed consistent with the 2002/657/EC European decision criteria. An extraction and clean-up method was developed for milk samples. The identification limit for the proposed method appears to be under 1 ng/mL. The developed methodology was applied to various milk samples, and the occurrence of isoflavones (particularly equol) was demonstrated in the concentration range 1-30 ng/mL. The efficiency of the proposed analytical method permitted evaluation of a new and promising approach to a global risk assessment of natural estrogenic active substances including phytoestrogens and their metabolites.     

A simple method for the characterization and quantification of soy isoflavone metabolites in the serum of MMTV-Neu mice using high-performance liquid chromatography/electrospray ionization mass spectrometry with multiple reaction monitoring

In a study aimed at elucidation of the possible role of dietary phytoestrogens in the growth of breast cancer, it was necessary to develop a convenient, accurate, and reproducible method for the characterization and quantification of isoflavone metabolites in the serum of MMTV mice that were fed diets containing different amounts of these polyphenols. The analytical method is based on liquid chromatography with mass spectrometry in multiple reaction monitoring mode, using deuterated genistein as internal standard. The identified metabolites were genistein and dihydrodaidzein (DHD); their average concentrations in serum were 0.71 and 0.21 microM, respectively, for animals fed 1.5 mg/day of genistein and 0.7 mg/day of daidzein. This method assured a limit of quantification of 0.04 microM for genistein and 0.08 microM for DHD, and a limit of detection of 0.018 microM for genistein and 0.035 microM for DHD. The coefficient of variation was 8.9% for genistein and lower than 15% for DHD. This study provides the first data indicating the presence of dihydrodaidzein in serum of mice fed diets containing soy proteins.     

A study of selected phytoestrogens retention by reverse osmosis and nanofiltration membranes - the role of fouling and scaling

Fouling and scaling are common phenomena that accompany membrane filtration and are caused by the presence of organic and inorganic matter in water, which may affect the removal of low-molecular mass organic micropollutants. Comparative filtration of deionized water containing selected phytoestrogens (biochanin A, daidzein, genistein, and coumestrol) was carried out using one new membrane and one contaminated with organic or inorganic matter. Two commercial Osmonics DS membranes were selected for the research, reverse osmosis DS3SE and nanofiltration DS5DK. Filtration was carried out in the dead-end mode. Higher removal of phytoestrogens was caused by reverse osmosis and retention depended on the molar mass of the compound. The decrease in membrane efficiency associated with fouling or scaling brings about an increase in the retention coefficient of phytoestrogens during both reverse osmosis and nanofiltration. The highest increase in phytoestrogen retention was found for the nanofiltraton membrane which was more susceptible to fouling than the osmotic one. This confirms the effect of membrane porosity on the phenomenon studied. The increase in micropollutants removal observed after fouling or scaling was caused by the modification of the membrane surface, hindered diffusion of the compound, and intensified or limited adsorption of micropollutants on the membrane surface.     

Lipophilicity and bio‐mimetic properties determination of phytoestrogens using ultra‐high‐performance liquid chromatography

This paper presents lipophilicity and bio‐mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein‐4,7‐dimethylether, prunetin, 3,4,7‐trihydroxyisoflavon, 4,6,7‐trihydroxyisoflavon, 4,6,7‐trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High‐performance liquid chromatography with fast gradient elution and Caco‐2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl‐sylane stationary phase using pH 2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α‐1‐acid‐glycoprotein (AGP) stationary phases. Caco‐2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco‐2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco‐2 permeability.     

Optimization of a rapid microwave assisted extraction method for the liquid chromatography-electrospray-tandem mass spectrometry determination of isoflavonoid aglycones in soybeans

A very fast chromatographic separation of isoflavonoids genistein, daidzein, formononetin and biochanin A was developed on a C18 high-speed column under isocratic conditions. The method was validated in terms of detection limits, quantitation limits (LOQs), linearity and precision. LOQs in 0.04-0.2 microg/g range were calculated, making feasible the determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three concentration orders of magnitude for each analyte (r2 0.990-1.000). The intra-day and inter-day repeatability was evaluated in terms of relative standard deviation (RSD%) at two concentration levels for each analyte (RSD% <9%). An optimization strategy was adopted to find the best conditions for the extraction of isoflavonoid aglycones from yellow soybeans using microwave-assisted extraction. The most relevant parameters resulted to be the microwave power, the extraction time and the acid concentration, optimal values being 600 W, 1 min and 12 M, respectively. When performing sample treatment on a fortified soybean sample, high recovery percentage was obtained for both compounds (94+/-8% for daidzein and 97+/-5% (n = 4) for genistein). The concentration level at which daidzein and genistein were found in the soybean sample were 1.21+/-0.15 mg/g and 2.38+/-0.09 mg/g (n=4), respectively.     

The experimental design approach to the optimization of a simple quick easy cheap effective rugged and safe method for the analysis of phytoestrogens in complex soy‐based food

The present paper deals with the multivariate optimization of an extraction‐purification strategy for the determination of phytoestrogens (daidzein, genistein, coumestrol, formononetin, and biochanin A) in soy‐based meat substitutes by high performance liquid chromatography with tandem mass spectrometry. For a reliable quantitation of these new concerning compounds in such a complex matrix, recovery and matrix effect must be carefully evaluated. Therefore, two sequential experimental designs were used to optimize the sample‐pretreatment of soy‐based burgers: the chosen technique was the quick, easy, cheap, effective, rugged and safe methodology, which does not require any particular facility or instrumentation. Thanks to the first screening design (Plackett‐Burman), the significant factors influencing the studied responses were identified and further investigated through a response surface design (Box‐Behnken). The optimal values of the variables (volume of extraction solvent mix/sample mass ratio and two clean‐up sorbents) led to quantitative recoveries (97–104%) and low ion suppression (matrix effect 60–93%) for all analytes. This optimized method was characterized by low detection limits (0.2–1.5 ng/g) and excellent intraday precision (RSD 2–4%). It was applied to the determination of the considered compounds in several soy‐burgers from the Italian market, detecting low ng/g levels (up to 40 ng/g) of coumestrol, formononetin, and biochanin A, and high concentrations (7.9–78 µg/g) of genistein and daidzein.     

Quantitative analysis using gas chromatography/combustion/isotope ratio mass spectrometry and standard addition of intrinsically labelled standards (SAIL)--application to isoflavones in foods

We have investigated a novel application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) for the quantitative analysis of the isoflavones in food matrices. Previous methods have been hampered by the lack of analytical standards to introduce early enough in the extraction procedure to allow compensations for losses at all stages of the procedure. In this work we have produced standard materials that can be added at the initial extraction, by intrinsically labelling soya plants by growing them in an atmosphere enriched in the stable isotope of carbon in CO(2). On analysis these plants were shown to contain phytoestrogens at a high (up to 20%) level of enrichment. The dried plant material has been used to estimate the isoflavone concentrations of a set of spiked flours. For daidzein the methodology was shown to produce results comparable to those achieved by GC/MS techniques. The method was less successful for genistein, possibly due to the greater fragility of this compound under the conditions required for the analysis.