High Genistein Research Articles

A highly selective cell-based fluorescent biosensor for genistein detection

Genistein, an isoflavone found mainly in legumes, has been shown to have numerous health benefits for humans. Therefore, there is substantial interest in producing it using microbial cell factories. To aid in screening for high genistein producing microbial strains, a cell-based biosensor for genistein was developed by repurposing the Gal4DBD-ERα-VP16 (GEV) transcriptional activator in Saccharomyces cerevisiae. In the presence of genistein, the GEV sensor protein binds to the GAL1 promoter and activates transcription of a downstream GFP reporter. The performance of the biosensor, as measured by fold difference in GFP signal intensity after external genistein induction, was improved by engineering the sensor protein, its promoter and the reporter promoter. Biosensor performance increased when the weak promoter REV1p was used to drive GEV sensor gene expression and the VP16 transactivating domain on GEV was replaced with the tripartite VPR transactivator that had its NLS removed. The biosensor performance further improved when the binding sites for the inhibitor Mig1 were removed from and two additional Gal4p binding sites were added to the reporter promoter. After genistein induction, our improved biosensor output a GFP signal that was 20 times higher compared to the uninduced state. Out of the 8 flavonoids tested, the improved biosensor responded only to genistein and in a somewhat linear manner. The improved biosensor also responded to genistein produced in vivo, with the GFP reporter intensity directly proportional to intracellular genistein concentration. When combined with fluorescence-based cell sorting technology, this biosensor could facilitate high-throughput screening of a genistein-producing yeast cell factory.

Intraperitoneal injection of genistein affects the distribution and metabolism of cholesterol in female yellow catfish Tachysurus fulvidraco.

Genistein is an abundant phytoestrogen in soybean. This study aimed to determine the effects of genistein on cholesterol distribution and metabolism in female yellow catfish. Three hundred fish (49.2 ± 1.4g) were randomly divided into five treatments and received intraperitoneal injections as follows: (1) blank, no injection; (2) control, vehicle only; (3) E2, 17β-estradiol at 10μg·g-1 body weight; (4) low genistein doses, genistein at 10μg·g-1 body weight; (5) high genistein doses, genistein at 100μg·g-1 body weight. Both high and low genistein doses significantly reduced (p < 0.05) serum TC and LDL-C 24h after injection. Moreover, the high genistein doses significantly reduced (p < 0.05) serum HDL-C. Both high and low doses of genistein significantly increased (p < 0.05) hepatic TC. Only high genistein doses significantly increased (p < 0.05) ovary TC. In the liver, both high and low genistein doses significantly increased (p < 0.05) protein and mRNA expression of ldlr. Meanwhile, high genistein doses significantly decreased (p < 0.05) mRNA expression of hmgcr. In ovary tissue, high genistein doses significantly decreased (p < 0.05) mRNA expression of cyp11a1. These results suggested that genistein affected the cholesterol distribution in female yellow catfish. Both high and low doses of genistein reduced cholesterol content in blood and increased its content in the liver by increasing the uptake of blood cholesterol. Meanwhile, high genistein doses may inhibit hepatic cholesterol synthesis. Additionally, high genistein doses could increase cholesterol transfer from serum into the ovary and disturb cholesterol conversion to pregnenolone.

Perbandingan Kandungan Genistein Pada Berbagai Varietas Kedelai (Glycine max) Di Indonesia

Soybeans is a functional food containing higher isoflavone compared to that of other food crops. Genistein, daidzein and glisitein are components of soybean isoflavones. Genistein to function as a protective agent against cancer and cardiovascular diseases. The content of genistein in soybean can be influenced by soybean varieties and plant maturities. This study was aimed to determine the genistein content of soybean varieties in Indonesia. The experiment was carried out at Biology Pharmacy laboratory of Indonesian School of Pharmacy and at Instrument Laboratory of School of Pharmacy, of Bandung Institute of Technology. Total of nine soybean varieties were used in the experiment. Extraction was carried out using heat treatment, and the extract yield was calculated on the base of grain soybean being used. The highest was 11.19% obtained from Dering 1 variety. The genistein was monitored using the Thin Layer Chromatography (TLC), but the result was not clear. Fractionation was performed using gravity column chromatography, and the fraction results were analyzed using a spectrophotodensitometer. Results showed that genistein content of Anjasmoro and Argomulyo soybean varieties, each was late and medium maturity, had high genistein content of 15.996 ± 2.51 µg/gram and 14.175 ± 2.03 µg/gram, respectively. In contrast, soybean varieties Detam 1, medium maturity, had low genistein content of 5.313 ± 4.77 µg/gram. This finding is important for the functional food industry in selecting raw material.

Overexpressing GmCHI1A increases the isoflavone content of transgenic soybean (Glycine max (L.) Merr.) seeds

Isoflavones, which are secondary metabolites synthesised through the phenylpropanoid pathway, play important roles in many essential physiological processes of the soybean plant. Isoflavones are well-known for their antioxidant, antimicrobial, anti-inflammatory and anticancer activities; however, soybean plants contain very low amount of isoflavone. Therefore, increasing isoflavone content is one of major concerns in soybean research. An effective approach to enhance isoflavone content in soybean is by overexpression of the Glycine max chalcone isomerase 1A (GmCHI1A) gene. In this study, the GmCHI1A gene was transformed into soybean plants and the T2 generation was selected for high isoflavone content (daidzein, genistein) in transgenic soybean germs. GmCHI1A gene expression was enhanced in T1 transgenic lines, which led to an increase in recombinant CHI1A (rCHI1A) protein content. In soybean germs of the T2 transgenic lines, the content of daidzein and genistein increased from 166.46 to 187.23% and from 329.77 to 463.93%, respectively. Four T2 transgenic soybean lines (T2-1, T2-4, T2-21 and T2-24) with high daidzein and genistein content were selected for future progeny evaluation.

Genistein Prevents Development of Spontaneous Ovarian Cancer and Inhibits Tumor Growth in Hen Model.

Genistein, the major isoflavone in soybean, has been reported to exert anticancer effects on various types of cancer including ovarian cancer; however, its chemopreventive effects and mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously developing ovarian cancer models. In this study, we demonstrated the preventive effects and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer at high incidence rates. Laying hens were randomized to three groups: control (3.01 mg/hen, n = 100), low (52.48 mg/hen n = 100), and high genistein supplementation (106.26 mg/hen/day; per group). At the end of 78 weeks, hens were euthanized and ovarian tumors were collected and analyzed. We observed that genistein supplementation significantly reduced the ovarian tumor incidence (P = 0.002), as well as the number and size of the tumors (P = 0.0001). Molecular analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde, a marker for oxidative stress and the expression of NFκB and Bcl-2, whereas it upregulated Nrf2, HO-1, and Bax expression at protein level in ovarian tissues. Moreover, genistein intake decreased the activity of mTOR pathway as evidenced by reduced phosphorylation of mTOR, p70S6K1, and 4E-BP1. Taken together, our findings strongly support the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of the genistein on the molecular pathways involved in ovarian tumorigenesis.

Estrogenic properties of genistein acting on FSHR and LHR in rats with PCOS.

The purpose of the study was to study the activity of the phytoestrogen genistein (GEN) act- ing on FSHR and LHR in rat ovaries with polycystic ovary syndrome (PCOS). Sixty rats were di- vided into six groups. Rats in the dose group received genistein at a concentration of either 5 (low genistein dose group, L-gen), 10 (middle genistein dose group, M-Gen) or 20 (high genistein dose group, H-Gen) mg per kg of body weight per day. Estrogen group (EG, received 0.5 mg/kg Dieth- ylstilbestrol). Concentration of sex hormones in serum was quantified by enzyme-linked immuno- sorbent assay (ELISA). Expressions of follicle-stimulating hormone receptor (FSHR) and lutein- izing hormone receptor (LHR) protein were determined by immunohistochemistry. Treatment with genistein resulted in a strong stimulation of the concentration of sex hormone in serum. The concentration of progesterone and FSH was significantly higher in H-Gen when compared to the PCOS model control group (MG) (P ⟨ 0.01). In contrast, the concentration of testosterone, LH and the ratio of LH/FSH decreased in GEN treatment groups compared to MG, the effect was statistically significant, tested by the ANOVA test (p⟨0.01). For hormone receptor activity, treat- ment with genistein resulted in an improvement of ovarian function with LHR protein expression being enhanced and FSHR protein expression being suppressed. Our results demonstrate that Genistein played a significant role in regulating FSH and LH receptor expression to improve perimenopausal ovarian and uterine function.

Autophagy-dependent mechanism of genistein-mediated elimination of behavioral and biochemical defects in the rat model of sporadic Alzheimer's disease

Alzheimer's disease is one of severe neurological diseases for which no effective treatment is currently available. The use of genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been proposed previously as one of approaches to improve the disease symptoms, as some positive effects of this compound in cellular and animal models were reported. Inhibition of apoptosis and antioxidative functions were suggested as causes of these effects. Here, we demonstrate that high genistein dose (150 mg/kg/day; the dose significantly higher than those used previously in AD studies by others) can activate autophagy in the streptozotocin-induced rat model of the sporadic form of AD. We found that this dose of genistein led to complete degradation of β-amyloid and hyperphosphorylated tau protein in the brain, while experiments with cell cultures demonstrated that these effects require autophagy stimulation, which has never been shown before. Importantly, behavior of high dose genistein-treated AD rats was completely corrected, i.e. it was indistinguishable from that of healthy animals. This was observed in all performed behavioral tests: Morris water maze test, elevated plus-maze test, open field test, and locomotor measurements in an actometer. We conclude that autophagy-dependent mechanism is responsible for genistein-mediated correction of AD when this isoflavone is used at the high dose.

High dose genistein aglycone in Sanfilippo syndrome: Results of a randomised, double-blinded, placebo controlled clinical trial

High dose genistein aglycone in Sanfilippo syndrome: Results of a randomised, double-blinded, placebo controlled clinical trial

The regulatory effect of Genistein on granulosa cell in ovary of rat with PCOS through Bcl-2 and Bax signaling pathways.

The effect of genistein on Bcl-2 and Bax protein expression in the ovarian tissue of rats with polycystic ovarian syndrome (PCOS) was evaluated. Sixty rats were divided into six groups. Rats in the Dose group received genistein at a concentration of either 5 (L-gen), 10 (M-Gen) or 20 (H-Gen) mg per kg of body weight per day. The expression of Bcl-2 mRNA and Bax mRNA was determined by in situ hybridization. Bcl-2 and Bax protein concentration was quantified by ELISA. The results showed that the expression of Bcl-2 mRNA and Bcl-2 protein was significantly higher in the high genistein Dose group (H-Gen) when compared to the Model group (MG) (P<0.05). Genistein induced higher expression of the Bcl-2 gene at the transcriptional and translational level. Treatment with genistein resulted in an improvement of ovarian function with Bcl-2 expression being enhanced and Bax expression being suppressed. These alterations may be due to the structural and functional modifications that take place in these cells, and could be related to apoptotic changes that occur in rats with PCOS.

Effects of the soya isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: Thyroid, estrogenic and metabolic biomarkers

This study examines the effects induced by environmentally relevant concentrations of the isoflavone genistein (3mg/L and 10mg/L) during early life stages of the Senegalese sole. Throughout the hypothalamus-pituitary-thyroid (HPT) axis, several neurohormonal regulatory thyroid signalling patterns (thyroglobulin/Tg, thyroid peroxidase/TPO, transthyretin/TTR, thyroid receptors/TRβ, and iodothrynonine deiodinases, Dio2 and Dio3) were analysed. Furthermore, the expression patterns of estrogen receptor ERβ and haemoprotein Cyp1a were also evaluated. In the control larvae, progressive increases of constitutive hormonal signalling pathways have been evidenced from the pre-metamorphosis phase onwards, reaching the highest expression basal levels at the metamorphosis (Tg, TPO, Dio2) and/or during post-metamorphosis (TTR, TRβ, ERβ). When the early larvae were exposed to both genistein concentrations (3mg/L and 10mg/L), a statistically significant down-regulation of TPO, TTR and Tg mRNA levels was clearly detected at the metamorphic stages. In addition, the Dio2 and Dio3 transcript expression levels were also down and up-regulated when exposed to both genistein concentrations. In the larvae exposed to genistein, no statistically significant responses were recorded for the TRβ expression patterns. Nevertheless, the ERβ and Cyp1a transcript levels were up-regulated at the middle metamorphic stage (S2, at 16 dph) in the larvae exposed to high genistein concentrations and, only the ERβ was down-regulated (S1, at 12dph) at the lower doses. Finally, all these pointed out imbalances were only temporarily disrupted by exposure to genistein, since most of the modulated transcriptional signals (i.e. up or down-regulation) were quickly restored to the baseline levels. Additionally, the control and genistein-exposed Senegalese sole specimens showed characteristic ontogenetic patterns and completely suitable for an optimal development, metamorphosis, and growth.

Clinical Study: Effect of Supplementation with High Genistein Soybean Isoflavones and Pumpkin Standardized Extract on Urinary Incontinence in Western Perimenopausal Women

Clinical Study: Effect of Supplementation with High Genistein Soybean Isoflavones and Pumpkin Standardized Extract on Urinary Incontinence in Western Perimenopausal Women

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BackgroundPhytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts.MethodsThus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed.ResultsExposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells.ConclusionFrom these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Morphology and immunological activities of genistein-modified poly(ethylene glycol) diacrylate networks

Abstract By virtue of well-known antioxidant, anti-inflammatory, and antibacterial activities, a phytochemical called ‘genistein’ is incorporated into polymer network membrane for wound dressing. Binary phase diagram of genistein/polyethylene glycol diacrylate precursor (PEGDA) blends was established both experimentally and theoretically. It was found that genistein crystals developed in the matrix of the PEGDA network, especially at high genistein contents. Biological activities including cytotoxicity, antioxidant, and anti-inflammatory of the networks were evaluated. The genistein-modified PEGDA network revealed improved antioxidant and anti-inflammatory functions desirable for multifunctional wound dressing.

Phytoestrogens and risk of prostate cancer: a meta-analysis of observational studies.

BackgroundEpidemiologic studies have reported various results relating phytoestrogens to prostate cancer (PCa). The aim of this study was to provide a comprehensive meta-analysis on the extent of the possible association between phytoestrogens (including consumption and serum concentration) and the risk of PCa.MethodsEligible studies were retrieved via both computer searches and review of references. The summary relative risk ratio (RR) or odds ratio (OR) and 95 % confidence interval (CI) were calculated with random effects models.ResultsA total of 11 studies (2 cohort and 9 case–control studies) on phytoestrogen intake and 8 studies on serum concentration were included in the meta-analysis. The pooled odds ratio (OR) showed a significant influence of the highest phytoestrogens consumption (OR 0.80, 95 % CI 0.70–0.91) and serum concentration (OR 0.83, 95 % CI 0.70–0.99) on the risk of PCa. In stratified analysis, high genistein and daidzein intake and increased serum concentration of enterolactone were associated with a significant reduced risk of PCa. However, no significant associations were observed for isoflavone intake, lignans intake, or serum concentrations of genistein, daidzein, or equol.ConclusionsThe overall current literature suggests that phytoestrogen intake is associated with a decreased risk of PCa, especially genistein and daidzein intake. Increased serum concentration of enterolactone was also associated with a significant reduced risk of PCa. Further efforts should be made to clarify the underlying biological mechanisms.

Isoflavone supplement composition and equol producer status affect gene expression in adipose tissue: a double-blind, randomized, placebo-controlled crossover trial in postmenopausal women

Isoflavone supplements, consumed by women experiencing menopausal symptoms, are suggested to have positive effects on menopause-related adiposity and cardiovascular disease risk profile, but discussions about their safety are still ongoing. The objective was to study the effects of an 8-wk consumption of 2 different isoflavone supplements compared with placebo on whole-genome gene expression in the adipose tissue of postmenopausal women. This double-blind, randomized, placebo-controlled crossover intervention consisted of 2 substudies, one with a low-genistein (LG) supplement (56% daidzein + daidzin, 16% genistein + genistin, and 28% glycitein + glycitin) and the other with a high-genistein (HG) supplement (49% daidzein + daidzin, 41% genistein + genistin, and 10% glycitein + glycitin). Both supplements provided ∼ 100 mg isoflavones/d (aglycone equivalents). After the 8-wk isoflavone and placebo period, whole-genome arrays were performed in subcutaneous adipose tissue of postmenopausal women (n = 26 after LG, n = 31 after HG). Participants were randomized by equol-producing phenotype, and data analysis was performed per substudy for equol producers and nonproducers separately. Gene set enrichment analysis showed downregulation of expression of energy metabolism-related genes after LG supplementation (n = 24) in both equol-producing phenotypes and oppositely regulated expression for equol producers (down) and nonproducers (up) after HG supplementation (n = 31). Expression of inflammation-related genes was upregulated in equol producers but downregulated in nonproducers, independent of supplement type. Only 4.4-7.0% of the genes with significantly changed expression were estrogen responsive. Body weight, adipocyte size, and plasma lipid profile were not affected by isoflavone supplementation. Effects of isoflavones on adipose tissue gene expression were influenced by supplement composition and equol-producing phenotype, whereas estrogen-responsive effects were lacking. LG isoflavone supplementation resulted in a caloric restriction-like gene expression profile for both producer phenotypes and pointed toward a potential beneficial effect, whereas both supplements induced anti-inflammatory gene expression in equol producers. The study was registered at clinicaltrials.gov as NCT01556737.

Asymmetric porous membranes formed by coagulation-induced phase separation in poly(ether sulfone)/poly(vinyl pyrrolidone)/genistein blends

Development of asymmetric channel morphology driven by coagulation-induced phase separation of genistein (G) modified poly(ether sulfone)/poly(vinyl pyrrolidone) (PES/PVP) blends has been examined in relation to their miscibility phase diagram. PES/G pairs turned out to be miscible in the amorphous state, whereas solid–liquid phase separation occurred at high genistein compositions. The solid–liquid phase diagram involving the liquidus and solidus lines were computed self-consistently in the framework of the combined free energy of Flory-Huggins for liquid–liquid phase separation and phase field free energy for crystal solidification. The ternary phase diagram of PES/PVP/G blends was subsequently established that consisted of various coexistence regions. The actual amounts of genistein incorporated in the PES/PVP membranes were determined as a function of weight percent of genistein in feed. On the basis of UV-vis spectroscopy, the extent of genistein leaching during incubation in human blood was evaluated in conjunction with the PVP leaching from the blend membrane.

Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression

OBJECTIVE:High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator.METHODS:Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry.RESULTS:The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780.CONCLUSION:Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause.

Isoflavone Genistein Induces Fluid Secretion and Morphological Changes in the Uteri of Post-Pubertal Rats

A reported increase in the incidence of infertility following high genistein intake could be related to alteration in the normal fluid volume and morphology of the uterus in adult female. In view of this, we investigated the effect of this compound on fluid secretion, fluid volume and morphology of the uterus in post-pubertal rats. Methods: Ovariectomised SD rats were treated with 17-β oestradiol (E) (0.8 X 10-4 mg/kg/day) and genistein (0.5, 5, 10, 25, 50 and 100 mg/kg/day) for three days. Following drug treatment, in-vivo uterine perfusion was performed and the rate of fluid secretion and the volume of fluid in the uterus were determined via changes in weight (μl/min) and F-dextran concentration of the perfusate respectively. The animals were then sacrificed and the uteri were removed for weight determination, morphological analyses and proliferative cell nuclear antigen (PCNA) expression analyses by Western blotting. Results: Subcutaneous genistein treatment resulted in a dose-dependent increase in fluid secretion rate, fluid volume and uterine wet weight. Treatment with 100 mg/kg/day genistein resulted in a remarkable increase in the rate of uterine fluid secretion, the volume of the uterine luminal fluid as well as the circumference of the uterine and uterine glandular lumen suggesting an excessive fluid accumulation. Meanwhile, there were evidence of glandular hyperplasia and an increase in the expression of PCNA following treatment with 50 and 100 mg/kg/day genistein. Conclusion: High genistein intake could potentially cause adverse effects on the uterus by inducing excessive fluid secretion and accumulation as well as hyperplasia.

Effects of Dietary Genistein on Hormone-Dependent Rat Mammary Carcinogenesis Induced by Ethyl Methanesulphonate

Genistein, a major soy isoflavone having weak estrogenic activities, has been suggested to reduce the risk of breast cancer incidence. However, many studies have yielded inconsistent results. We investigated the effects of dietary genistein on the development of breast cancer using ethyl methanesulphonate (EMS) chemically induced rat model of hormone-dependent mammary carcinoma. Female Wistar King A rats were orally given EMS for 12 wk and fed isoflavone-free NIH-07PLD diets with or without genistein, beginning immediately after weaning period. All EMS-treated rats fed either diet developed estrogen and/or progesterone receptor-positive mammary carcinoma by 24 wk. The addition of either low or high genistein, which produced the plasma concentrations comparable with those observed in humans consuming high soy diets, did not show any preventive activity. Soy-containing pellet food, exhibiting substantial plasma concentrations of isoflavones such as genistein, daidzein, equol, and glycitein, significantly increased the latency periods, compared to either NIH-07PLD diet with low (P = 0.027) or high (P = 0.034) genistein. Body weights, total EMS uptakes, and urinary estradiol concentrations were not significantly different among groups. These data indicate that genistein does not exert clear preventive effects and that isoflavone components other than genistein might be preventive against hormone-dependent mammary carcinogenesis.

Modulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity and Genistein Binding by Cytosolic pH

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 μA/cm(2) and K(d) = 32.7, 56.6 and 71.9 μm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 μm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.