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A pharmaceutical formulation of genistein, produced as an amorphous solid dispersion by hot melt extrusion (genistein HME), has been developed that can be administered prophylactically to improve outcomes and survival following radiation exposure. Here, genistein HME was evaluated in a phase 1, open-label, single ascending dose (SAD) and multiple single dose (MSD) study enrolling 34 healthy volunteers. In the SAD study, participants were administered a single dose (500, 1000, 2000, or 3000 mg) and in the MSD study, participants were administered a single daily dose for six consecutive days (3000 mg/day). The overall adverse event profile and pharmacokinetics of genistein HME were determined. Additionally, biomarkers of genistein HME were evaluated by profiling whole blood for changes in gene expression by RNA sequencing. Genistein HME was found to be safe at doses up to 3000 mg. Most toxicities were mild to moderate gastrointestinal events, and no dose-limiting toxicities were reported. The maximum tolerated dose was not determined and the no observable adverse effect level was 500 mg. Genistein HME bioavailability greatly increased between the 2000 mg and 3000 mg doses. RNA sequencing analysis revealed that the majority of drug-related changes in gene expression occurred 8-12 hours after the sixth dose in the MSD study. Based on these results, the putative effective dose in humans is 3000 mg., (© 2022, The American College of Clinical Pharmacology.)

Two high-performance liquid chromatography-diode array detection methods have been developed and validated for the simultaneous quantification of genistein (GNS) and all trans retinoic acid (ATRA) as a novel anticancer combination therapy in their co-formulated nanoparticles and in rat plasma. Separation was performed on C18 column (250 × 4.6 mm, 5 μm) using celecoxib as internal standard. A mobile phase containing acetonitrile and water adjusted to pH 3 using 1% trifluoroacetic acid was delivered in gradient elution modes with time programmed UV detection. For extraction of the drugs and the internal standard from rat plasma, liquid- liquid extraction was applied. The proposed methods were validated as per International Conference on Harmonisation (ICH) guidelines (in the range 0.1-10 μg/mL for analysis of GNS and ATRA in nanoparticles) or according to Food and Drug Administration (FDA) guidance on bioanalytical method validation (in the range 0.025-20 μg/mL for analysis of GNS and ATRA in rat plasma). Pharmacokinetic study in six rats was performed following intravenous (IV) administration of a single dose of 0.5 mg/Kg of GNS and ATRA. The drugs' concentrations were measured up to 24 hours, and different pharmacokinetic parameters were calculated. The obtained parameters were comparable with the reported values for IV administration of each drug alone in rats. This confirms the applicability of the proposed method in monitoring the levels of the two drugs in vivo following their coadministration and indicating that the two drugs could be coadministered as a promising novel combination therapy for the treatment of lung cancer without great alteration in their pharmacokinetic parameters compared with their individual IV administration., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Lignans are associated with improved postmenopausal breast cancer (BC) survival, but whether these associations, particularly with enterolactone (major lignan metabolite), persist over time is unclear. Little is known about other phytoestrogens on prognosis in long-term survivors. The study examines associations of prognosis with 1) circulating postdiagnosis enterolactone, 2) eight circulating phytoestrogen metabolites, and 3) changes in enterolactone and genistein. In a German cohort of 2,105 postmenopausal BC patients with blood samples collected at recruitment 2002-2005 (baseline) and re-interview in 2009 (follow-up), delay-entry Cox proportional hazards regression was used. Landmark analysis showed that circulating enterolactone (log2) associations with 5-year survival changed over time, with strongest hazard ratios of 0.89 (95% CI, 0.80-0.99) at blood draw (BD) and 0.86 (0.77-0.97) at 2 years post-BD for BC mortality, and 0.87 (0.80-0.95) at BD and 0.84 (0.76-0.92) at 3 years post-BD for all-cause mortality, which attenuated thereafter. In long-term survivors, increasing concentrations of genistein (1.17, 1.01-1.36), resveratrol (1.19, 1.02-1.40), and luteolin (1.96, 1.07-3.58) measured in follow-up blood samples were associated with poorer subsequent prognosis. Neither enterolactone at follow-up nor changes in enterolactone/genistein were associated with prognosis. Large long-term longitudinal studies with multiple phytoestrogen measurements are required to understand long-term effects of phytoestrogens after BC.

Although flavonoid phytoestrogens have been suggested to be associated with reduced risk of colorectal cancer (CRC), their influence on CRC prognosis remains uncertain. A population-based cohort of 2051 patients diagnosed with stage I⁻III CRC in southwest Germany in 2003⁻2010 were followed for five years. Post-diagnostic serum concentration of genistein and luteolin were measured using Ultra-Performance Liquid Chromatography with mass spectrometry. Multivariable Cox regression analysis was conducted to calculate the Hazard Ratios (HRs) and 95% confidence interval (CI) for the association between flavonoids concentration and overall morality, CRC-specific mortality, CRC recurrence, and disease-free survival (DFS). Median (interquartile range) serum concentration of genistein and luteolin was 11.90 ng/µL (10.08⁻14.13) and 7.20 ng/µL (6.40⁻8.16), respectively. Neither serum genistein nor luteolin was associated with CRC prognosis. There was no clear evidence of departure from linearity. However, the association might be differential by adjuvant chemotherapy. Associations pointed towards lower risk in patients who received chemotherapy and higher risk in those without chemotherapy for overall mortality regarding serum genistein (P-interaction = 0.02) and correspondingly for CRC recurrence (P-interaction: 0.03) and DFS (P-interaction: 0.01) with respect to luteolin. Our study provides little evidence that serum genistein and luteolin are associated with colorectal cancer prognosis. Future studies are warranted to evaluate the potential interaction with adjuvant chemotherapy.

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4-99.2% (median: 97.3%), ER-β, 82.7-97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phytoestrogens may influence prostate cancer development. This study aimed to examine the association between prediagnostic circulating concentrations of isoflavones (genistein, daidzein, equol) and lignans (enterolactone and enterodiol) and the risk of prostate cancer. Individual participant data were available from seven prospective studies (two studies from Japan with 241 cases and 503 controls and five studies from Europe with 2,828 cases and 5,593 controls). Because of the large difference in circulating isoflavone concentrations between Japan and Europe, analyses of the associations of isoflavone concentrations and prostate cancer risk were evaluated separately. Prostate cancer risk by study-specific fourths of circulating concentrations of each phytoestrogen was estimated using multivariable-adjusted conditional logistic regression. In men from Japan, those with high compared to low circulating equol concentrations had a lower risk of prostate cancer (multivariable-adjusted OR for upper quartile [Q4] vs. Q1 = 0.61, 95% confidence interval [CI] = 0.39-0.97), although there was no significant trend (OR per 75 percentile increase = 0.69, 95 CI = 0.46-1.05, p trend = 0.085); Genistein and daidzein concentrations were not significantly associated with risk (ORs for Q4 vs. Q1 = 0.70, 0.45-1.10 and 0.71, 0.45-1.12, respectively). In men from Europe, circulating concentrations of genistein, daidzein and equol were not associated with risk. Circulating lignan concentrations were not associated with the risk of prostate cancer, overall or by disease aggressiveness or time to diagnosis. There was no strong evidence that prediagnostic circulating concentrations of isoflavones or lignans are associated with prostate cancer risk, although further research is warranted in populations where isoflavone intakes are high., (© 2018 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background & Aims: To evaluate the relationship between phytoestrogen and colon cancer risk, we quantified plasma isoflavones (Genistein and Daidzein) and lignan (enterolactone) in a Korean nested case-control study and conducted replication study in a Vietnamese case-control study., Methods: Study populations of 101 cases and 391 controls were selected from the Korean Multicenter Cancer Cohort which was constructed from 1993 to 2004. For replication study, Vietnamese hospital-based case-control subjects of 222 cases and 206 controls were selected from 2003 to 2007. The concentrations of plasma genistein, daidzein, and enterolactone were quantified by liquid chromatography-mass spectrometry. Logistic regression models were used to compute odds ratios (ORs) and 95% confidence intervals (CIs), and meta-analysis was conducted to estimate combined ORs (CORs) and 95% Cis of Korean and Vietnamese population in 2014., Results: Genistein showed a continual decrease in colorectal cancer risk according to level up of the concentration categories in Korean and Vietnamese population (P for trend = 0.032, and 0.001, respectively) and a significantly decreased risk was found at the highest concentration of genistein and daidzein (for the highest category compared to the lowest: COR (95% CI) = 0.46 (0.30-0.69), and COR (95% CI) = 0.54 (0.36-0.82)). When the study population was stratified, the beneficial relationship of genistein with colorectal cancer was observed regardless of sex and anatomical subtype. However, enterolacton level was not associated with colorectal cancer risk., Conclusions: High plasma levels of isoflavones had relationship with a decreased risk of colorectal cancer, regardless of different ethnic background., (Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Doenjang (DJ), a fermented soybean product used in soups, stews, and sauces, contains high quality proteins, fats, vitamins, minerals, and other functional ingredients, including isoflavones and saponins. This study investigated whether DJ improves the bioavailability of isoflavones compared to boiled soybean (BS) in sham-operated or ovariectomized (OVX) rats. We also examined the effects of ovariectomy on the differences in bioavailability of isoflavones. BS and DJ were administered in sham-operated and OVX rats, and blood samples were collected. Twenty-six isoflavone-derived metabolites were identified. Pharmacokinetic analysis revealed that T 1/2 values of the individual isoflavone metabolites were most different in sham and OVX rats, even after the same sample treatment; however, T max values were significant different in a few metabolites such as daidzein 4'-glucuronide, daidzein 4'-sulfate, 2-(4-hydroxyphenyl)propionic acid, and benzoic acid. For most of the individual metabolites, C max was higher in both sham and OVX rats administered BS than those administered DJ. The AUC was generally lower in OVX rats than in sham rats. The AUC of daidzein and genistein in BS-fed sham rats was approximately 1.7-fold higher than those administered DJ, whereas glycitein was detected only in the DJ group. No significant differences in AUC of daidzein and genistein were observed between BS and DJ administration in OVX rats, although the total isoflavone content of DJ was lower; thus, DJ-mediated isoflavone bioavailability was more effective in OVX rats. Similar tendencies were observed for phase II and gut-mediated metabolites. These results suggested that DJ enhanced isoflavone bioavailability under estrogen deficiency, even when the total isoflavone content was decreased by fermentation., (© 2018 Institute of Food Technologists®.)

Objective: Selected estrogen receptor β-selective phytoestrogen (phytoSERM), a preparation of genistein, daidzein, and S-equol, has an 83-fold selective affinity for estrogen receptor (ER) β, and may promote neuronal survival and estrogenic mechanisms in the brain without exerting feminizing activity in the periphery. The aim of this study was to assess the safety, tolerability, and single-dose pharmacokinetics of the phytoSERM formulation in perimenopausal and postmenopausal women., Methods: Eighteen women aged 45 to 60 years from a 12-week clinical trial evaluating cognitive performance and vasomotor symptoms were randomly assigned to placebo, 50 mg, or 100 mg phytoSERM treatment groups. Plasma levels of the three parent phytoestrogens and their metabolites were measured before and at 2, 4, 6, 8, and 24 hours after ingestion by isotope dilution high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry., Results: Plasma concentrations of genistein, daidzein, and S-equol peaked at 9, 6, and 4 hours, respectively, for the 50-mg dose, and at 6, 6, and 5 hours, respectively, for the 100-mg dose. The maximum concentration (Cmax) and area under the curve (AUC) for the three parent compounds were greater in the 100-mg dose group, indicating a dose-dependent change in concentration with the phytoSERM treatment. No adverse events were elicited., Conclusions: A single-dose oral administration of the phytoSERM formulation was well-tolerated and did not elicit any adverse events. It was rapidly absorbed, reached high plasma concentrations, and showed a linear dose-concentration response in its pharmacokinetics. These findings are consistent with previously reported parameters for each parent compound (Clinicaltrials.gov NCT01723917).

Scope: Cheonggukjang (CGJ) is a soybean-based quick-fermented food popular in Korea that contains a variety of biologically active compounds including isoflavones and saponins. Isoflavone bioavailability may be important for the bone health of postmenopausal women; therefore, the aim of this study is to evaluate the influence of fermentation on the isoflavone metabolite nutrikinetic profile after single dose CGJ or unfermented soybean administration in ovariectomized (OVX) and sham mice., Methods and Results: We identify 34 isoflavone metabolites using UPLC-QTOF-MS and analyze their nutrikinetics at different time points (0.25, 0.5, 1, 2, 4, 8, 16, and 24 h) to understand their fermentation- and OVX-mediated time-dependent concentration changes. Nutrikinetics analysis shows that genistein, daidzein, genistein 4'-sulfate, dihydrodaidzein sulfate, equol 4'-sulfate, and equol-7-glucuronide are present at high concentrations in all groups based on area-under-the-curve analysis. OVX mice appear to show lower isoflavone bioavailability than mice in the sham group. CGJ enhances various isoflavone metabolite bioavailability including genistein, 3-hydroxygenistein, and equol 7-glucuronide, compared to the unfermented soybean-treated group. Among these metabolites, intact isoflavones, 3-hydroxygenistein, genistein 4'-sulfate, and equol 7-glucuronide promote osteoblastogenesis and inhibit osteoclast formation., Conclusions: CGJ has good isoflavone bioavailability and may be beneficial for the bone health of postmenopausal women., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Phytoestrogens (PE) may improve breast cancer prognosis by modifying tumor prognostic markers, such as cell proliferation marker Ki-67 and human epidermal growth factor receptor 2 (HER2). Epidemiological evidence linking lignans and isoflavones to Ki-67 and HER2 is limited. We examined associations between the major metabolites of lignans and isoflavones - enterolactone (ENL) and genistein (GEN) - respectively, and Ki-67 expression and HER2 in tumor tissue of breast cancer patients., Methods and Results: Data from 1060 invasive breast cancer patients from the population-based MARIE study were used. Multivariate-adjusted linear (Ki-67 log-transformed) and quantile regression, and logistic regression analyses (HER2, Ki-67 dichotomized) were performed to calculate β estimates and ORs, respectively. Median post-diagnostic ENL and GEN concentrations were 19.5 and 4.8 nmol/L, respectively. Median Ki-67 was 12.0%, and 21.2% of the tumors were HER2+. After adjustment, there was an inverse association between GEN and Ki-67 at high expression levels (OR for Ki-67 ≥20% versus <20% of 0.93 (95%CI [0.87;0.99]) per 10 nmol/L GEN increment)., Conclusion: Our findings indicate an inverse association between GEN and Ki-67 at high levels of Ki-67 expression. Additional investigations are recommended to confirm our findings and to further elucidate mechanisms linking PE metabolites to breast cancer survival., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Purpose: The in vivo oestrogenicity of genistein and its glycoside genistin is still under debate. The present study aimed to develop a physiologically based kinetic (PBK) model that provides insight in dose-dependent plasma concentrations of genistein aglycone and its metabolites and enables prediction of in vivo oestrogenic effective dose levels of genistein and genistin in humans., Experimental Approach: A PBK model for genistein and genistin in humans was developed based on in vitro metabolic parameters. The model obtained was used to translate in vitro oestrogenic concentration-response curves of genistein to in vivo oestrogenic dose-response curves for intake of genistein and genistin., Key Results: The model predicted that genistein-7-O-glucuronide was the major circulating metabolite and that levels of the free aglycone were generally low [0.5-17% of total plasma genistein at oral doses from 0.01 to 50 mg (kg·bw) -1 ]. The predicted in vivo benchmark dose for 5% response values for oestrogenicity varied between 0.06 and 4.39 mg kg -1 genistein. For genistin, these values were 1.3-fold higher. These values are in line with reported human data and show that oestrogenic responses can be expected at an Asian dietary and a supplementary intake, while intake resulting from a Western diet may not be effective., Conclusions and Implications: The present study shows how plasma concentrations of genistein and its metabolites and oestrogenic dose levels of genistein in humans can be predicted by combining in vitro oestrogenicity with PBK model-based reverse dosimetry, eliminating the need for human intervention studies., (© 2017 The British Pharmacological Society.)

Purpose: Whether it is safe for estrogen receptor-positive (ER+) patients with breast cancer to consume soy isoflavone genistein remains controversial. We compared the effects of genistein intake mimicking either Asian (lifetime) or Caucasian (adulthood) intake patterns to that of starting its intake during tamoxifen therapy using a preclinical model., Experimental Design: Female Sprague-Dawley rats were fed an AIN93G diet supplemented with 0 (control diet) or 500 ppm genistein from postnatal day 15 onward (lifetime genistein). Mammary tumors were induced with 7,12-dimethylbenz(a)anthracene (DMBA), after which a group of control diet-fed rats were switched to genistein diet (adult genistein). When the first tumor in a rat reached 1.4 cm in diameter, tamoxifen was added to the diet and a subset of previously only control diet-fed rats also started genistein intake (post-diagnosis genistein)., Results: Lifetime genistein intake reduced de novo resistance to tamoxifen, compared with post-diagnosis genistein groups. Risk of recurrence was lower both in the lifetime and in the adult genistein groups than in the post-diagnosis genistein group. We observed downregulation of unfolded protein response (UPR) and autophagy-related genes (GRP78, IRE1α, ATF4, and Beclin-1) and genes linked to immunosuppression (TGFβ and Foxp3) and upregulation of cytotoxic T-cell marker CD8a in the tumors of the lifetime genistein group, compared with controls, post-diagnosis, and/or adult genistein groups., Conclusions: Genistein intake mimicking Asian consumption patterns improved response of mammary tumors to tamoxifen therapy, and this effect was linked to reduced activity of UPR and prosurvival autophagy signaling and increased antitumor immunity. Clin Cancer Res; 23(3); 814-24. ©2017 AACR., (©2017 American Association for Cancer Research.)

In this study, effects of estradiol, progesterone and genistein on uterine aquaporin (AQP)-1, 2, 5 and 7 expression were investigated in sex-steroid deficient state which could help to elucidate the mechanisms underlying uterine fluid volume changes that were reported under these hormone and hormone-like compound influences., Methods: Uteri from ovariectomized, female Sprague-Dawley rats receiving seven days estradiol, progesterone or genistein (25, 50 and 100mg/kg/day) were harvested and levels of AQP-1, 2, 5 and 7 proteins and mRNAs were determined by Western blotting and Real-time PCR (qPCR) respectively. Distribution of these proteins in uterus was observed by immunohistochemistry., Results: Genistein caused a dose-dependent increase in uterine AQP-1, 2, 5 and 7 protein and mRNA expression, however at the levels lower than following estradiol or progesterone stimulations. Effects of genistein were antagonized by estradiol receptor blocker, ICI 182780. Estradiol caused the highest AQP-2 protein and mRNA expression while progesterone caused the highest AQP-1, 5 and 7 protein and mRNA expression in uterus. AQP-1, 2, 5 and 7 protein were found to be distributed in the myometrium as well as in uterine luminal and glandular epithelia and endometrial blood vessels. In conclusion, the observed effects of estradiol, progesterone and genistein on uterine AQP-1, 2, 5 and 7 expression could help to explain the differences in the amount of fluid accumulated in the uterus under these different conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Isoflavones may play a role in the prevention of hormone-related cancers. Equol is an isoflavone metabolized from daidzein in the presence of certain intestinal bacteria. Slackia sp. strain NATTS, a newly identified equol-producing bacterium, was recently isolated from human feces in Japan. We investigated the association of serum levels and dietary intake of isoflavones and Slackia sp. strain NATTS with the risk of prostate cancer in a case-control study among Japanese men., Methods: Fifty-six patients with newly diagnosed prostate cancer and 56 hospital controls were enrolled in this study. Isoflavones were assessed by measurement of serum levels and administration of a food frequency questionnaire. Slackia sp. strain NATTS in feces was also measured. The odds ratios (ORs) and 95 % confidence intervals (CIs) for prostate cancer were then determined using a logistic regression model., Results: The adjusted ORs for prostate cancer in comparison with the highest to lowest categories were 0.06 (95 % CI 0.02-0.24) for serum genistein, 0.18 (95 % CI 0.06-0.52) for daidzein, 0.16 (95 % CI 0.06-0.46) for glycitein, 0.52 (95 % CI 0.22-1.22) for equol, 0.86 (95 % CI 0.30-2.48) for dietary genistein, and 0.80 (95 % CI 0.28-2.28) for dietary daidzein. The adjusted OR for prostate cancer in comparison with values above versus below the median was 0.95 (95 % CI 0.42-2.16) for Slackia sp. strain NATTS., Conclusion: Our study findings suggest that high serum levels of genistein, daidzein, and glycitein are significantly associated with a decreased risk of prostate cancer among Japanese men.

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC-MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4'-glucuronides (39-49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33-41 %) and monoglucuronides (30-40 %) predominated. In male rats the disulfates (23-62 %) and 7-sulfo-4'-glucuronides (19-54 %) were predominant, while in female rats the 7-glucuronides (81-93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5-1.3 %) and rats (0.5-3.1 %) but comparatively high in mice (3.1-26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

Background: Vegetarian and vegan diets have become more popular among adolescents and young adults. However, few studies have investigated the nutritional status of vegans, who may be at risk of nutritional deficiencies., Objective: To compare dietary intake and nutritional status of Finnish long-term vegans and non-vegetarians., Methods: Dietary intake and supplement use were estimated using three-day dietary records. Nutritional status was assessed by measuring biomarkers in plasma, serum, and urine samples. Vegans' (n = 22) data was compared with those of sex- and age-matched non-vegetarians (n = 19)., Results: All vegans adhered strictly to their diet; however, individual variability was marked in food consumption and supplementation habits. Dietary intakes of key nutrients, vitamins B12 and D, were lower (P < 0.001) in vegans than in non-vegetarians. Nutritional biomarker measurements showed lower concentrations of serum 25-hydroxyvitamin D3 (25(OH)D3), iodine and selenium (corrected for multiple comparisons, P < 0.001), Vegans showed more favorable fatty acid profiles (P < 0.001) as well as much higher concentrations of polyphenols such as genistein and daidzein (P < 0.001). Eicosapentaenoic acid proportions in vegans were higher than expected. The median concentration of iodine in urine was below the recommended levels in both groups., Conclusions: Long-term consumption of a vegan diet was associated with some favorable laboratory measures but also with lowered concentrations of key nutrients compared to reference values. This study highlights the need for nutritional guidance to vegans.

The isoflavone genistein has been shown to act as topoisomerase II poison in various cell lines. Here, we address the question whether genistein is able to affect topoisomerase II in vivo. Juvenile male Wistar rats received either a single dose of genistein subcutaneously (s.c.; 10 mg/kg BW) or a lifelong isoflavone-rich diet encompassing in utero, lactation phase and 10 days of oral consumption, whereas genistein was mainly taken up as glycosides (25-50 mg/kg BW). The effects on the level of covalent topoisomerase II-DNA-complexes in the duodenum and colon were measured using the "Isolation of in vivo complexes of enzyme to DNA" (ICE)-bioassay. Simultaneously, serum as well as tissue concentrations of genistein and its metabolites were quantified by LC-MS. Genistein (s.c.) significantly increased the amount of covalent topoisomerase IIα and β-DNA complexes in the gut, showing more persistent effects in the colon than in the duodenum. In case of a lifelong dietary isoflavone exposure, no effects on the stabilization of cleavage complexes was observed, except a slight increase of topoisomerase IIα-DNA-complexes in the colon. The differences between the exposure routes might be attributed to the higher serum concentration of the genistein aglycon after subcutaneous treatment probably due to circumvention of first-pass metabolism compared to oral consumption of an isoflavone-rich diet. These data indicate that subcutaneously administrated genistein clearly possesses topoisomerase poisoning properties in vivo, whereas an isoflavone-rich diet containing genistein only caused a slight effect which relevance has to be clarified in further studies., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Epidemiological studies indicate lower prevalences of breast and prostate cancers and cardiovascular disease in Southeast Asia where vegetarianism is popular and diets are traditionally high in phytoestrogens. This study assessed plasma isoflavones in vegetarian and non-vegetarian Malaysian men according to age. Daidzein, genistein, equol (a daidzein metabolite), formononetin, biochanin A, estrone, estradiol and testosterone were measured by validated liquid chromatography tandem mass spectrometry (LCMSMS). Plasma isoflavone and sex hormone concentrations were measured in 225 subjects according to age (18-34, 35-44 and 45-67 years old). In all age groups, vegetarians had a higher concentration of circulating isoflavones compared with non-vegetarians especially in the 45-67 year age group where all isoflavones except equol, were significantly higher in vegetarians compared with omnivores. By contrast, the 18-34 year group had a significantly higher concentration of daidzein in vegetarians and significantly higher testosterone and estrone concentrations compared with non-vegetarians. In this age group there were weak correlations between estrone, estradiol and testosterone with some of the isoflavones. This human study provides the first Malaysian data for the phytoestrogen status of vegetarian and nonvegetarian men.

Purpose: Several nutrients act as phytoestrogens, being anti-adipogenic when consumed with a fat-rich diet. Their effect on a low-fat diet (LFD) background is unknown. We tested soy and genistein effects on adipose tissue in LFD-fed mice and genistein activity in the 3T3-L1 adipogenesis model., Methods: C57BL/6 J male mice were fed an 8.5% soy-supplemented LFD (SS-LFD) or a soy-free LFD (SF-LFD) for 147 days. Groups of 3-week-old (pubertal) and 6-week-old (adult) mice on the SF-LFD were also treated with 17ß-estradiol (E2, 5 µg/kg/day) ip or pure genistein (5 mg/kg/day) by gavage for 15 days. Body fat deposition and gene expression profiles were evaluated. E2 and genistein effects on ERα, ERβ and PPARγ transcriptional activities were characterized in ERα- or ERβ-transfected 3T3L1 cells during differentiation, by the use of reporter plasmids., Results: The SS-LFD group increased fat mass compared with the SF-LFD group. Genistein alone increased while E2 decreased fat pads in the 15-day-treated mice. In visceral fat, genistein differentially regulated 13 metabolic pathways compared to E2. PPARγ-controlled genes were downregulated by E2, while they were upregulated by genistein. In 3T3-L1 cells, genistein activated ERβ-driven transcription, differentiation and lipid accumulation, while inhibited ERα-driven transcription, without effects on lipid accumulation. E2 activated both ERs only in preadipocytes. In differentiated untransfected cells, genistein inhibited PPARγ, while activated PPARγ in the presence of ERβ., Conclusions: Soy and genistein at nutritional doses induce fat development in LFD-fed mice and adipogenesis in 3T3-L1 cells, with a mechanism that involves, at least in vitro, ERβ and is dependent on cell differentiation stage.

Most orally administered polyphenols are metabolized, with very little absorbed as aglycones and/or unchanged forms. Metabolic and pharmacokinetic studies are therefore necessary to understand the pharmacological mechanisms of polyphenols. Jumihaidokuto (JHT), a traditional Japanese medicine, has been used for treatment of skin diseases including inflammatory acne. Because JHT contains various types of bioactive polyphenols, our aim was to clarify the metabolism and pharmacokinetics of the polyphenols in JHT and identify active metabolites contributing to its antidermatitis effects. Orally administered JHT inhibited the increase in ear thickness in rats induced by intradermal injection of Propionibacterium acnes. Quantification by LC-MS/MS indicated that JHT contains various types of flavonoids and is also rich in hydrolysable tannins, such as 1,2,3,4,6-penta-O-galloyl glucose. Pharmacokinetic and antioxidant analyses showed that some flavonoid conjugates, such as genistein 7-O-glucuronide and liquiritigenin 7-O-glucuronide, appeared in rat plasma and had an activity to inhibit hydrogen peroxide-dependent oxidation. Furthermore, 4-O-methylgallic acid, a metabolite of Gallic acid, appeared in rat plasma and inhibited the nitric oxide reaction. JHT has numerous polyphenols; it inhibited dermatitis probably via the antioxidant effect of its metabolites. Our study is beneficial for understanding in vivo actions of orally administered polyphenol drugs.

Background: Obesity is one of the well-known risk factors of breast cancer. Accumulating evidence suggests that adiponectin, an obesity-related hormone, is inversely associated with breast cancer risk, particularly in postmenopausal women. Obesity is also associated with high levels of insulin. In addition, studies have suggested that the soy isoflavones present in the traditional Japanese diet have been associated with decreased risk of breast cancer. However, there is no study that has assessed associations between serum levels of isoflavones, insulin, adiponectin and the risk of breast cancer all together with menopausal status., Methods: In a case-control study of 63 histologically confirmed breast cancer patients and 76 controls, serum isoflavone, insulin, and high-molecular-weight (HMW) adiponectin levels and breast cancer risk were examined for their association with breast cancer risk after adjustment for various risk factors., Results: Women in the highest tertile of serum HMW adiponectin levels were associated with a statistically significant decreased risk for breast cancer compared with women in the lowest tertile [odds ratio (OR), 0.09; 95 % confidence interval (CI) 0.03-0.33]. This association was observed in postmenopausal women (OR 0.06; 95 % CI 0.01-0.28), but not in premenopausal women. The observed associations were independent of possible effects of insulin, body mass index, and known risk factors for breast cancer. Serum isoflavones and insulin levels were not associated with breast cancer risk., Conclusions: This study suggests that high serum HMW adiponectin levels are significantly associated with a decreased risk for breast cancer. Our result support the hypothesis that serum adiponectin may act as a potential biomarker for breast cancer.

The soy isoflavones daidzein and genistein produce several biological activities related to health benefits. A number of isoflavone extracts are commercially available, but there is little information concerning the specific isoflavone content of these products or differences in their bioavailability and pharmacokinetics. This study describes the development and validation of an analytical method to detect and quantify daidzein, genistein, and equol in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was applied in a crossover, randomized, bioavailability study. Twelve healthy volunteers were administered the same total isoflavones dose from two isoflavone supplement preparations (Super-Absorbable Soy Isoflavones (Life Extension, USA) and Fitoladius (Merck, Spain)). The pharmacokinetic parameters (AUC0-24/dose and Cmax/dose) of the isoflavones from the two preparations differed significantly. Such differences in bioavailability and kinetics may have relevant effects on the health benefits derived from their intake.

Background: The study aimed to examine the association between genes encoding molecules in the ornithine decarboxylase (ODC)-polyamine pathway (ODC1, AMD1, NQO1, NOS2A, and OAZ2) and gastric cancer risk and whether the gene-phytoestrogen interaction modifies gastric cancer risk., Methods: Among 76 gastric cancer cases and their 1:4 matched controls within the Korean Multi-center Cancer Cohort, a total of 30 SNPs in five genes involved in the ODC pathway were primarily analyzed. The second-stage genotyping in 388 matched case-control sets was conducted to reevaluate the significant SNPs interacting with phytoestrogens during the primary analysis. The summary odds ratios (ORs) [95 % confidence intervals (CIs)] for gastric cancer were estimated. Interaction effects between the SNPs and plasma concentrations of phytoestrogens (genistein, daidzein, equol, and enterolactone) were evaluated., Results: In the pooled analysis, NQO1 rs1800566 showed significant genetic effects on gastric cancer without heterogeneity [OR 0.83 (95 % CI 0.70-0.995)] and a greater decreased risk at high genistein/daidzein levels [OR 0.36 (95 % CI 0.15-0.90) and OR 0.26 (95 % CI 0.10-0.64), respectively; p interaction < 0.05]. Risk alleles of AMD1 rs1279599, AMD1 rs7768897, and OAZ2 rs7403751 had a significant gene-phytoestrogen (genistein and daidzein) interaction effect to modify the development of gastric cancer. They had an increased gastric cancer risk at low isoflavone levels, but a decreased risk at high isoflavone levels (p interaction < 0.01)., Conclusions: Our findings suggest that common variants in the genes involved in the ODC pathway may contribute to the risk of gastric cancer possibly by modulating ODC polyamine biosynthesis or by interaction between isoflavones and NQO1, OAZ2, and AMD1.

Soybean isoflavones are beneficial for treating hormone-related diseases. Simultaneous consumption of soybean isoflavones and Liuwei Dihuang pills (LWPs) is effective for treating perimenopausal period syndrome. However, why the combination of isoflavones and LWPs is more effective than ingestion of each component alone remains unknown. Here, we show that enhanced estrogenic activities would appear when the ovariectomized rats were fed with a soybean diet in combination of LWPs treatment. Our further studies explored enhancements of Lactobacillus (19-fold) and Bifidobacterium (12-fold) contents in the intestine of rat and 1.84-fold higher intestinal β-glucosidase activity in LWPs treatment group compared with the control group. As a result, steady-state concentrations of genistein (1.20-fold), daidzein (1.36-fold), and equol (1.43-fold) in serum were significantly elevated in the combination group compared with the soybean alone group. The results present the first evidence of the mechanism of enhanced estrogenic activity of dietary soybean isoflavones in combination with LWPs. Our study indicates that alterations of gut bacteria after LWPs treatment play a key role in the enhanced estrogenic effect of dietary soybean, suggesting a direct relationship between dietary soybean, LWPs, and gut flora., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objectives: Genistein is one of the main soy isoflavones in our daily diet. There were studies proving that high-dietary intake of genistein may relate to the low morbidity and mortality of prostate cancer (PCa) in the Asian population. Since there were few studies of plasma genistein level in the Chinese population, we performed this study to preliminarily evaluate the associations among plasma genistein, epidemiologic factors and PCa in a Chinese population., Methods: Between 2012 and 2013, 100 men over the age of 40 underwent prostate biopsy for PCa at Huashan Hospital, Shanghai, China. Clinical information, epidemiologic information and blood samples were collected prior to biopsy for each patient. All patients underwent 10-core ultrasound-guided transperineal prostate biopsy, and the pathology results were collected after biopsy. We measured the plasma genistein concentration of the blood samples and analyzed the results along with the clinical and epidemiologic information., Results: Among the 100 patients, 46 (46.0 %) were diagnosed with PCa. The median plasma genistein concentration of non-PCa patients (728.6 ng/ml) was significantly higher than that of PCa patients (513.0 ng/ml) (P < 0.05). In the univariate analysis, we found that age and smoking history were related to PCa (P < 0.05). In the multivariate analysis, we found that age, smoking history and plasma genistein were related to PCa (P < 0.05). The age-adjusted odds ratio of PCa risk comparing plasma genistein level above median to that below median was 0.31 (95 % CI 0.13-0.71)., Conclusion: Our study suggested that high concentration of plasma genistein level may contribute to the low incidence of prostate cancer in Chinese population.

Importance: Soy isoflavone supplements are used to treat several chronic diseases, although the data supporting their use are limited. Some data suggest that supplementation with soy isoflavone may be an effective treatment for patients with poor asthma control., Objective: To determine whether a soy isoflavone supplement improves asthma control in adolescent and adult patients with poorly controlled disease., Design, Setting, and Participants: Multicenter, randomized, double-blind, placebo-controlled trial conducted between May 2010 and August 2012 at 19 adult and pediatric pulmonary and allergy centers in the American Lung Association Asthma Clinical Research Centers network. Three hundred eighty-six adults and children aged 12 years or older with symptomatic asthma while taking a controller medicine and low dietary soy intake were randomized, and 345 (89%) completed spirometry at week 24., Interventions: Participants were randomly assigned to receive soy isoflavone supplement containing 100 mg of total isoflavones (n=193) or matching placebo (n=193) in 2 divided doses administered daily for 24 weeks., Main Outcomes and Measures: The primary outcome measure was change in forced expiratory volume in the first second (FEV1) at 24 weeks. Secondary outcome measures were symptoms, episodes of poor asthma control, Asthma Control Test score (range, 5-25; higher scores indicate better control), and systemic and airway biomarkers of inflammation., Results: Mean changes in prebronchodilator FEV1 over 24 weeks were 0.03 L (95% CI, -0.01 to 0.08 L) in the placebo group and 0.01 L (95% CI, -0.07 to 0.07 L) in the soy isoflavone group, which were not significantly different (P = .36). Mean changes in symptom scores on the Asthma Control Test (placebo, 1.98 [95% CI, 1.42-2.54] vs soy isoflavones, 2.20 [95% CI, 1.53-2.87]; positive values indicate a reduction in symptoms), number of episodes of poor asthma control (placebo, 3.3 [95% CI, 2.7-4.1] vs soy isoflavones, 3.0 [95% CI, 2.4-3.7]), and changes in exhaled nitric oxide (placebo, -3.48 ppb [95% CI, -5.99 to -0.97 ppb] vs soy isoflavones, 1.39 ppb [95% CI, -1.73 to 4.51 ppb]) did not significantly improve more with the soy isoflavone supplement than with placebo. Mean plasma genistein level increased from 4.87 ng/mL to 37.67 ng/mL (P < .001) in participants receiving the supplement., Conclusions and Relevance: Among adults and children aged 12 years or older with poorly controlled asthma while taking a controller medication, use of a soy isoflavone supplement, compared with placebo, did not result in improved lung function or clinical outcomes. These findings suggest that this supplement should not be used for patients with poorly controlled asthma., Trial Registration: clinicaltrials.gov Identifier: NCT01052116.

Background: The isoflavone genistein, a natural soy product with receptor tyrosine kinase-inhibiting activity, as well as phytoestrogenic and other potential anticarcinogenic effects, is being studied as an anticancer agent. Since isoflavones are commonly consumed in food products containing soy proteins, a method to control for baseline isoflavone consumption is needed., Methods: HPLC was used to evaluate baseline plasma and urine concentrations of isoflavone in fifty-four participants with bladder cancer enrolled on a phase II chemoprevention study of G-2535. The soy food frequency questionnaire was used to assess participant's baseline soy intake. The association between baseline isoflavone concentrations and intakes for genistein and daidzein was assessed by the Spearman's rank correlation coefficient., Results: The majority of participants had no detectable genistein or daidzein in plasma at baseline. The median and range of values were 0 (0-1480) nmol/L for genistein, and 0 (0-1260) nmol/L for daidzein. In urine, the median and range of values were 91.0 (0-9030) nmol/L for genistein and 623 (0-100,000) nmol/L for daidzein. The median and range of weekly estimated genistein intake was 0 (0-236) mg/wk; the median and range of weekly estimated daidzein intake was 0 (0-114) mg/wk. There was no relationship to soy intake as measured by the food frequency questionnaire and baseline isoflavone levels in plasma or urine and the Spearman's rank correlation coefficients were not significant., Conclusion: The soy food frequency questionnaire did not correlate with plasma or urine concentrations of either isoflavone., Impact: Alternative methods for controlling for soy consumption, including measuring plasma and urine concentrations, in isoflavone chemoprevention trials should be considered., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Aims/hypothesis: To examine the association between soybean products and risk of type 2 diabetes, we measured four isoflavone biological markers--genistein, daidzein, glycitein and equol--in a nested case-control study., Methods: The study population was composed of 693 cases (316 women and 377 men) and 698 matched controls (317 women and 381 men) within the Korean Genome and Epidemiology Study. The concentrations of isoflavone biomarkers were measured using HPLC-MS/MS on plasma samples that were collected at baseline. A stratified analysis was undertaken to examine the association between plasma isoflavone concentrations and risk of type 2 diabetes according to sex and equol production. Logistic regression models were used to compute ORs and 95% CIs adjusted for confounders., Results: In women, compared with the lowest quartile of plasma concentration of genistein, the highest quartile exhibited a significantly decreased risk of diabetes (OR 0.58, 95% CI 0.35, 0.95). When stratified by equol-producing status in women, the OR for diabetes in the highest vs the lowest quartile of genistein concentration was 0.31 (95% CI 0.16, 0.60) in equol producers, but genistein concentration was not associated with risk of diabetes in equol non-producers (p for interaction = 0.013). In men, isoflavone concentrations were not associated with risk of diabetes, regardless of equol-producing status., Conclusions/interpretation: High plasma concentrations of genistein were associated with a decreased risk of type 2 diabetes in women. This inverse association was prominent in equol-producing participants. These results suggest a beneficial effect of a high intake of soybean products on risk of type 2 diabetes in women.

Bone is one of the most common sites for metastasis in breast cancer (BC). Micro-metastasis in bone marrow was detected in 30% of patients with stage I, II, or III BC at primary surgery and is a strong indicator of poor prognosis. The role dietary soy isoflavones play in BC with bone micro-metastasis is unclear. In this study, we examined the effects of genistein, daidzein, (-)-equol or a mixture of soy isoflavones on BC with bone micro-metastasis using an experimental model of murine mammary cancer 4T1 cells engineered with luciferase. A small number (1000) of 4T1 cells were injected into the tibia of female Balb/c mice to establish micro-tumors in bone. Soy isoflavones were supplemented in the AIN-93G diet at 750 mg/kg and were provided to mice from 3 weeks before to 3 weeks after cell injection. Bioluminescent imaging was conducted on day 2 (D2), D6, D8, D16 and D20 post cell injection and the results indicated dietary soy isoflavones enhanced the growth of bone micro-tumors on D8. Furthermore, dietary soy isoflavones stimulated metastatic tumor formation in lungs and increased Ki-67 protein expression in these metastasized tumors. In vitro, soy isoflavones (<10 µM) had limited effects on the growth, motility or invasion of 4T1 cells. Thus, the in vivo stimulatory effect could be likely due to systemic effects between the host, 4T1 tumors and soy isoflavones. In conclusion, soy isoflavones stimulate BC with bone micro-metastasis in mice and further investigations are needed regarding their consumption by BC survivors.

A combination of soy isoflavones and Liu Wei Di Huang Wan (LWDHW) is potentially effective for postmenopausal women with intolerable vasomotor episodes who are not suitable candidates for hormonal therapy. The objective of this open-label, three-phase, crossover study was to determine the influence of both single and multiple oral doses of LWDHW on isoflavone pharmacokinetics in healthy postmenopausal women. Eleven subjects were assigned to receive the following regimens in a fixed sequence with washout periods of at least one week: Phase A, a single oral dose of soy milk; Phase B, a single oral dose of soy milk coadministered with LWDHW; and Phase C, multiple oral doses of LWDHW for 14 days followed by a single oral dose of soy milk. Blood samples were collected and mixed with β-glucuronidase/sulfatase to hydrolyze isoflavone conjugates to their respective aglycones (i.e., daidzein and genistein) and were determined using high performance liquid chromatography. The pharmacokinetic parameters analyzed were maximal plasma concentration (C max), time to reach peak concentration (T max), area under the plasma concentration-time curve (AUC), and half-life (t 1/2). The results found no statistically significant differences in pharmacokinetic parameters of daidzein and genistein among the three regimens.

Scope: Genistein from foods or supplements is metabolized by the gut microbiota and the human body, thereby releasing many different metabolites into systemic circulation. The order of their appearance in plasma and the possible influence of food format are still unknown. This study compared the nutrikinetic profiles of genistein metabolites., Methods and Results: In a randomized cross-over trial, 12 healthy young volunteers were administered a single dose of 30 mg genistein provided as a genistein tablet, a genistein tablet in low fat milk, and soy milk containing genistein glycosides. A high mass resolution LC-LTQ-Orbitrap FTMS platform detected and quantified in human plasma: free genistein, seven of its phase-II metabolites and 15 gut-derived metabolites. Interestingly, a novel metabolite, genistein-4'-glucuronide-7-sulfate (G-4'G-7S) was identified. Nutrikinetic analysis using population-based modeling revealed the order of appearance of five genistein phase II metabolites in plasma: (1) genistein-4',7-diglucuronide, (2) genistein-7-sulfate, (3) genistein-4'-sulfate-7-glucuronide, (4) genistein-4'-glucuronide, and (5) genistein-7-glucuronide, independent of the food matrix., Conclusion: The conjugated genistein metabolites appear in a distinct order in human plasma. The specific early appearance of G-4',7-diG suggests a multistep formation process for the mono and hetero genistein conjugates, involving one or two deglucuronidation steps., (© 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background/objectives: Isoflavones are present in soy foods and soy-based supplements. Despite low plasma isoflavone concentrations in the general Western population, concentrations in supplement users exceed those suggested to be beneficial for health in Asian populations, raising concerns for adverse effects. To aid risk assessment, quantification of the relation between isoflavone intake and plasma concentrations is essential., Subjects/methods: Plasma samples were collected from postmenopausal women in three placebo-controlled crossover studies with 8-week periods for supplements (two studies, ~100 mg isoflavones/day, n=88) or 4-week periods for soy foods (one study, ~48 mg isoflavones/day, n=15). Plasma isoflavone concentrations (daidzein, equol, genistein and glycitein) were quantified using high-performance liquid chromatography and electrochemical detection. The association between plasma concentrations and isoflavone intake, equol producer status, intake-producer interaction and background dietary intake was assessed based on the assumption of a log-linear relation., Results: Median plasma total isoflavone concentrations after the soy food and supplement interventions were respectively 2.16 and 3.47 μmol/l for equol producers and 1.30 and 2.39 μmol/l for non-producers. Regression analysis showed that doubling isoflavone intake increased plasma concentrations by 55-62% (±s.e. 1-2%, R(2)>0.87) for daidzein, genistein, equol (only for producers) and total isoflavones; for glycitein the association was weaker (15±1%, R(2)=0.48). Adjustments for energy, carbohydrate and fat intake did not affect these estimates. Inter-individual variation, estimated based on repeated measures in one of the studies, was 30-96%., Conclusions: Although the relation between isoflavone intake and plasma concentrations was adequately quantified, the use of isoflavone intake data for risk assessment needs caution due to large inter-individual variation in plasma concentrations.

Twenty-one-day-old female Wistar rats were treated daily with orally administered soy isoflavones (SIFs) at concentrations of 50, 100, or 200 mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian follicle development was evaluated. At the end of the treatment period, the ultrastructure of the ovarian granulosa cells was examined by transmission electron microscopy. The apoptotic cell death of ovarian granulosa cells was detected using TUNEL staining. The mRNA expression levels of caspase-3, caspase-8, caspase-9, Bcl2, Bax, and Fas were determined by real-time quantitative PCR. The protein expression levels of caspase-3, Bcl2, Bax, and Fas were determined by western blotting. Our data showed that exposure to SIFs resulted in morphological changes consistent with ovarian granulosa cell apoptosis. The percentage of TUNEL-positive granulosa cells was increased. The mRNA expression levels of the apoptosis-related genes caspase-3, caspase-8, caspase-9, Bax, and Fas increased significantly. The protein levels of Bax, Fas, and cleaved caspase-3 were also increased. These results indicate that the exposure of rats to modest doses of SIFs from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis. The mechanism of SIF-induced alterations in ovarian follicle development may involve the activation of Fas-mediated and Bcl2/Bax-mediated apoptotic signaling pathways., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: There are conflicting reports on the impact of soy on breast carcinogenesis. This study examines the effects of soy supplementation on breast cancer-related genes and pathways., Methods: Women (n = 140) with early-stage breast cancer were randomly assigned to soy protein supplementation (n = 70) or placebo (n = 70) for 7 to 30 days, from diagnosis until surgery. Adherence was determined by plasma isoflavones: genistein and daidzein. Gene expression changes were evaluated by NanoString in pre- and posttreatment tumor tissue. Genome-wide expression analysis was performed on posttreatment tissue. Proliferation (Ki67) and apoptosis (Cas3) were assessed by immunohistochemistry., Results: Plasma isoflavones rose in the soy group (two-sided Wilcoxon rank-sum test, P < .001) and did not change in the placebo group. In paired analysis of pre- and posttreatment samples, 21 genes (out of 202) showed altered expression (two-sided Student's t-test, P < .05). Several genes including FANCC and UGT2A1 revealed different magnitude and direction of expression changes between the two groups (two-sided Student's t-test, P < .05). A high-genistein signature consisting of 126 differentially expressed genes was identified from microarray analysis of tumors. This signature was characterized by overexpression (>2-fold) of cell cycle transcripts, including those that promote cell proliferation, such as FGFR2, E2F5, BUB1, CCNB2, MYBL2, CDK1, and CDC20 (P < .01). Soy intake did not result in statistically significant changes in Ki67 or Cas3., Conclusions: Gene expression associated with soy intake and high plasma genistein defines a signature characterized by overexpression of FGFR2 and genes that drive cell cycle and proliferation pathways. These findings raise the concerns that in a subset of women soy could adversely affect gene expression in breast cancer., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Summary: Postmenopausal estrogen decline is implicated in several age-related physical and psychological changes in women, including decreases in perceived quality of life. The phytoestrogen genistein at a dose of 54 mg daily in osteopenic postmenopausal women after 2 years implies an improvement on quality of life and depression symptoms., Introduction: Postmenopausal estrogen decline is implicated in several age-related physical and psychological changes in women, including decreases in perceived quality of life (QoL). A number of trials with hormone therapy showed beneficial effects of the intervention on quality of life parameters. However, because of known or suspected serious side effects of conventional hormone therapy, there is a need for alternatives., Methods: We conducted a double-blind randomized placebo-controlled trial using the isoflavone genistein, 54 mg, or placebo for 2 years. In this trial, we recruited 262 postmenopausal women aged 49 to 67 years., Results: At baseline, after 1 year, and at final visit, participants filled in the Short Form of 36 questions (SF-36) and the Zung Self-rating Depression Scale (ZSDS). For the placebo group, scores on all dimensions of the SF-36 decreased after 1 and 2 years. The genistein group showed increases on all dimensions of the SF-36 at the end of the study. There were, however, statistically significant differences in changes of scores between the two intervention groups. For the ZSDS, similarly, significant differences were found between groups., Conclusion: In conclusion, the findings of this randomized trial showed that genistein improves quality of life (health status, life satisfaction, and depression) in osteopenic postmenopausal women.

Several studies have demonstrated that a balanced diet can contribute to better human health. For this reason, soy-based food and pure isoflavones (pills) are one of the most consumed. The association of this consumption and lower risks of chronic diseases and cancer is well established for the Asian population and has been attracting the attention of people worldwide, especially women at menopause who seek to alleviate the symptoms associated with the lack of estrogen. Despite positive epidemiological data, concerns still exist because of conflicting results found in scientific literature with relation to the role of isoflavones in breast and hormone-related cancers. The aim of our study was to investigate the cytotoxicity, induction of apoptosis, and changes in apoptosis-related genes of maximal physiological serum levels of the isoflavone genistein (Gen) in MCF-7 tumoral cells and in HB4a non-tumoral cells. In addition, induction of cell cycle arrest was also investigated. Only supraphysiological levels of Gen (50 and 100 μM) were cytotoxic to these cell lines. Concentrations of 10 and 25 μM did not induce apoptosis and significant changes in expression of the studied genes. Positive results were found only in cell cycle analysis: G0/G1 delay of MCF-7 cells in both concentrations of Gen and at 25 μM in HB4a cells. It is the first study investigating effects of Gen in the HB4a cell line. Thus, despite the lack of apoptosis induction (generally found with high concentrations), Gen at physiologically relevant serum levels still exerts chemopreventive effects through the modulation of cell cycle.

Scope: The effect of food combination on metabolic profile in postprandial plasma has hardly been reported. We investigated the absorption and metabolism of quercetin and soy isoflavones in humans after combination meal consumption., Methods and Results: Five healthy volunteers ingested sautéed onion and tofu, and the plasma metabolites of quercetin and isoflavones were analyzed. Quercetin and genistein were incubated with human intestinal Caco-2 cells and human hepatoma HepG2 cells to further analyze the influence of simultaneous supply to the small intestine and the liver. Glucuronosyl conjugates of quercetin and methylated quercetin were the major plasma metabolites in the case of onion intake. Plasma metabolites with the single serving of tofu were both glucuronide and sulfate metabolites of isoflavones. Interestingly, quercetin sulfate was only detected after the combined intake of sautéed onion and tofu, accompanied with a decrease in sulfated isoflavones. Besides, quercetin was shown as the preferential substance for phase II enzymes over genistein in both Caco-2 and HepG2 cells., Conclusion: These results indicate that, when flavonoids and isoflavonoids were ingested together, the metabolic conversions in the small intestine and/or the liver could be altered, resulting in the variation of the postprandial profiles of the plasma metabolites., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To develop and validate a food frequency questionnaire (FFQ) to estimate the genistein intake in a Malaysian population of pregnant women., Method: A single 24-h dietary recall was obtained from 40 male and female volunteers. A FFQ of commonly consumed genistein-rich foods was developed from these recalls, and a database of the genistein content of foods found in Malaysia was set up. The FFQ was validated against 7-d food diary (FD) kept by 46 pregnant women and against non-fasting serum samples obtained from 64 pregnant women. Reproducibility was assessed by comparing the responses on two FFQs administered approximately 1 month apart., Results: The Pearson correlation coefficient between FFQ1 and FD was 0.724 and that between FFQ2 and FD was 0.807. Classification into the same or adjacent quintiles was 78% for FFQ1 versus FD and 88% for FFQ2 versus FD. A significant dose -- response relation was found between FFQ-estimated genistein intake and serum levels., Conclusion: The FFQ developed is a reliable, valid tool for categorising people by level of genistein intake.

Objective: This study aims to evaluate the effects of soy isoflavones on breast tissue in postmenopausal women., Methods: In this randomized, double-blind, placebo-controlled study, 80 women (aged ≥ 45 y and with amenorrhea >12 mo) with vasomotor symptoms were randomized to receive either 250 mg of standardized soy extract corresponding to isoflavone 100 mg/day (n = 40) or placebo (n = 40) for 10 months. Breasts were evaluated through mammographic density and breast parenchyma using ultrasound (US) at baseline and 10-month follow-up. Independent t test, analysis of variance, Mann-Whitney U test, and χ2 trend test were used in statistical analysis., Results: Baseline clinical characteristics showed no significant differences between the isoflavone group and the placebo group, with mean (SD) age of 55.1 (6.0) and 56.2 (7.7) years, mean (SD) menopause duration of 6.6 (4.8) and 7.1 (4.2) years, and mean (SD) body mass index of 29.7 (5.0) and 28.5 (4.9) kg/m2, respectively (P > 0.05). The study was completed by 32 women on isoflavone and 34 women on placebo. The groups did not differ in mammographic density or breast parenchyma by US (P > 0.05). Within each group, the baseline and final moments did not differ in mammography or US parameters significantly (P > 0.05)., Conclusions: The use of soy isoflavone extract for 10 months does not affect breast density, as assessed by mammography and US, in postmenopausal women.

The incidence of gastric cancer throughout the world is ~2-3 times higher in men than in women. Previous research suggested that isoflavones, which are structurally similar to 17β-estradiol, may prevent gastric cancer. Based on a large, population-based, prospective study, we recently reported a null association between dietary isoflavone intake and gastric cancer. However, epidemiologic studies using blood concentrations of isoflavones might better reflect the effect of isoflavones on gastric cancer carcinogenesis than dietary assessment. We therefore conducted a nested case-control study within the Japan Public Health Center-Based Prospective Study. Participants were followed-up from 1990 to 2004. Among 36,745 participants who answered the baseline questionnaire and provided blood samples, 483 gastric cancer cases matched to 483 controls were used in the analysis. ORs and 95% CIs were estimated with a conditional logistic regression model. The overall distribution of plasma isoflavone concentrations was not associated with the development of gastric cancer. Compared with groups with the lowest plasma concentrations (reference groups), the groups with the highest daidzein and genistein concentrations had adjusted ORs and 95% CIs of 1.11 (0.74-1.66; P-trend = 0.6) and 0.96 (0.64-1.44; P-trend = 0.9), respectively. The results did not change when analysis was based on sex, subsite, or histological type. We found no association of plasma isoflavone concentrations with gastric cancer risk. Our data support the previously observed null association between isoflavone intake and gastric cancer risk.

A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.

Objective: Estrogen deficiency after menopause results in rapid bone loss, predisposing women to osteoporotic fractures. Genistein, a phytoestrogen present in high concentrations in soy, is an ingredient in dietary supplements aggressively marketed for bone health. However, in a recent long-duration clinical trial in postmenopausal women, the efficacy of soy extracts in reducing bone loss was disappointing. To better understand the failure of soy extracts to consistently induce a robust skeletal response in women, we investigated the long-term (5 mo) efficacy of genistein, administered as a daily oral supplement, (1) in preventing cancellous bone loss in skeletally mature virgin Long-Evans rats ovariectomized at 7 months of age and (2) in improving cancellous bone mass and architecture in aged retired-breeder rats ovariectomized at 16 or 22 months of age., Methods: Rats within each age group were randomly assigned into one of three treatment groups (n = 7-12 rats/group): (1) vehicle control, (2) genistein 485 μg/day, or (3) genistein 970 μg/day, resulting in mean (SE) serum genistein levels of 0.18 (0.10), 0.76 (0.15), and 1.48 (0.31) μM, respectively. Total tibia bone mass and density were evaluated using dual-energy x-ray absorptiometry, whereas cancellous bone mass and architecture in the tibial metaphysis, as well as cortical bone mass and architecture in the tibial diaphysis, were evaluated by micro-CT., Results: Oral genistein administered as a dietary supplement did not influence the cumulative effects of ovariectomy, aging, and/or reproductive history on cancellous and cortical bone mass and architecture., Conclusions: Serum levels of genistein similar to those in women consuming a high-soy diet are ineffective in preventing or treating bone loss in rat models for postmenopausal osteoporosis.

We tested the hypothesis that the phytoestrogen genistein protects the brain against ischemic stroke by improving the circulatory function in terms of reduced production of thromboxane A2 and leukocyte-platelet aggregates, and of preserved vascular reactivity. Ischemia-reperfusion (90 min-3 days, intraluminal filament) was induced in male Wistar rats, and functional score and cerebral infarct volume were the end points examined. Genistein (10mg/kg/day) or vehicle (β-cyclodextrin) was administered at 30 min after ischemia or sham-operation. Production of thromboxane A2 and leukocyte-platelet aggregates, as well as reactivity of carotid artery to U-46619 (thromboxane A2 analogue) and to platelet releasate was measured. At 3 days post-ischemia, both improvement in the functional examination and reduction in the total infarct volume were shown in the ischemic genistein-treated group. Genistein significantly reverted both the increased thromboxane A2 concentration and the increased leukocyte-platelet aggregates production found in samples from the ischemic vehicle-treated group. Both U-46619 and platelet releasate elicited contractions of the carotid artery, which were significantly lower in the ischemic vehicle-treated group. Genistein significantly restored both the decreased U-46619- and the decreased platelet releasate-elicited contractile responses. In conclusion, genistein protects the brain against an ischemia-reperfusion challenge, at least in part, by its beneficial effects on the circulatory function., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An accurate and sensitive analytical method has been developed for the quantification of genistein in dog plasma using high-performance liquid chromatography/tandem mass spectrometry. Genistein and diclofenac (internal standard) were extracted from the plasma sample using methyl tert-butyl ether and then separated on an Agilent Zorbax C18 column using a gradient mobile phase. The detector was a Q-trap mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. The assay was linear over the concentration range of 0.10-500 ng/mL with a lower limit of quantification of 0.10 ng/mL. The method was shown to be reproducible and reliable, with inter-day and intra-day accuracy and precision within ±15%. The method was successfully applied to a pharmacokinetic comparison of immediate and extended release tablets in beagle dogs after oral administration. Immediate release tablets showed rapid genistein absorption, with mean peak concentration of 726 ± 199 ng/mL reached at 0.2 ± 0.0 h. However, the absorption of genistein was considerably slower and more sustainable for extended release tablets. The relative bioavailability of the extended release tablet over the immediate release formulation was estimated to be 134 ± 47% based on the AUCInf values from non-compartmental analysis.

Some felid diets contain isoflavones but the metabolic capacity of cats toward isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet (n = 6) or as part of a dietary matrix (n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and disulfate as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and disulfate, dihydrodaidzein, and O-desmethylangolensin. No β-glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared with cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein and daidzein, in domestic cat plasma after dietary ingestion has been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of typical dietary intake.

Endocrine disruptors that mimic natural hormones and inhibit the action of hormones have recently attracted attention as one of the main cause of precocious puberty. In this study, the levels of 7 EDCs and 3 isoflavones that exhibit estrogen-like actions were measured in the plasma of precocious puberty patients and compared to control subjects to determine if there is an association between the onset of precocious puberty and the levels of EDCs in the plasma. EDCs examined in this study were bisphenol-A (BPA), di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), mono(2-ethylhexyl) phthalate (MEHP), monobutyl phthalate (MBP), n-nonyl phenol (n-NP), and t-octylphenol (t-OP), and whereas the isoflavones were equol, genistein, and diadzein. The level of MBP in the plasma of patients was 1.3 times higher than that of the controls. The levels of t-OP and n-NP in the plasma of patients were respectively 1.15 and 1.2 times higher than those of the control group. Finally, the levels of the diadzein, equol and genistein were 1.37, 1.3 and 2.67 times higher than those of the control group, and genistein showed a statistically meaningful result (P = 0.0008). The results suggest that these six substances (MBP, t-OP, n-NP, daidzein, equol, and genistein) have an effect on precocious puberty.

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the quantification of isotoosendanin, an important bioactive component isolated from Meliae cortex. A Capcell PAK C18 column (100 × 4.6 mm) was used for the chromatographic elution using methanol-10 mM ammonium acetate-formic acid (80:20:0.1, v/v/v) as mobile phase at the flow rate of 0.6 mL/min. MS-MS analysis was performed on a triple quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization source in positive ion mode. Extraction of isotoosendanin and genistein (internal standard, IS) from rat plasma was determined by precipitating protein treatment. Quantification was performed by MS in the multiple reaction monitoring mode with positive ionization at m/z 557 → 437 for the analyte and m/z 271 → 215 for IS, respectively. Linear isotoosendanin calibration curves were obtained between 2.0-2,000 ng/mL with a correlation coefficient greater than 0.99. Acceptable precision and accuracy were acquired for concentrations over the standard curve range. Satisfactory results were achieved for sensitivity, specificity, recovery, freeze/thaw and stability. This analytical method was successfully applied to determine the pharmacokinetic parameters of isotoosendanin after an oral administration of 200 mg/kg to rats.