Phytoestrogen Metabolites Research Articles

Blackcurrant Anthocyanins Attenuate Estrogen -Deficiency-Induced Bone Loss through Modulating Microbial-Derived Short-Chain Carboxylic Acids and Phytoestrogen Metabolites in Peri- and Early Postmenopausal Women.

The present study aimed to assess the effects of blackcurrant (BC) anthocyanins on concentrations of microbial-derived short-chain carboxylic acids (SCCAs) and metabolites of phytoestrogens. We then examined their associations with six-month changes in whole-body bone mineral density (BMD) and biomarkers of bone metabolism. Fecal and blood samples from a pilot randomized controlled trial were collected and analyzed from 37 eligible peri- and early postmenopausal women aged 45-60 years who were randomized into one of three treatment groups consuming one placebo capsule (control), 392 mg BC (low BC) or 784 mg BC (high BC) daily for six months. Significant differences were observed between groups at baseline in acetic, propionic, valeric, caproic and heptanoic acids (p < 0.05). Isobutyric acid significantly decreased from baseline (0 months) to six months in the control group (p < 0.05) and the high BC group had a significantly greater concentration than the control group at six months (p < 0.05). Butyric acid was significantly greater in the high BC group than low BC at six months (p < 0.05). Six-month changes in caproic and isobutyric acids showed weak correlations with changes in whole-body BMD (r = 0.3519, p < 0.05 and r = 0.3465, p < 0.05, respectively). Isovaleric and valeric acids displayed weak correlations with BALP (r = 0.3361, p < 0.05) and OPG (r = 0.3593, p < 0.05), respectively. Enterodiol was positively correlated with BALP (r = 0.6056, p < 0.01) while enterolactone was positively correlated with osteocalcin (r = 0.5902, p < 0.001) and negatively correlated with sclerostin (r = -0.3485, p < 0.05). The results suggest that BC may be a potential dietary agent to reduce postmenopausal bone loss through modulating microbially-derived SCCAs and phytoestrogen metabolites.

Urinary phytoestrogens and the risk of uterine leiomyomata in US women

BackgroundUterine leiomyomata (UL) is a common gynecological disease in women. Studied on the relationship between single metabolites of urinary phytoestrogens and UL, especially for the combined effects of mixed metabolites on UL still are insufficient.MethodsIn this cross-sectional study, we included 1,579 participants from the National Health and Nutrition Examination Survey. Urinary phytoestrogens were assessed by measuring urinary excretion of daidzein, genistein, equol, O-desmethylangolensin, enterodiol and enterolactone. The outcome was defined as UL. Weighted logistic regression was used to analyze the association between single metabolites of urinary phytoestrogens and UL. Notably, we adopted the weighted quantile sum (WQS) regression, Bayesian kernel machine regression (BKMR), and quantile g-computation (qgcomp) models, to investigate the combined effects of six mixed metabolites on UL.ResultsThe prevalence of UL was approximately 12.92%. After adjusting age, race/ethnicity, marital status, drinking status, body mass index, waist circumference, menopausal status, ovary removed status, use of female hormones, hormones/hormone modifiers, total energy, daidzein, genistein, O-desmethylangolensin, enterodiol, and enterolactone, the association of equol with UL was significant [Odds ratio (OR) = 1.92, 95% confidence interval (CI): 1.09–3.38]. In the WQS model, mixed metabolites of urinary phytoestrogen had a positive association with UL (OR = 1.68, 95%CI: 1.12–2.51), with the highest weighted chemical of equol. In the gpcomp model, equol had the largest positive weight, followed by genistein and enterodiol. In the BKMR model, equol and enterodiol have positive correlation on UL risk, while enterolactone has negative correlation.ConclusionOur results implied a positive association between the mixed metabolites of urinary phytoestrogen and UL. This study provides evidence that urinary phytoestrogen-metabolite mixture was closely related to the risk of female UL.

Urinary Phytoestrogen Metabolites Positively Correlate with Serum 25(OH)D Level Based on National Health and Nutrition Examination Survey 2009-2010.

Studies showed that vitamin D (25-hydroxyvitamin D) level in the human blood circulation could be affected by exogenous estrogen exposure. This study aims to explore the relationships between urinary phytoestrogens metabolites and serum total 25(OH)D in general population, urinary phytoestrogens metabolites (daidzein, enterodiol, enterolactone, equol, genistein and o-desmethylangolensin). Totally 2,609 adults ≥6 y old from the 2009-2010 National Health and Nutrition Examination Surveys (NHANES) were recruited into the cross-sectional analyses and information including demographic, socioeconomic, examinations and laboratory test were collected. All analyses were performed using Stata13.0, one-way analysis of variance and multivariable regression were utilised according to data characteristics, respectively. It showed that age, race, education level, body mass index (BMI), and sampling season had significant effects on serum 25(OH)D level (all p<0.001). In the whole population, urinary enterodiol and equol were significantly positively associated with serum total 25(OH)D level (β=0.86, 95%CI=0.08-1.65, p<0.05; β=1.68, 95%CI=0.91-2.45, p<0.001). Equol was also found significantly positively correlated with total 25(OH)D in both female and male separately (β=1.69, 95%CI=0.51-2.87, p<0.05; β=1.66, 95%CI=0.63-2.69, p<0.05). Phytoestrogen concentrations in the urinary and 25(OH)D levels in the serum had proved a positive correlation in our study, which provide theoretical basis and reference for the dietary nutrient intake in the population.

Urinary equol levels are positively associated with urinary estradiol excretion in women

Isoflavones found in soy products are a promising class of nutrients that may have a positive effect on human health. In particular, the phytoestrogen metabolite equol is associated with a reduced risk of developing female hormone-related diseases. However, the effect of equol on estrogen remains unclear. Equol can modify blood and urinary estradiol (E2) levels. The aim of this cross-sectional study was to examine the associations between urinary estrogen levels, equol levels, and equol production status in Japanese women. We analyzed urine samples from 520 women by gas chromatography-mass spectrometry. Urinary E2 and 4-hydroxylated E2 levels were higher in equol producers (EQP) than in non-EQPs (P < 0.0001 and P=0.00112, respectively). After adjusting for age and tobacco use by analysis of covariance, the association remained significant (β = 0.299, P < 0.0001). Analysis of covariance demonstrated that equol levels in urine were also positively associated with urinary E2 (β = 0.597, P < 0.0001). The log equol concentration showed a significant, but moderate, negative association with the serum E2 concentration (β = − 0.0225, P = 0.0462). Our findings suggest that equol may promote urinary E2 excretion and modify blood E2 levels in women.

Biological effects of phytoestrogens: clinical effectiveness and potential application in the treatment of climacteric syndrome

Aim. To review scientific publications on clinical effectiveness and potential application of phytoestrogens (PE) in the treatment of climacteric syndrome. Materials and methods. The PRISMA approach was used. The systematic search was performed in three electronic scientific databases: Cochrane library, PubMed/MEDLINE, and eLIBRARY.ru. The publications were also manually searched for in the reference lists of the relevant articles. Results. The filters were used to select 79 publications; 26 of them were included in the final review. The main method of treatment for the manifestations of the climacteric syndrome is menopausal hormonal therapy (MHT). One of the alternative methods for the control of its symptoms is the application of phytoestrogens (PE), primarily, isoflavones, lignans, and coumestans. Active metabolites of PE are expressed under the influence of gut microflora that is different in various groups of women, which can determine the differences in the effectiveness. Taking into account multidirectional character of the studies as well as numerous publications of low quality, it is still impossible to come to the final conclusion on the effectiveness of PE-containing food supplements (FS) when it comes to a decrease in the occurrence rate and expression of such symptoms as hot flushes, night sweats, and cognitive deterioration. The conclusions on the prevention of cardio-vascular diseases are not definite either. Conclusions . The review showed that scientists could not come to the conclusion on the effectiveness and safety of PE-containing FS. Probably, the most prospective means of the control of climacteric syndrome are FS that contain isoflavones of red clover and soy in the maximum effective concentration. Considering free access to such FS on the market, they must meet high safety requirements.

Beyond Metabolism: The Complex Interplay Between Dietary Phytoestrogens, Gut Bacteria, and Cells of Nervous and Immune Systems.

The human body has a large, diverse community of microorganisms which not only coexist with us, but also perform many important physiological functions, including metabolism of dietary compounds that we are unable to process ourselves. Furthermore, these bacterial derived/induced metabolites have the potential to interact and influence not only the local gut environment, but the periphery via interaction with and modulation of cells of the immune and nervous system. This relationship is being further appreciated every day as the gut microbiome is researched as a potential target for immunomodulation. A common feature among inflammatory diseases including relapsing-remitting multiple sclerosis (RRMS) is the presence of gut microbiota dysbiosis when compared to healthy controls. However, the specifics of these microbiota-neuro-immune system interactions remain unclear. Among all factors, diet has emerged as a strongest factor regulating structure and function of gut microbial community. Phytoestrogens are one class of dietary compounds emerging as potentially being of interest in this interaction as numerous studies have identified depletion of phytoestrogen-metabolizing bacteria such as Adlercreutzia, Parabacteroides and Prevotella in RRMS patients. Additionally, phytoestrogens or their metabolites have been reported to show protective effects when compounds are administered in the animal model of MS, Experimental Autoimmune Encephalomyelitis (EAE). In this review, we will illustrate the link between MS and phytoestrogen metabolizing bacteria, characterize the importance of gut bacteria and their mechanisms of action in the production of phytoestrogen metabolites, and discuss what is known about the interactions of specific compounds with cells immune and nervous system. A better understanding of gut bacteria-mediated phytoestrogen metabolism and mechanisms through which these metabolites facilitate their biological actions will help in development of novel therapeutic options for MS as well as other inflammatory diseases.

Detection of Phytoestrogen Metabolites in Breastfed Infants' Urine and the Corresponding Breast Milk by Liquid Chromatography-Tandem Mass Spectrometry.

To date, there has been limited information on phytoestrogen (PE) exposure and metabolism in breastfed infants. In the present work, 50 sample pairs of Chinese breastfed infants' urine and the corresponding breast milk were collected. The contents of the relevant PE metabolites in the biosamples were detected via liquid chromatography-tandem mass spectrometry. The correlations between the PE metabolite contents in breastfed infants' urine and those in the corresponding breast milk were analyzed. The average concentrations of total PE metabolites in breast milk and urine were 0.27 and 0.23 nmol/mL, respectively. Genistein and enterolactone levels in the infant urine were positively correlated with their concentrations in the corresponding breast milk samples, which implies that urine excretion can be utilized as a noninvasive parameter for precise genistein and enterolactone intake assessment. Additionally, the efficiency of PE urine excretion showed significant differences across infants with different ages, genders, and durations of pregnancy.

Prognostic associations of circulating phytoestrogens and biomarker changes in long-term survivors of postmenopausal breast cancer

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.

Are dietary genistein and equol potent enhancers of eicosapentaenoic acid and docosahexaenoic acid levels in rainbow trout ( Oncorhynchus mykiss )?

Phytoestrogens are putatively able to enhance the biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but have also been shown to affect fish growth dose dependently. The aim of the present study was to identify a concentration for the phytoestrogen genistein and the phytoestrogen metabolite equol that further increases the endogenous biosynthesis of EPA and DHA without impairing fish growth. Juvenile rainbow trout (87.2 ± 0.3 g) were fed seven diets on a fixed ratio for 8 weeks. A vegetable oil-based diet served as a control diet and was supplemented with equol (EQ) and genistein (G), respectively, at 0.1%, 0.2% and 0.3% of feed dry matter (1, 2 and 3). Growth and nutrient composition of whole body homogenates were not affected by dietary treatments. EPA and DHA levels in liver, fillet and whole body samples were not significantly increased by EQ and G diets. Fish fed EQ diets showed dose dependently increased liver weights and C18:0 liver levels, indicating estrogen-like effects at increased dietary dosages. In conclusion, the utilization of equol and genistein in plant oil-based diets in order to enhance the biosynthesis of EPA and DHA seems not reasonable in rainbow trout.

Linking diet, gut microbiota and autoimmunity: A phytoestrogen diet alters the gut microbiota and influences Experimental Autoimmune Encephalomyelitis (EAE)

Abstract The etiology of multiple sclerosis (MS) involves genetic predisposition and an unknown environmental trigger. The gut microbiome may be an environmental contributing factor. Previously, we have shown that certain gut bacteria especially those with ability to metabolize phytoestrogen are less abundant in MS patients compared to healthy individuals. We hypothesize that phytoestrogen metabolism via microbial flora induces an anti-inflammatory state in the intestine. Therefore their reduced levels might predispose to disease development and/or severity. To test this, we placed C57Bl/6 mice on a diet with or without phytoestrogens and induced Experimental Autoimmune Encephalomyelitis (EAE), an experimental model of MS. We observed that mice on a phytoestrogen diet developed milder disease compared to those on a phytoestrogen free diet; a phenomenon dependent on the presence of phytoestrogen-metabolizing bacteria in the gut. We also found that a phytoestrogen diet alters the gut microbiota compared to a phytoestrogen-free diet with the gut microbiota of phytoestrogen group closely resembling the gut microbiota of healthy individuals. The gut microbiota of mice on a phytoestrogen-free diet closely resembles the gut microbiota of MS patients. Furthermore, mice on a phytoestrogen-diet exhibit an altered immune profile in the colonic lamina propria and mesenteric lymph nodes. Finally, we found that phytoestrogen-metabolites altered the cytokine-producing function of human colonic epithelial cells in vitro. Our results suggest that dietary phytoestrogen metabolites produced by commensal bacteria can alter the phenotype and function of the intestinal immune system, which may influence central nervous system autoimmunity.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species-the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota's transformation of dietary phytoestrogens in captive SWR females.IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

Byproducts of aqueous chlorination of equol and their estrogenic potencies

While the phytoestrogen metabolite equol has been reported to exist in surface water, its behavior in drinking water treatment plants remains unrevealed. In this study, eight products including four chlorinated equols (monochloro-equol, dichloro-equol, trichloro-equol, and tetrachloro-equol) were identified in an aqueous chlorinated equol solution by UHPLC-quadrupole-orbitrap-HRMS. Two main pathways of chlorination reaction are proposed: (1) chlorine-substitution reactions on the aromatic ring and subsequent dehydration to form the chlorine-substituted equols, and (2) break-up of the heterocyclic ring with oxygen followed by oxidation of aldehyde to carboxyl. The human estrogen receptor (hER) activating activity for monochloro-equol (EC50 = 3456 nM) and dichloro-equol (EC50 = 2456 nM) were slightly stronger than that of equol (EC50 = 3889 nM). This is the first report on the behavior of equol in drinking water chlorination, which provided an important information on the risk assessment of equol in drinking water.

Performance and egg quality of laying hens fed flaxseed: highlights on n-3 fatty acids, cholesterol, lignans and isoflavones

Flaxseed is a rich source of α-linolenic acid and phytoestrogens, mainly lignans, whose metabolites (enterodiol and enterolactone) can affect estrogen functions. The present study evaluated the influence of dietary flaxseed supplementation on reproductive performance and egg characteristics (fatty acids, cholesterol, lignans and isoflavones) of 40 Hy-Line hens (20/group) fed for 23 weeks a control diet or the same diet supplemented with 10% of extruded flaxseed. The flaxseed diet had approximately three times the content of lignans (2608.54 ng/g) as the control diet, mainly secoisolariciresinol diglucoside (1534.24 v. 494.72 ng/g). When compared with the control group, hens fed flaxseed showed a similar deposition rate (72.0% v. 73.9%) and egg yield. Furthermore, there was no effect of flaxseed on the main chemical composition of the egg and on its cholesterol content. Estradiol was higher in the plasma of the control group (1419.00 v. 1077.01 pg/ml) probably due to the effect of flaxseed on phytoestrogen metabolites. The plasma lignans were higher in hens fed flaxseed, whereas isoflavones were lower, mainly due to the lower equol value (50.52 v. 71.01 ng/ml). A similar trend was shown in eggs: the flaxseed group had higher level of enterodiol and enterolactone, whereas the equol was lower (198.31 v. 142.02 ng/g yolk). Secoisolariciresinol was the main lignan in eggs of the flaxseed group and its concentration was three times higher then control eggs. Flaxseed also improved the n-3 long-chain polyunsaturated fatty acids of eggs (3.25 v. 0.92 mg/g egg), mainly DHA, however, its oxidative status (thiobarbituric reactive substances) was negatively affected. In conclusion, 10% dietary flaxseed did not affect the productive performance of hens or the yolk cholesterol concentration, whereas the lignans and n-3 polyunsaturated fatty acid content of eggs improved. Further details on the competition between the different dietary phytoestrogens and their metabolites (estrogen, equol, enterodiol and enterolactone) should be investigated.

Cardiometabolic risk and gut microbial phytoestrogen metabolite phenotypes.

Recent evidence supports that the gut microbial community, independently and/or interactively with dietary intake, is a target for reducing cardiovascular disease risk through its effects on cardiometabolic risk factors. Dietary phytoestrogens may be a source for interactive effects. Phytoestrogens, such as isoflavones, lignans, and flavonoids, are compounds found in plants that have estrogenic or antiestrogenic activities, as well as antioxidant, antiproliferative, or apoptotic actions. Given these physiological activities, phytoestrogens may have a role in cardiometabolic health. Some phytoestrogens consumed in the diet undergo biotransformation through gut bacterial metabolism to other compounds that may exhibit similar or different physiological activity than the parent compound. There is interindividual variability in the capability to metabolize phytoestrogens to their metabolites, and there is a resulting phenotype that can be evaluated based on urinary metabolite excretion. Evidence suggests that phytoestrogen metabolites and/or phenotypes are associated with cardiometabolic risk factors, such as blood pressure, abdominal obesity, and serum lipids, triglycerides, glucose, and inflammatory markers. The objective of this review was to provide an overview of the observed associations between gut microbial phytoestrogen metabolites and metabolite phenotypes with cardiometabolic risk factors, with focus on the more extensively studied isoflavone metabolites.

Urinary Concentrations of Phytoestrogens Are Not Associated with Risk of Type 2 Diabetes: The Singapore Chinese Health Study

ObjectiveWe evaluated the relation between urine concentrations of phytoestrogens and risk of incident type 2 diabetes (T2D) in middle‐aged and older Chinese.Methods: Urine phytoestrogen metabolites (including isoflavones and lignans) were assayed by HPLC among 564 T2D cases and 564 controls in a nested case‐control study within the Singapore Chinese Health Study. Morning urine samples were collected from participants free of diagnosed diabetes, cardiovascular disease and cancer (1999‐2004). Incident self‐reported T2D cases were identified during the follow‐up at 2006‐2010, and controls were matched for age and date of urine collection, sex and dialect group.Results: The multivariate‐adjusted odds ratios (ORs) and 95% confidence intervals (CIs) across quartiles of urine isoflavones were 1.00, 0.76 (0.52‐1.11), 0.78 (0.53‐1.14) and 0.79 (0.54‐1.15), respectively (P‐trend=0.54); and the ORs (95% CIs) for lignans were 1.00, 0.87 (0.60‐1.27), 1.10 (0.77‐1.56) and 0.93 (0.63‐1.37), respectively (P‐trend=0.93). No significant associations were found for individual metabolite of isoflavones (genistein, daidzein, glycetin, and equol) or lignans (enterodiol and enterolactone).ConclusionsThe present study did not find a significant relation between urine concentrations of phytoestrogen metabolites andT2D risk in Chinese population.Funding sources: The study was supported by NIH R01 CA144034 and UM1 CA182876, and Singapore Tier 1 grant.

Effects of equol on gene expression in female cynomolgus monkey iliac arteries

Background and aimsTo examine effects of equol, the soy phytoestrogen metabolite, on gene expression in the monkey iliac artery. Methods and resultsA high fat/high cholesterol diet was fed to eight ovariectomized cynomolgus monkeys for 6.5 years. After biopsy of the left iliac artery, the animals were randomized to two treatment groups for 8 months; the treatment groups were equol (23.7 mg/100 g diet, n = 4) and vehicle (n = 4). The right iliac artery was removed at necropsy. Gene expression in the iliac arteries in response to equol was determined by DNA microarray. Comparison of atherosclerotic lesions and plasma lipids at pre-versus post-equol treatment time points and in vehicle versus equol treatment groups did not identify any significant differences. Despite the lack of effect of equol on these parameters, 59 genes were down-regulated and 279 were up-regulated in response to equol. Comparison of these data to previous work identified 10 genes regulated in opposite directions by equol compared to presence of atherosclerosis plaque (Menopause 2011; 18:1087–1095) and 55 genes differentially expressed in the same direction in response to both equol and estradiol (Eyster et al., Menopause 2014;21:143–152.). ConclusionsSimilar responses of genes to both equol and estradiol may reflect the extent to which equol serves as a natural selective estrogen receptor modulator in the arteries. Opposite responses of 10 genes to equol versus the presence of atherosclerosis implicates those genes in the potential protective effects of equol in arteries.

The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth

BackgroundIncreased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions.ResultsIn an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P < 0.01).Conclusions6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

A rapid 2 min liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/ z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 ( O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1–5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm × 2.0 mm i.d.) column at 50 °C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC–MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.

Joint Effects of BMI, Diet, and Urinary Biomarkers of Environmental Exposures in Relation to Female Pubertal Maturation

ISEE-0327 Background: Environmental exposures may alter timing of puberty, a factor related to later reproductive function, breast cancer, and other chronic diseases. The Breast Cancer and Environment Research Centers (BCERCs) are investigating environmental exposures and puberty in females. We hypothesize that hormonally active exposures because of their weak biologic activity may alter age at pubertal onset, although their effects may be modified by adiposity (the major source of prepubertal estrogen). Methods: A multi-ethnic cohort of 6-8 year old girls was established at three national BCERC sites. At baseline, urine specimens from 1151 girls were analyzed for biomarker panels of hormonally active chemicals (9 phthalate, 8 phenol, and 3 phytoestrogen metabolites). Breast development stages from physical examination, and covariate information including body mass index (BMI), diet, and selected characteristics were collected. We calculated multivariate adjusted prevalence ratios (aPR) for the association of baseline biomarker measurements with breast stage (stage B2+ [any] vs. B1 [none]) at first annual follow-up (N = 948). Results: Breast development (B2+) was present in 25% of girls. Three biomarkers had small but significant effects on breast stage in BMI-biomarker interaction models. For example, the likelihood of being B2+ decreased monotonically across quintiles of enterolactone (aPR = 0.92 [0.83-0.98] vs 1st [reference]) among girls with BMI-percentile ≥ median. BP-3 showed an inverse association and 2,5-dichlorophenol a positive association among high BMI-percentile girls; there was no association in low-BMI girls. Modification of the biomarker-B2+ association was observed with selected dietary factors that may be related to hormonal mechanism of the exposures. Conclusion: Preliminary results from this longitudinal study suggest that environmental exposures may be associated with pubertal developmental if considered jointly with body size distribution or dietary factors. Longer follow-up may provide further insight on effects of weak hormonal agents in relation to female puberty.

Food choice and phytoestrogen consumption in women previously treated for postmenopausal breast cancer

Background Phytoestrogens are plant-derived, bioactive substances with a chemical structure similar to that of 17-oestradiol. Women previously treated for breast cancer may increase their phytoestrogen intake to avoid conventional hormone replacement therapy or because of a belief that they may help avoid recurrence [1,2]. There is no recommended daily intake and there are some concerns about phytoestrogen safety in this group, although the evidence is conflicting and more research is needed [3,4]. Methods Three hundred and sixteen women each completed a 4- day food and drink diary (14 of whom also completed a 7-day weighed intake diary 6 weeks previously). The 55 most recently recruited women collected their urine for 24 hours whilst completing their diaries and were interviewed by telephone regarding their food choices since diagnosis. Results A new dietary analysis database was created using peerreviewed published data and analysing 34 additional foods and beverages for which there were no published results. The urinanalysis results contributed validation data. A summary of the dietary intake data is shown in Table 1. There was a lack of primary analytical data on the phytoestrogen profile of many foods and beverages routinely consumed by this study population. However, food frequency data from the highest quartile show the important contribution of nonsoya foods to high intakes (Table 2). Telephone interviews were completed by 39 subjects. For most women, having breast cancer had not changed their diet. Health concerns unrelated to cancer, the needs of other family members, cooking on a budget and physical appearance all seemed more important than the impact of the cancer diagnosis. Discussion Variation in phytoestrogen intakes and metabolite excretion reflect food preferences, dietary analysis database limitations and likely variations in existing knowledge combined with a lack of routine access to dietary information. In the absence of definitive advice, more immediate health and social concerns influence food choice rather than past breast cancer diagnosis. Conclusion No data previously existed on intake in this potentially vulnerable group and these data will help evaluate the health implications related to such phytoestrogen consumption patterns. Acknowledgement Funded by the Food Standards Agency, UK.