Search

We found detectable levels of three phytoestrogens (enterolactone, daidzein and genistein) and bisphenol A (BPA) in 21 residual amniotic fluid specimens that were collected before 20 weeks gestation. Samples were obtained by amniocentesis from women who were referred to the Mount Sinai Medical center because of advanced maternal age. Phytoestrogens were present in higher concentrations than BPA. Enterolactone was detected at the highest concentration (median 95.9 microg/L), followed by daidzein and genistein (9.5 and 1.4 microg/L, respectively). BPA was present at very low concentrations (10%>LOD of 0.5 microg/L). The relative concentration of the chemicals measured in amniotic fluid were identical to those in urine reported by other studies, i.e. enterolactone>daidzein>genistein>>BPA. Amniotic fluid is a source of fetal exposure to polar xenobiotics that come from the mother.

Epidemiologic and experimental data suggest that consumption of diets that are rich in isoflavones may decrease cancer risk in the breast, prostate, and other tissues. Isoflavones such as genistein and daidzein are structurally similar to endogenous estrogens, and demonstrate both estrogenic and weak anti-estrogenic activities; these activities may underlie the impaired fertility and reproductive tract disorders reported in animals exposed to high doses of isoflavones. To identify possible effects of isoflavones on male fertility, we evaluated reproductive parameters in Wistar-Unilever rats receiving dietary exposure to PTI G-2535, a characterized mixture of soy-derived isoflavones containing 45% genistein, 23% daidzein, and 4% glycitein. Beginning at 10 weeks of age, rats received chronic dietary exposure to the soy isoflavone mixture (200 or 2000 mg/kg diet) for a minimum of 12 months. Controls received unsupplemented chow diet only for the same period. Dietary exposure to isoflavones induced no gross toxicity or alterations in body weight gain. Absolute and relative weights of the testis and epididymis in groups receiving high or low doses of isoflavones were comparable to those of controls, and histopathologic evaluations demonstrated that testicular morphology was similar in all study groups. Isoflavone exposure had no significant effects on spermatid count, sperm production, or sperm morphology in any group. These data suggest that the reproductive system of adult male rats is relatively insensitive to isoflavone toxicity at dose levels that demonstrate significant activity in cancer chemoprevention, and that male reproductive function is unlikely to be affected by long-term administration of isoflavones for cancer prevention or other purposes. The results of this study conducted in adult male rats differ from the significant alterations in reproductive parameters that have been reported in female rats receiving prenatal or juvenile exposure to isoflavones.

Soy infant formulas are widely used, but few studies have evaluated long-term safety or examined specific forms of toxicity, such as to the endocrine or immune systems. This review focuses on newer experimental studies of the effects on estrogen activity, immune function, and thyroid economy of genistein and daidzein, two isoflavones in soy infant formula, and existing human studies of soy formula use. In order to judge the likelihood that an endpoint seen in laboratory studies might occur in soy-fed infants, we examined the doses and the resulting serum or plasma concentrations from the laboratory studies and compared them with doses and concentrations seen in soy-fed infants. We also summarized the estimates of the potency of the isoflavone compounds relative to estradiol. Given the scarcity and inconsistency of existing human data and the substantial laboratory evidence of hormonal and other activity at doses relevant to the soy-fed infant, we conclude that more clinical and epidemiological study is warranted.

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P < 0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P < 0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P < 0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P < 0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.

The objective of the Vegetal Estrogens in Nutrition and the Skeleton (VENUS) project was to evaluate existing data on dietary exposure to compounds with oestrogenic and anti-oestrogenic effects present in plant foods as constituents or contaminants, and to identify and disseminate in vitro and in vivo methodologies to analyse the effects of such compounds on bone. To permit the assessment of exposure to isoflavones in European populations (Italy, the UK, Ireland, The Netherlands), the VENUS database of phyto-oestrogen levels in foods was established. Data on the isoflavone (genistein and daidzein) content of 791 foods, including almost 300 foods commonly consumed in Europe, were collected. Levels of coumestrol, formononetin and biochanin A in a limited number of foods were also included. Lignan levels (secoisolariciresinol and matairesinol) in 158 foods were incorporated into the database, which also contains information on the references sourced for the compositional data, on the analytical methods used by each author and on the number of foods analysed in each reference. The VENUS database was constructed in Microsoft Access 2000, which is widely available as part of Microsoft Office Professional. This paper outlines the procedures used for the selection and evaluation of existing literature data for incorporation into the database. In addition, the design of the database is described, along with the data entry and quality control procedures used in its construction. Limitations of the data are discussed and guidelines for its use are provided.

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.

Isoflavones and lignans are biologically active plant-food constituents that have potential chemopreventive properties. Quantitation of isoflavones and lignans in humans is necessary to establish the benefits and risks of exposure to these compounds in populations and to determine which components of a mixed diet contribute to the exposure. Isoflavones and lignans are metabolized by colonic bacteria to more biologically active metabolites; thus both the parent compounds and the metabolites are measured routinely. Isoflavonoids (genistein, daidzein, dihydrodaidzein, O-desmethylangolensin and equol) and lignans (enterolactone, enterodiol, matairesinol and secoisolariciresinol) can be quantified in various body fluids. Typically, high concentrations of isoflavonoids in urine and serum are associated with soy consumption, and high concentrations of lignans are associated primarily with intake of whole grains and other fiber-containing plant foods. Controlled feeding studies and nutritional epidemiologic studies demonstrate a linear dose response between dietary intake and urinary excretion of isoflavones. Lignan excretion is associated positively with dietary fiber intake as well as with diets that are on average higher in fiber and carbohydrate and lower in fat; thus lignans have also been proposed as a marker of healthier dietary patterns. The complex interactions between the colonic environment and the external and internal factors that modulate it contribute to significant variation in serum and urinary phytoestrogen levels among individuals. Understanding these sources of variation is important to be able to use these measures effectively as dietary biomarkers.

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.

An efficient one-pot synthesis is described of 5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b'']triindole (CTr), a potent estrogen agonist from food plants. For the procedure, gramine is treated with dimethyl sulfate and sodium in ethanol at room temperature. Quenching of the reaction with water and workup of the product provides CTr in approximately 75% yield.

In an international context of promoting scientific research on food safety, the interest in molecules having potential hormonal disrupting effects is growing. While industrial endocrine disruptors (phthalates, alkylphenols, PCBs, etc.) have been studied for several years, natural compounds like phytoestrogens remain less investigated. Accordingly, a research project was initiated with its main objectives to develop efficient analytical methods for a wide range of phytoestrogens in various food matrices, and to evaluate their occurrence in food products. Electrospray ionization with tandem mass spectrometric (MS/MS) analysis of isoflavones (genistein, daidzein, equol, formononetin, biochanin A), lignans (enterolactone, enterodiol), and coumestans (coumestrol) was investigated. This study revealed the formation of a large number of fragment ions in both positive and negative modes, corresponding to specific cleavages of the hydroxyl, carbonyl, and/or methoxy groups, and to Retro-Diels-Alder reactions. An LC/ESI-MS/MS method was developed consistent with the 2002/657/EC European decision criteria. An extraction and clean-up method was developed for milk samples. The identification limit for the proposed method appears to be under 1 ng/mL. The developed methodology was applied to various milk samples, and the occurrence of isoflavones (particularly equol) was demonstrated in the concentration range 1-30 ng/mL. The efficiency of the proposed analytical method permitted evaluation of a new and promising approach to a global risk assessment of natural estrogenic active substances including phytoestrogens and their metabolites., (Copyright 2003 John Wiley & Sons, Ltd.)

A variety of effects have been attributed to phytoestrogens, which are present in variable concentrations in soy-based laboratory animal diets. The authors review the inherent complexities in evaluating the effects of soy and dietary phytoestrogens on various in vitro and in vivo systems.

Objective: To analyse 75 cereals and three soy flours commonly eaten in Europe for the phytoestrogens daidzein and genistein., Design: The phytoestrogens daidzein and genistein were extracted from dried foods, and the two isoflavones quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards., Setting: Foods from the Cambridge UK area were purchased, prepared for eating, which included cooking if necessary, and freeze dried. Three stock soy flours were also analysed., Results: Eighteen of the foods assayed contained trace or no detectable daidzein or genistein. The soy flours were rich sources, containing 1639-2117 mg/kg. The concentration of the two isoflavones in the remaining foods ranged from 33 to 11,873 micro g/kg., Conclusion: These analyses will supply useful information to investigators determining the intake of phytoestrogens in cereal products in order to relate intakes to potential biological activities., Sponsorship: This work was supported by the United Kingdom Medical Research Council, Ministry of Agriculture Fisheries and Food (contract FS2034) and the United States of America Army (contract DAMD 17-97-1-7028).

Dietary phytoestrogens have been implicated in the prevention of chronic diseases. However, it is uncertain whether the phytoestrogens or the foods associated with phytoestrogens account for the observed effects. We report here a new liquid chromatography photodiode array mass spectrometry (LC-PDA-MS) assay for the determination of nanomolar amounts of the most prominent dietary phytoestrogens (genistein, dihydrogenistein, daidzein, dihydrodaidzein, glycitein, O-desmethylangolensin, hesperetin, naringenin, quercetin, enterodiol, enterolactone) in human plasma or serum and urine. This assay was found to be suitable for the assessment of quercetin exposure in an onion intervention study by measuring urinary quercetin levels. Other successful applications of this assay in clinical and epidemiologic studies validated the developed method and confirmed previous results on the negative association between urinary isoflavone excretion and breast cancer risk.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.

We review the methods used to measure phytoestrogens (genistein, daidzein, lignans and their derivatives) in foods and biological fluids, and discuss advantages and disadvantages of each. The range of detection limits reported varies widely between individual laboratories, but generally the best reported sensitivity is as follows: immunoassay>HPLC-mass spectrometry=HPLC-multichannel electrochemical detection (coularray)>GC-single ion monitoring-mass spectrometry>HPLC-UV diode array>HPLC-single channel electrochemical detection. The best sensitivity reported so far is 0.002 pmol per assay for daidzein by radioimmunoassay. HPLC with UV diode array detection is the most commonly employed, but is the least sensitive and specific. GC and HPLC coupled with mass spectrometry or electrochemical detection are the most accurate and reproducible methods for a wide variety of analytes. Generally most methods, with the exception of immunoassay, have not been correlated with other methods. Recoveries from extraction methods, limits of detection, nature of compounds analysed and the internal standards used are summarised for more than 90 reports in the literature. From this data, it is clear that an inter-laboratory validation and correlation between a wide range of methods for phytoestrogen analysis is required. One underdeveloped area that requires particular attention is the analysis of plant lignans.

Interest in analytical methods for plant estrogens (phytoestrogens) has risen sharply in the past 10 years. In this review, we examine the existing analytical methods based on separations by gas-liquid chromatography, high-performance liquid chromatography and capillary electrophoresis in addition to methods of detection by ultraviolet absorption, fluorescence, electrochemical oxidation/reduction and mass spectrometry. These methods are compared with other methods of phytoestrogen analysis utilizing immunoassay approaches. The advantages and disadvantages of each of these methods are highlighted and potential areas for further development identified.

Isotopically and isomerically pure polydeuterated flavonoids and isoflavonoids have been prepared for quantitation of these compounds in biological matrices. Various deutero-labeling techniques are presented and methods for establishing the isotopical and isomerical purity of deuterated products are discussed.

Background: Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells., Methods and Results: Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation)., Conclusions: In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Kudzu-root and soy phytoestrogens have been associated with anti-cancer and anti-intoxication activities. Sales of capsules containing kudzu-root and soy extracts through health food stores and the Internet are unregulated. To compare efficacy, the amount of phytoestrogens present in commercial preparations and their fate in biological samples must be determined. In this study, the levels and composition of phytoestrogens in kudzu-root and soy extracts were studied using high-performance liquid chromatography with ultraviolet light detection. The bioavailability of phytoestrogens was studied by measuring red blood cell (RBC) uptake and serum protein binding ability. Phytoestrogen levels in acidified kudzu-root samples were 5- to 10-fold greater than those in nonacidified samples. Puerarin accounted for 80% of total phytoestrogens in kudzu-root. In soy extract, puerarin was absent while genistin, glycetein, and daidzin or daidzein were the major phytoestrogens. The RBC uptake depended on the phytoestrogen's polarity and molecular length. When serum was dialyzed with phytoestrogen standards in a buffer, the protein binding of phytoestrogens correlated negatively with their polarity. However, when serum was dialyzed with kudzu-root or soy extract, almost all of the phytoestrogens present in the extract bound to serum protein. Therefore, this study suggests differences in the bioavailability of phytoestrogens when they are ingested as purified compounds compared with crude plant extract. The differential composition of phytoestrogens in kudzu-root and soy may account for the differences in their therapeutic activities.

Higher consumption of phytoestrogens might be protective against certain chronic diseases. Accurate quantification of habitual phytoestrogen intake is important for assessing associations between phytoestrogens and risk for certain diseases. The aim of this study was to estimate dietary intake of phytoestrogens in Dutch middle-aged and elderly women and to describe their main sources. Women were recruited between 1993 and 1997 and aged 50-69 y at enrollment (Prospect-EPIC; n = 17,357). A detailed food frequency questionnaire referring to the preceding year was filled in at recruitment. A literature search was conducted to obtain data regarding content of the isoflavones daidzein, genistein, formononetin, biochanin A, the coumestan coumesterol and the lignans matairesinol and secoisolariciresinol in relevant food items. Concentrations of each phytoestrogen in each food item were subsequently grouped by seven categories; group scores were multiplied by daily intakes of food items and then summed across food items to produce for each participant a total daily intake score for each phytoestrogen. Approximately 75% of participants were postmenopausal at recruitment. The mean age was 57 y. Geometric means of daily intake of daidzein, genistein, formononetin, biochanin A, coumesterol, matairesinol and secoisolariciresinol were 0.15, 0.16, 0.08, 0.001, <0.001, 0.07 and 0.93 mg, respectively. The main sources for isoflavones were peas and beans, nuts, grain products, coffee, tea and soy products. The main sources for coumestans were peas, beans and other vegetables. The main sources of lignans were grain products, fruit and alcoholic beverages (red and white wines). We conclude that intake levels of phytoestrogen in our study population are low; however, they are comparable with intake levels previously reported for other Western cohorts. In this population, phytoestrogen intake consisted largely of lignans.

We have investigated a novel application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) for the quantitative analysis of the isoflavones in food matrices. Previous methods have been hampered by the lack of analytical standards to introduce early enough in the extraction procedure to allow compensations for losses at all stages of the procedure. In this work we have produced standard materials that can be added at the initial extraction, by intrinsically labelling soya plants by growing them in an atmosphere enriched in the stable isotope of carbon in CO(2). On analysis these plants were shown to contain phytoestrogens at a high (up to 20%) level of enrichment. The dried plant material has been used to estimate the isoflavone concentrations of a set of spiked flours. For daidzein the methodology was shown to produce results comparable to those achieved by GC/MS techniques. The method was less successful for genistein, possibly due to the greater fragility of this compound under the conditions required for the analysis., (Copyright 2002 John Wiley & Sons, Ltd.)

A hop-based dietary supplement, marketed for natural breast enhancement, was analysed to determine the identity and biological activity of active constituents and potential biological effects in man. Extracts of the dietary supplement were analysed by LC-MS(n) and phytoestrogens identified and quantitated by reference to appropriate standards. Only hop-associated phytoestrogens were found in the dietary supplement at significant concentrations as follows (mean+/-1 S.D.); 8-prenylnaringenin 10.9+/-0.3, 6-prenylnaringenin 27.4+/-1.2, 6,8-diprenylnaringenin 0.9+/-0.1, xanthohumol 321+/-17 and isoxanthohumol 81.1+/-1.6 microg/g of dietary supplement. The oestrogenic activity of extracts in an ERalpha reporter gene assay was equivalent to 48+/-6.3 ng 17beta-oestradiol/g supplement and consistent with the 8-prenylnaringenin content. The dietary supplement extract also inhibited reductive 17beta-hydroxysteroid oxidoreductase activity, but to a greater extent than a concentration matched reference mixture of hop phytoestrogens. However, the supplement was only weakly active in mouse uterotrophic assays following administration in feed or after subcutaneous injection of extract at doses of 8-PN up to 250 times higher than that recommended for women. These preliminary findings suggest that the dietary supplement is unlikely to produce oestrogenic effects in vivo at the level of the uterus; supporting evidence is still required to demonstrate efficacy.

We present a study on the mass spectral as well as the binding properties of three triterpene glycosides (cimicifugoside, cimiracemoside F, 27-deoxyactein) contained in black cohosh to the ligand binding domain of estrogen receptor beta (ER-beta). Using affinity ultrafiltration and LC/ MS detection, initial experiments using estradiol and the phytoestrogens daidzein and genistein (compounds known to bind ER-beta) were performed to serve as positive controls. The same affinity techniques and LC/MS procedures were then employed to show that neither the triterpene glycosides nor their enzymatically prepared aglycons bound significantly to ER-beta, except for 27-deoxyactein aglycon, which showed weak binding affinity (4%). Additionally, metabolites of the aglycons were prepared by incubation with female human liver microsomes and subjected to binding experiments with ER-beta. No significant binding of the metabolites to the receptor was observed. Further studies are needed to fully characterize whether these triterpene glycosides as well as other components of black cohosh in this plant extract bind to the estrogen receptor alpha (ER-alpha).

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.

It is generally not known that most commercial rodent diets are formulated with soy protein and deliver large daily doses of isoflavones to animals throughout their lifespan, including the in utero period. Here, we demonstrate that isoflavones are bioavailable and show that commercial rodent diets universally used by animal facilities lead to very high steady-state serum isoflavone concentrations in adult rats (2613 +/- 873 ng/mL) and mice (2338 +/- 531 ng/mL), exceeding the animal's endogenous estrogen level by 30,000- to 60,000-fold. We demonstrate the maternal-fetal intrauterine transfer of isoflavones in animals fed a standard Purina 5001 soy-containing diet and show that newborn rat pups have high serum isoflavones levels (540 +/- 174 ng/mL) that are maintained throughout the suckling period by passage of isoflavones into maternal milk. These findings have profound implications for all animal experiments, including multigenerational studies and studies of transgenic animals, especially if biochemical or morphological end-points are influenced by the hormonal or nonhormonal properties of phytoestrogens. These compounds have the potential to modulate genotypic and phenotypic expression in general, and therefore, all investigators should be vigilant to the phytoestrogen composition of commercial rodent diets because there is a history of potent biological effects in larger animals and in humans from high circulating isoflavone concentrations.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estradiol. We report the associations of two major phytoestrogens, genistein and enterolactone, with breast cancer risk, using urinary specimens collected 1-9 years before breast cancer was diagnosed. The subjects were 88 breast cancer cases and 268 controls, selected from a cohort of postmenopausal women (n = 14,697) who participated in a breast cancer screening program. Mean levels of urinary genistein and enterolactone were determined by time resolved fluoroimmunoassay, using an average of two overnight urinary samples obtained from each participant on the first and the second screening rounds with a time interval of approximately 1 year. Odds ratios (ORs) of the highest to the lowest tertile of urinary phytoestrogen/creatinine concentrations and 95% confidence intervals (CIs) were computed. Higher urinary genistein excretion was weakly and nonsignificantly associated with a reduced breast cancer risk. OR for the highest tertile compared with lowest tertile was 0.83; 95% CI, 0.46-1.51. Higher urinary enterolactone excretion was weakly and nonsignificantly associated with an increased breast cancer risk. OR for the highest tertile compared with the lowest tertile was 1.43; 95% CI, 0.79-2.59. Tests for trends for both phytoestrogens were nonsignificant. We were not able to detect the previously reported protective effects of genistein and enterolactone on breast cancer risk in our postmenopausal population of Dutch women. Such an effect may be smaller than expected and/or limited to specific subgroups of the population.

It has been suspected that beer drinking may change the hormonal status of men caused by phytoestrogens. Five different Austrian lager beers have been investigated for estrogenic activity by a yeast two-plasmid system harboring the human estrogen receptor alpha, after concentration by solid phase extraction. The beer concentrate was further fractionated by reversed phase HPLC, and then the fractions were characterized by the biological assay and GC-MS. The most potent fraction did not contain a known phytoestrogen. The total activity corresponded to an average of 43 ng of 17beta-estradiol/L of beer. It was concluded that the human health hazard of beer drinking originating from compounds activity on the estrogen receptor alpha is negligible.

Dietary phytoestrogens have a number of biological effects, including endocrine disruption, antioxidant potential, and protein tyrosine kinase inhibition. Secoisolariciresinol, matairesinol, and shonanin are lignan phytoestrogens found in foodstuffs, especially flaxseed. Normally they are glycosidically linked to carbohydrates and in the large intestine are deconjugated from the carbohydrate portion by bacteria. The aglycone lignans can be further modified to form the mammalian phytoestrogens enterodiol, enterolactone, and enterofuran, which are absorbed into the body and excreted in urine. To assess the health implications of phytoestrogens in general populations, knowledge of the quantity in the foods eaten is necessary. This article describes a simple preparative procedure for the assay of secoisolariciresinol, matairesinol, and shonanin in foodstuffs after hydrolytic removal of any conjugated carbohydrate. The difficulties in the practical application of the assay procedure are illustrated and discussed. Analytical results indicating the concentration of secoisolariciresinol, matairesinol, and shonanin in a number of foodstuffs are presented. Also, the mass spectral data of a putative mammalian phytoestrogen, called enterofuran, identified in urine are presented., (Copyright 2000 Academic Press.)

Food samples (n 114) were prepared from vegetables commonly eaten in Europe. The glycosidic forms of the phyto-oestrogens daidzein and genistein were extracted from the dried foods into aqueous methanol. The isoflavones were quantified by GC-MS after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 114 foods assayed, at a limit of quantification of 0.1 microg/kg dry weight, forty-eight contained no detectable daidzein or genistein, forty-one contained less than 100 microg/kg of the two isoflavones combined and the remaining twenty-five contained more than this amount. Soyabean products contained between 470 and 1420 mg (average of 960 mg) daidzein and genistein combined per kg wet weight of food, and legumes contained between 20 and 5750 microg/kg wet weight of food, with an average of 620 microg/kg. Cooking by boiling in water caused a decrease in the daidzein and genistein content of food in twenty-four of twenty-eight foods. The extent of the decrease was variable and warrants further investigation. The present paper comprises the first comprehensive description of the content of daidzein and genistein in vegetables.

Aim: To present the currently available evidence on the cardiovascular benefits and risks associated with phyto-oestrogens. DATA-SYNTHESIS: Medline search from 1966-1999 updated with cross-check of references of papers with keywords such as phyto-oestrogens, isoflavones, lignans, genistein, daidzein, enterolactone, enterodiol, cardiovascular disease, cardiovascular disease risk factors., Conclusions: Phyto-oestrogens are plant chemicals divided into three main classes: isoflavones, coumestans, and lignans that display oestrogen-like activity due to their ability to bind to the oestrogen receptor. They are found in grains, beans, green vegetables, fruits, nuts, and grasses. Isoflavones are primarily found in soybeans and soy foods. For epidemiological studies of the relation between phyto-oestrogen intake and disease parameters, intake is estimated with several measures, such as biomarkers (concentrations in urine or blood) or dietary questionnaires, though the optimal method is not yet clear. Phyto-oestrogens are considered to act as selective oestrogen receptor modulators (SERM), exerting both oestrogen agonist and antagonist action. Supplementation with isolated soy protein containing the isoflavones genistein and daidzein reduces serum total and LDL-cholesterol and triglycerides in animals and in humans. Vascular reactivity might be improved by supplementation with isolated soy protein or isoflavones isolated from red clover. Studies on atherosclerosis in animals indicate a potential for risk reduction. Evidence in humans is still scanty. The little we know of the effects of regular dietary phyto-oestrogen intake comes from studies in which phyto-oestrogens were added to the usual diet. Most supplementation studies have been conducted with soy isoflavones, whereas the importance of lignans has not been determined, though they could be more important sources than isoflavones in Western populations. Research has been focused on risk factors. Studies of clinically manifest endpoints are urgently needed.

Background and Objective: Phytoestrogens (weak estrogens found in plants or derived from plant precursors by human metabolism) have been hypothesized to reduce the risk of a number of cancers. However, epidemiologic studies addressing this issue are hampered by the lack of a comprehensive phytoestrogen database for quantifying exposure. The purpose of this research was to develop such a database for use with food-frequency questionnaires in large epidemiologic studies., Methods: The database is based on consumption patterns derived from semistructured interviews with 118 African-American, Latina, and white women residing in California's San Francisco Bay Area. HPLC-mass spectrometry was used to determine the content of seven specific phytoestrogenic compounds (i.e. the isoflavones: genistein, daidzein, biochanin A, and formononetin; the coumestan: coumestrol; and the plant lignans: matairesinol and secoisolariciresinol) in each of 112 food items/groups., Results: Traditional soy-based foods were found to contain high levels of genistein and daidzein, as expected, as well as substantial amounts of coumestrol. A wide variety of "hidden" sources of soy (that is, soy protein isolate, soy concentrate, or soy flour added to foods) was observed. Several other foods (such as various types of sprouts and dried fruits, garbanzo beans, asparagus, garlic, and licorice) were also found to be substantial contributors of one or more of the phytoestrogens analyzed., Conclusions: Databases, such as the one described here, are important in assessing the relationship between phytoestrogen exposure and cancer risk in epidemiologic studies. Agencies, such as the United States Department of Agriculture (USDA), that routinely provide data on food composition, on which epidemiologic investigations into dietary health effects are based, should consider instituting programs for the analysis of phytochemicals, including the phytoestrogens.

Objective: We recently described the development of a comprehensive database for assessing phytoestrogen exposure in epidemiologic studies. This paper describes the first application of this database and the primary sources of phytoestrogen consumption in non-Asian women., Methods: Four hundred and forty-seven randomly selected African-American, Latina, and white women, ages 50-79 years, residing in California's San Francisco Bay Area and participating as controls in an ongoing population-based case-control study of breast cancer, were included in the present analysis. Average daily consumption of each of seven phytoestrogenic compounds was determined for each woman by combining the values from the new database with food consumption reported on a food-frequency questionnaire., Results: Phytoestrogens in the non-Asian Bay Area diet appear to come primarily from: (1) traditional soy-based foods (e.g. tofu and soy milk); (2) "hidden" sources of soy (e.g. foods containing added soy protein isolate, soy concentrate, or soy flour, e.g. many brands of doughnuts and white bread); and (3) a variety of foods which contain only low to moderate amounts of phytoestrogens per 100 grams but which are frequently consumed (e.g. coffee and orange juice)., Conclusions: In the absence of a comprehensive assessment of various phytoestrogens in a wide variety of foods, epidemiologic studies could suffer from the effects of uncontrolled confounding by unmeasured sources of phytoestrogen exposure potentially leading to biased estimates of effect and misinterpretation of findings.

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Background: To verify the availability of foods containing phytoestrogens, the quality of available products, the degree of knowledge of these foods by dealers, the willingness of dealers to give cooperation in preparation of the MENFIS study, a study assessing the efficacy of a phytoestrogen rich diet on the long term effects of menopause., Methods: Perspective research by questionnaires carried out in the herbalist's shops of Bari., Results: For the aims of the study the herbalist's shops resulted in being the most suitable, because they were the most supplied with these products and with natural foods, with a high degree of knowledge of these foods and a helpfulness towards the users, perhaps because of the strong motivation towards this type of diet., Conclusions: The easy availability of these products, the good preparation of managers of the herbalist's shops and their good experience is a sound basis for those intending to start dietary programs with phytoestrogen rich foods.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

We analyzed the intake of selected foods that contain phytoestrogens in relation to breast cancer (BC) risk using data from a hospital-based case-control study performed in Mexico City from 1994 to 1995. A total of 198 women with BC, aged 21-79 years, were individually age matched to an identical number of women with no breast disease. By a direct interview, information on socioeconomic characteristics and diet was obtained. A semiquantitative questionnaire was used to estimate the frequency of consumption of 95 foods. The effect of selected foods that contain phytoestrogens on BC risk was estimated using logistic regression models. The adjusted odds ratio for the consumption of more than one slice of onion per day and BC was 0.27 (95% confidence interval = 0.16-0.47), with a statistically significant trend (p < 0.001). This protective effect remained after adjustment for known risk factors of BC. Among premenopausal women, there was also a protective and significant effect due to the intake of lettuce and spinach and nonsignificant protective effects for the consumption of apples and herbal tea. Additional studies aimed at evaluating the potential protective effect of particular phytoestrogens on BC risk are needed.

The compositional analyses and safety assessment of glyphosate-tolerant soybeans (GTS) were previously described. These analyses were extensive and included addressing the potential effects on seed composition from the genetic modification. Detailed compositional analyses established that GTS, which had not been treated with glyphosate, were comparable to the parental soybean line and to other conventional soybeans. In this study, two GTS lines, 40-3-2 and 61-67-1, were treated with commercial levels of glyphosate, the active ingredient in Roundup herbicide. The composition of the seed from soybeans sprayed with glyphosate was compared to that of a nonsprayed parental control cultivar, A5403. The nutrients measured in the seed included protein, oil, ash, fiber, carbohydrates, and amino acids. The concentration of isoflavones (also referred to as phytoestrogens) was also measured as these compounds are derived from the same biochemical pathway that was engineered for glyphosate tolerance. The analytical results from these studies demonstrate that the GTS soybeans treated with glyphosate were comparable to the parental soybean cultivar, A5403, and other conventional soybean varieties.

The female flowers of the hop plant are used as a preservative and as a flavoring agent in beer. However, a recurring suggestion has been that hops have a powerful estrogenic activity and that beer may also be estrogenic. In this study, sensitive and specific in vitro bioassays for estrogens were used for an activity-guided fractionation of hops via selective solvent extraction and appropriate HPLC separation. We have identified a potent phytoestrogen in hops, 8-prenylnaringenin, which has an activity greater than other established plant estrogens. The estrogenic activity of this compound was reflected in its relative binding affinity to estrogen receptors from rat uteri. The presence of 8-prenylnaringenin in hops may provide an explanation for the accounts of menstrual disturbances in female hop workers. This phytoestrogen can also be detected in beer, but the levels are low and should not pose any cause for concern.

Phytoestrogens are plant compounds that are structurally or functionally similar to steroidal estrogens produced by the body, such as estradiol. Phytoestrogens are derived from dietary precursors. Methodologically rigorous studies demonstrate the benefit of phytoestrogens in addressing the climacteric syndrome--including vasomotor symptoms--and postmenopausal health risks. Studies suggest that phytoestrogen supplementation offers a potential alternative or complement to conventional HRT for osteoporosis prevention. Whether these effects will translate into reduced fracture rates or enhanced function and well-being remains for further study. The majority of evidence about the impact of plant estrogen consumption on the risk of cancer is epidemiologic. Rates of breast, endometrial and ovarian cancers are low in Asian cultures, where the diet is rich in soy isoflavones.

Plants abound in essential phytochemicals produced for their various vital functions. The same compounds seem also to be crucial for human health and disease. Recent human epidemiological and laboratory animal and cell studies on cancer and heart disease have highlighted the phytoestrogens--naturally occurring principles that share with steroidal oestrogens an ability to activate oestrogen receptors. The best known non-steroidal phytoestrogens include the isoflavones daidzein, genistein, formononetin and biochanin A, the coumestan coumestrol, and the lignans secoisolariciresinol and matairesinol. Acknowledging the potentially chemoprotective role of these non-nutrients, we have quantified all biologically important isoflavonoids and lignans in cereals, oilseeds and nuts, legumes, vegetables, fruits, berries and beverages such as tea, coffee and wine. In this chapter, we present a review of our studies on staple plant foods, indicating that plants contain, besides a wide range of chemicals with a number of biological properties, biologically active phytoestrogens--precursors of hormone-like compounds found in mammalian systems.

Soy-based infant formulas have been in use for >30 y. These formulas are manufactured from soy protein isolates and contain significant amounts of phytoestrogens of the isoflavone class. As determined by HPLC, the isoflavone compositions of commercially available formulas are similar qualitatively and quantitatively and are consistent with the isoflavone composition of soy protein isolates. Genistein, found predominantly in the form of glycosidic conjugates, accounts for >65% of the isoflavones in soy-based formulas. Total isoflavone concentrations of soy-based formulas prepared for infant feeding range from 32 to 47 mg/L, whereas isoflavone concentrations in human breast milk are only 5.6 +/- 4.4 microg/L (mean +/- SD, n = 9). Infants fed soy-based formulas are therefore exposed to 22-45 mg isoflavones/d (6-11 mg x kg body wt(-1) x d(-1)), whereas the intake of these phytoestrogens from human milk is negligible (<0.01 mg/d). The metabolic fate of isoflavones from soy-based infant formula is described. Plasma isoflavone concentrations reported previously for 4-mo-old infants fed soy-based formula were 654-1775 microg/L (mean: 979.7 microg/L: Lancet 1997:350;23-7), significantly higher than plasma concentrations of infants fed either cow-milk formula (mean +/- SD: 9.4 +/- 1.2 microg/L) or human breast milk (4.7 +/- 1.3 microg/L). The high steady state plasma concentration of isoflavones in infants fed soy-based formula is explained by reduced intestinal biotransformation, as evidenced by low or undetectable concentrations of equol and other metabolites, and is maintained by constant daily exposure from frequent feeding. Isoflavones circulate at concentrations that are 13,000-22,000-fold higher than plasma estradiol concentrations in early life. Exposure to these phytoestrogens early in life may have long-term health benefits for hormone-dependent diseases.

Objective: The purpose of this study was to investigate the phytoestrogen content of different foods, formulas and drinks that may be consumed by infants during their first year of life in an attempt to define levels of exposure on different feeding regimens., Methodology: High performance liquid chromatography (HPLC) was used to determine the levels of genistein, daidzein, biochanin A, formononetin and equol in samples purchased from Australian supermarkets. Single lots of duplicate or triplicate samples of soy beverages, cow's milks, infant formulas and infant yoghurts were analysed., Results: All foods tested contained isoflavones, at varying levels, suggesting that exposure to these compounds is almost ubiquitous. Casein-based infant formulas contained between 0.001 and 0.03 mg L(-1). Soy-based infant formulas ranged from 17.2 to 21.9 mg L(-1) with the values detected in yoghurt at similar levels to that of cow's milk. For comparison, the soy-based beverages (which are not recommended for use under 12 months of age) contained levels of isoflavones from 22.9 to 71.5 mg L(-1)., Conclusions: Given the relatively broad choice of infant foods becoming available, exposure to dietary isoflavones during the first year of life is virtually ubiquitous. The exposure may be higher if soy infant formulas are consumed, however, the levels attained appear to fall within normal physiological boundaries.