Estrogen receptors (ERs), comprising ER α and ER β, are crucial for regulating cell growth and differentiation via homo- and hetero-dimer formation. However, accurately detecting ER dimerization with precise spatiotemporal resolution remains a significant challenge. In this study, fluorescence resonance energy transfer-based biosensors to monitor ER dynamics in real-time, are developed and optimized. This approach involves comprehensive structural analysis, linker comparison, and the selection of optimal fluorescent protein pairs, resulting in three distinct biosensors capable of detecting all ER homo- and hetero-dimerizations within the nucleus. These biosensors are utilized to reveal interactions between ER α/β and calmodulin during dimer formation. Furthermore, by leveraging the ligand-binding domain (LBD) of ER β, ER ββ LBD biosensor is designed for real-time analysis of ER ββ homodimerization in the cytoplasm, enhancing the ability to screen ER dimerization-related drugs. Additionally, we developed a novel ER ββ translocation biosensor, which enables real-time observation of ER ββ translocation to the nucleus-a capability previously unavailable, is developed. This spatiotemporal analysis demonstrates the relevance of ER translocation in response to drug binding efficacy and extracellular matrix changes. Our biosensors offer transformative tools for studying ER dynamics, providing valuable insights for drug screening and the investigation of ER-related cellular processes.

Coumestrol is a phytoestrogen found in various plant foods. Increasing evidence ascertained its robust anti-inflammatory, anti-oxidative properties likewise ability to mitigate insulin resistance. Thus, it may be a potential medicine in the treatment of many metabolic disorders, including obesity, type 2 diabetes (T2D) as well as non-alcoholic fatty liver disease (NAFLD). In this study, we aimed to shed some light on its influence on the accumulation of certain lipid fractions and the expression of pro-inflammatory proteins in primary rat hepatocytes during the lipid-overload state. The cells were isolated from the male Wistar rat's liver with the use of collagenase perfusion. It was followed by incubation of the cells with the presence or absence of palmitic acid and/or coumestrol. The accumulation of lipid fractions was assessed by gas-liquid chromatography (GLC) whereas the expression of the proteins was evaluated by the Western blot technique. Treatment with coumestrol in the state of increased fatty acids availability led to the deposition of triacylglycerols rather than diacylglycerols, significantly decreased expression of proinflammatory and profibrotic cytokines, especially interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), as well as transforming growth factor β (TGF-β), and nuclear factor κβ (NF-κβ). Also, we observed a substantial diminution in proinflammatory enzymes expression. Taking into consideration the direction of the aforementioned changes, we may assume that coumestrol can ameliorate the array of factors leading to the development of steatosis, likewise counteracting progression to steatohepatitis, thus it may be a step forward to the long-awaited breakthrough in the treatment of NAFLD.

Coumestrol is a phytoestrogen widely known for its anti-diabetic, anti-oxidant, and anti-inflammatory properties. Thus, it gets a lot of attention as a potential agent in the nutritional therapy of diseases such as obesity and type 2 diabetes. In our study, we evaluated whether coumestrol affects insulin resistance development via the sphingolipid signaling pathway in primary rat hepatocytes. The cells were isolated from the male Wistar rat's liver with the use of collagenase perfusion. Next, we incubated the cells with the presence or absence of palmitic acid and/or coumestrol. Additionally, some groups were incubated with insulin. The sphingolipid concentrations were assessed by HPLC whereas the expression of all the proteins was evaluated by Western blot. Coumestrol markedly reduced the accumulation of sphingolipids, namely, ceramide and sphinganine through noticeable inhibition of the ceramide de novo synthesis pathway in insulin-resistant hepatocytes. Moreover, coumestrol augmented the expression of fatty acid transport proteins, especially FATP5 and FAT/CD36, which also were responsible for excessive sphingolipid accumulation. Furthermore, coumestrol altered the sphingolipid salvage pathway, which was observed as the excessive deposition of the sphingosine-1-phosphate and sphingosine. Our study clearly showed that coumestrol ameliorated hepatic insulin resistance in primary rat hepatocytes. Thus, we believe that our study may contribute to the discovery of novel preventive and therapeutic methods for metabolic disorders.

Genistein is an isoflavone abundant in soybean and infants are exposed to high levels of genistein in soy-based formula. It is known that genistein mediates estrogen receptor (ER) signaling, and exposure during neonatal development could cause acute and long term endocrine effects. We assayed genistein's impact on the neonatal mouse pituitary gland because it is an endocrine signaling hub and is sensitive to endocrine disruption during critical periods. Pituitary explant cultures, which actively proliferate and differentiate, were exposed to 0.06 μM-36 μM genistein and assayed for mRNA and protein changes. Genistein induced mRNA expression of the ERα regulated gene, Cckar, to the same magnitude as estradiol (E2) but with less potency. Interestingly, 36 μM genistein strongly inhibited pituitary proliferation, measured by a reduction in mKi67 mRNA and phospho-Histone H3 immunostaining. Examining cell cycle dynamics, we found that 36 μM genistein decreased Ccnb1 (Cyclin B1) mRNA; while mRNA for the cyclin dependent kinase inhibitor Cdkn1a (p21) was upregulated, correlated with an apparent increase in p21 immunostained cells. Strikingly, we observed a robust onset of cellular senescence, permanent cell cycle exit, in 36 μM genistein treated pituitaries by increased senescence activated β-galactosidase staining. We also found that 36 μM genistein decreased Bcl2 mRNA levels, a gene protective against apoptosis. Taken together these data suggest that genistein exposure during the neonatal period could initiate senescence and halt proliferation during a time when the proper numbers of endocrine cells are being established for mature gland function.

The development of biomarkers of reproductive medicine is still in its infancy because many black boxes are still present in reproductive medicine. Novel approaches to human infertility diagnostics and treatment must be developed because reproductive medicine has lagged behind in the implementation of biomarkers in clinical medicine. Despite the dearth of the available literature, the current rapid pace of publications suggests that this gap will soon be filled therefore; this review is a précis of the research that has been done so far and will provide a basis for the development of biomarkers in reproductive medicine.

Phytoestrogens are estrogenic compounds that occur naturally in plants. Phytoestrogens can cross the placenta, and animal studies have found associations between in utero exposure to phytoestrogens and markers of early puberty. We investigated the association between in utero exposure to phytoestrogens and early menarche (defined as <11.5 years of age at onset) using data from a nested case-control study within the Avon Longitudinal Study of Parents and Children, a longitudinal study involving families living in the South West of England. Concentrations of six phytoestrogens were measured in maternal urine samples collected during pregnancy. Logistic regression was used to explore associations between tertiles of phytoestrogen concentrations and menarche status, with adjustment for maternal age at menarche, maternal education, pre-pregnancy body mass index (BMI), child birth order, duration of breastfeeding, and gestational age at sample collection. Among 367 mother-daughter dyads, maternal median (interquartile range) creatinine-corrected concentrations (in µg/g creatinine) were: genistein 62.1 (27.1-160.9), daidzein 184.8 (88.8-383.7), equol 4.3 (2.8-9.0), O-desmethylangolensin (O-DMA) 13.0 (4.4-34.5), enterodiol 76.1 (39.1-135.8), and enterolactone 911.7 (448.1-1558.0). In analyses comparing those in the highest tertile relative to those in the lowest tertile of in utero phytoestrogen exposure, higher enterodiol levels were inversely associated with early menarche (odds ratio (OR)=0.47; 95% confidence interval (CI): 0.26-0.83), while higher O-DMA levels were associated with early menarche (OR=1.89; 95% CI: 1.04-3.42). These findings suggest that in utero exposure to phytoestrogens may be associated with earlier age at menarche, though the direction of association differs across phytoestrogens.

The aim of this work was to compare the effect of neonatal treatment with the phytoestrogens coumestrol (COU) and genistein (GEN), administered in equimolecular doses, on the sexual behavior and partner preference of male rats. Four groups of male rats were injected daily from day 1 to 5 with 150 µg of GEN, an equivalent amount of COU, 1 µg of β-estradiol 3-benzoato (EB), or olive oil (VEH) (control). A fifth group remained intact. In the GEN group, intromission and ejaculation latencies decreased, whereas ejaculatory frequency increased. Contrasting results were observed in COU males. EB males could not ejaculate and their mount and intromission latencies increased significantly. To determine sexual-partner preferences, a multiple partner preference arena was used and two types of tests were performed, the first one without allowing contact test (CT) with the stimulus animals, followed by a CT. COU and GEN groups did not show preference for any stimulus animal, whereas the EB males preferred the expert male. When CT with the stimulus animals was allowed, GEN-males preferred the receptive female, unlike the COU and EB groups. It is concluded that neonatal treatment with COU and GEN induced opposite effects, the effects of COU being more estrogenic.

The goal of this study was to investigate the impact of kudzu (Pueraria mirifica) and the isoflavone puerarin in functional toxicological tests on spermatozoa and to assess the affinity of extracts and pure isoflavones for estrogen receptor (ER)-alpha and -beta (ERα, ERβ) in receptor binding assays. Capacitation, acrosome reaction and chromatin decondensation in spermatozoa were analyzed using microscopic analysis. Kudzu, but not puerarin, reduced motility of sperm. Puerarin reduced the percent spontaneous acrosome reaction in spermatozoa. The pathways used by kudzu that affect sperm function are not fully mirrored by puerarin. Puerarin, kudzu and its other phytoestrogenic components displayed preferential affinity for ERβ, however the diverse effects of kudzu and puerarin on sperm function implicate the involvement of multiple signaling systems.

Plant physiological, epidemiological, and food science studies have shed light on lignans as healthy diets for the reduction of the risk of lifestyle-related noncommunicable diseases and, thus, the demand for lignans has been rapidly increasing. However, the low efficiency and instability of lignan production via extraction from plant resources remain to be resolved, indicating the requirement for the development of new procedures for lignan production. The metabolic engineering of lignan-biosynthesizing plants is expected to be most promising for efficient, sustainable, and stable lignan production. This is supported by the recent verification of biosynthetic pathways of major dietary lignans and the exploration of lignan production via metabolic engineering using transiently gene-transfected or transgenic plants. The aim of this review is to present an overview of the biosynthetic pathways, biological activities, and metabolic engineering of lignans and also perspectives in metabolic engineering-based lignan production using transgenic plants for practical application.

Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2α were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2α metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow.

Phytoestrogens are structurally similar to estrogens and may affect breast cancer risk by mimicking estrogenic/antiestrogenic properties. In Western societies, whole grains and possibly soy foods are rich sources of phytoestrogens. A population-based case-control study in German postmenopausal women was used to evaluate the association of phytoestrogen-rich foods and dietary lignans with breast cancer risk. Dietary data were collected from 2,884 cases and 5,509 controls using a validated food-frequency questionnaire, which included additional questions phytoestrogen-rich foods. Associations were assessed using conditional logistic regression. All analyses were adjusted for relevant risk and confounding factors. Polytomous logistic regression analysis was performed to evaluate the associations by estrogen receptor (ER) status. High and low consumption of soybeans as well as of sunflower and pumpkin seeds were associated with significantly reduced breast cancer risk compared to no consumption (OR = 0.83, 95% CI = 0.70-0.97; and OR = 0.66, 95% CI = 0.77-0.97, respectively). The observed associations were not differential by ER status. No statistically significant associations were found for dietary intake of plant lignans, fiber, or the calculated enterolignans. Our results provide evidence for a reduced postmenopausal breast cancer risk associated with increased consumption of sunflower and pumpkin seeds and soybeans.

Phytoestrogens have been thought to have favorable effects on women's health and perhaps in offsetting cancers. The possible adverse effects of phytoestrogens have not been evaluated.

Abnormal uterine bleeding with endometrial pathology in three women was found to be related to a high intake of soy products. The first woman had postmenopausal bleeding with uterine polyp, proliferative endometrium and a growing leiomyoma. The second woman presented with severe dysmenorrhea, abnormal uterine bleeding, endometriosis and uterine leiomyoma not responding to treatment. The third woman with severe dysmenorrhea, abnormal uterine bleeding, endometriosis and uterine leiomyomata presented with secondary infertility. All three women improved after withdrawal of soy from their diet.

Additional information on phytoestrogens is necessary to ascertain their safety before they can be routinely used as supplements.

The aim of this study was to investigate the potency of LH suppression and the uterotrophic effects of quercetin, a flavonoid widely present in our diet which in vitro has been shown to posses estrogenic properties.

Fifty-nine female Sprague-Dawley (SD) rats were ovariectomized (ovx) and fed with soy-free rodent chow with the addition of quercetin or estradiol-3 benzoate (E2B). Quercetin was added to the rodent chow at the dose of 200mg/kg (n=12) and 1000 mg/kg (n=11) which on average corresponded to 3.55 mg and 18.42 mg per animal per day, respectively, and E2B at the dose of 4.3mg/kg (n=12) or 17.3mg/kg (n=12) which corresponded to 0.07 mg and 0.20 mg per animal per day, respectively. The control group (n=12) received soy-free chow only. After three months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHbeta and uterine insulin like growth factor (IGF)-1, progesterone receptor (PR) and complement 3 protein (C3) mRNA levels were measured. Additionally, the in vitro binding capacity of quercetin with a porcine cytosolic ER preparation was evaluated.

In contrast to E2B, dietary quercetin did not decrease pituitary LH expression, had no effects on uterine weight and uterine expression of estrogen regulated genes. The binding capacity of quercetin with the ERs was also 35000-fold lower compared with 17beta-estradiol (E2).

Our study shows that quercetin does not show any estrogenic effects in the pituitary and the uterus of the ovx SD rats.

The aim of the present study was to evaluate the uterotropic effects of the administration of dietary equol, a metabolite of soy-derived daidzein or formononetin present in red clover, in an ovariectomized rat model of menopause.

Two doses of racemic equol were used (50 mg/kg of chow and 400 mg/kg of chow) and the results were compared with two doses of estradiol-3 benzoate (E2B) (4.3 mg/kg of chow and 17.3 mg/kg of chow). After 3 months, animals were sacrificed and the uteri were removed, weighed and paraffin-embedded for morphometrical and immunohistochemical evaluation. The expression of selected uterine estrogen-responsive genes was also measured using real-time reverse transcription-polymerase chain reaction.

Compared to controls, uterine weights in animals treated with high-dose equol were significantly higher, presented histologic features of mild estrogenic stimulation and had greater epithelial height and thickness of the uterine stroma and myometrium. Staining for the presence of the proliferating cell nuclear antigen (PCNA) also showed a greater prevalence of the PCNA-positive cells in the uterine stroma in animals treated with high-dose equol. Conversely, the percentage of PCNA-positive cells in the uterine epithelium was lower compared to the controls. Dietary high-dose equol treatment also increased significantly levels of uterine insulin-like growth factor 1, progesterone receptor and complement protein 3 mRNA. Although statistically significant, all these effects were, however, lower in magnitude compared to the effects of low- and high-dose E2B treatment. Low-dose equol did not have any effects on the above-studied parameters.

Long-term high-dose dietary equol administration to ovariectomized rats exerts uterotropic effects at the cellular and molecular level which question the safety of uncontrolled and unlimited consumption of soy or red clover supplements by postmenopausal women with intact uteri.

To investigate the potency of LH suppression, as an indirect measure of alleviation of postmenopausal vasomotor symptoms, as well as the uterotropic effects of two isoflavones: daidzein and puerarin in an ovariectomized (ovx) rat model and compare them with the effects of 17beta-estradiol benzoate (E2B).

Eighty female Sprague-Dawley rats were ovx and divided into six different treatment groups and one control group (11-12 animals per group). Daidzein, puerarin and E2B were added to the soy free rodent chow in low and high doses (250 mg and 1000 mg per kg, 600 mg and 3000 mg per kg and 4.3 mg and 17.3 mg per kg, respectively). After 3 months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHbeta and uterine IGF-1, PR and C3 mRNA levels were measured. Additionally serum LH levels were measured in a radioimmunoassay.

Both of our tested isoflavones at low and high doses had no effect on the expression of the pituitary LH at the mRNA and protein level. Only E2B at both doses significantly decreased pituitary LHbeta gene expression and serum LH levels. Daidzein and puerarin at high dose increased significantly uterine weights. Uterine IGF-1 gene expression was only upregulated in puerarin high group. Uterine PR mRNA levels were higher in animals fed with low dose daidzein and high dose puerarin. Uterine C3 gene expression was upregulated in animals fed with daidzein and puerarin at high doses. Although statistically significant, all these effects were however very discrete compared to those of E2B at low and high doses.

We speculate that due to the lack of LH suppressing effects in our model, it is very unlikely for daidzein and puerarin to alleviate vasomotor symptoms in postmenopausal women. In contrast, due to their uterotropic effects, high dose consumption of commercially available preparations containing daidzein or puerarin may expose women with an intact uterus to the risk of endometrial hyperplasia.

HOXA10 is necessary for normal development of the Müllerian duct, and continued adult expression in the uterus is necessary for female fertility. HOXA10 expression is altered by diethylstilbestrol, leading to uterine anomalies. Other endocrine disruptors may potentially lead to reproductive anomalies or dysfunction by altering HOXA10 expression. Here we investigated the effect of isoflavones on HOXA10 expression after in utero or adult exposure in the mouse. Genistein, but not diadzein, regulated HOXA10 mRNA and protein expression in the adult mouse uterus. In contrast, in utero genistein or diadzein exposure had no lasting effect on HOXA10 expression in the exposed offspring. Reporter gene expression driven by the HOXA10 estrogen response element was increased in a dose-responsive manner by genistein, but not daidzein. Neither estrogen receptor-alpha nor estrogen receptor-beta binding to the HOXA10 estrogen response element was affected by genistein or daidzein. In utero exposure to isoflavones is unlikely to result in HOXA10-mediated developmental anomalies. Adult genistein exposure alters uterine HOXA10 expression, a potential mechanism by which this agent affects fertility.

A method is presented for the estimation of 13C-chemical shifts for carbon atoms in protonated and deprotonated molecules; in principle, this method can be applied to ions in general. Experimental 13C-chemical shifts were found to vary linearly with computed atomic charges using the PM3 method. Pseudo-13C-chemical shifts for atoms in protonated and deprotonated molecules can be estimated from computed atomic charges for such atoms using the above linear relationship. The pseudo-13C-chemical shifts obtained were applied to the rationalization of product ion mass spectra of protonated and deprotonated molecules of flavone and 3-, 5-, 6-, 7-, 2'-, 3'-, and 4'-hydroxyflavones, where product ion formation is due to either cross-ring cleavage of the C-ring (retro-Diels-Alder reaction) or to cleavage of a C-ring bond followed by loss of either a small neutral molecule or a radical. The total product ion abundance ratio of C-ring cross cleavage to C-ring bond cleavage, gamma, varied by a factor of 660 for deprotonated monohydroxyflavones, i.e., from 0.014:1 to 9.27:1. The magnitude of gamma, which is dependent on the relative bond orders within the C-ring of the protonated and deprotonated molecules of monohydroxyflavones, can be rationalized on the basis of the magnitudes of the 13C- and 1H-chemical shifts as determined by nuclear magnetic resonance spectroscopy.

Many environmental chemicals are known endocrine disruptors (EDs). These have the potential to alter endocrine systems via various mechanisms that include binding to hormone receptors, thereby either mimicking or blocking the hormone actions and causing abnormal gene expression. Here, to elucidate the molecular mechanism(s) underlying the detrimental effects associated with the estrogenicity of these chemicals, we determined whether gene profiles were altered in rats exposed to 4-tert-octyphenol (OP) and diethylstilbestrol (DES) in utero. Pregnant rats were treated with a high dose of OP (600 mg/kg BW per day) or DES (500 microg/kg BW per day) at gestational days (GD) 17, 18 and 19. Both dams and neonates were euthanized at lactation day (LD) 5. The transcript profiles of uterine tissue were compared in treated versus control in both maternal and neonatal sites using cDNA microarray to determine the expression levels of approximately 13,000 genes and expressed sequence tags (ESTs). The expression levels of some known estrogen-responsive genes, i.e., complement component 3, epidermal growth factor receptor or c-fos oncogene and calbindin 3, as well as some other randomly selected genes, including general transcription factor IIa, transcription factor 4 and lymphocyte specific 1, were increased by OP and/or DES treatment in the uteri of both maternal and neonate groups. However, the magnitude of these alterations in gene expression differed markedly between dams and neonates, most likely reflecting the temporal susceptibility of the reproductive tract to estrogenic chemicals. Importantly, the altered gene patterns identified by microarray analysis were confirmed by RT-PCR and real-time RT-PCR. Fifteen primers were designed to amplify specific altered genes. These genes were selected for validation because of their markedly increased expression levels and they were classified on the basis of gene ontology. Overall, a high correlation was observed between microarray and real-time PCR data. Taken together, these results indicate that placental exposure to OP or DES may cause temporal changes in gene expression in the uteri of dams and neonates. Moreover, these findings may provide useful indicators of the adverse effects of EDs and prove particularly important in elucidating the effects of xenoestrogens on estrogen-responsive tissues, such as the developing reproductive tract.

Natural hormones and some synthetic chemicals spread into our surrounding environment share the capacity to interact with hormone action and metabolism. Exposure to such compounds can cause a variety of developmental and reproductive detrimental abnormalities in wildlife species and, potentially, in human. Many experimental and epidemiological data have reported that exposure of the developing fetus or neonate to environmentally relevant concentrations of some among these endocrine disrupters induces morphological, biochemical and/or physiological disorders in brain and reproductive organs, by interfering with the hormone actions. The impact of such exposures on the hypothalamic-pituitary-gonadal axis and subsequent sexual maturation is the subject of the present review. We will highlight epidemiological human studies and the effects of early exposure during gestational, perinatal or postnatal life in female rodents.

The incidence of hormone-related diseases such as prostatic, breast, ovarian, and endometrial cancer is lower in Asian populations compared to Western countries. High consumption of soybean products that are rich in phytoestrogens, predominantly genistein, is postulated to be responsible for the lower incidence of hormone-related disease, although the mechanism through which this effect might be mediated is unclear. In this study, microarray analysis was used to identify the changes in gene expression elicited by treatment of the human endometrial cancer cell line, Ishikawa, with genistein at both physiologically achievable and supraphysiological concentrations. Genistein treatment at 5 microM concentration induced multiple changes in gene expression including some implicated in oncogenesis. In contrast, treatment with a supraphysiological concentration of genistein predominantly activated stress response genes and showed very limited overlap with the genes regulated at lower concentrations. Of the genes regulated by genistein, 9.3% were also regulated by 17beta-estradiol suggesting that genistein exerts its response via the estrogen pathway. These results indicate that at physiological concentrations, genistein is able to elicit pleiotropic effects on a variety of pathways believed to be involved in tumorigenesis. Supraphysiological concentrations of genistein, such as those used in many previous studies, elicit changes in gene expression that are unlikely to occur in vivo.

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.

The goal of this study was to identify age-related changes in the expression of estrogen target genes in mouse uterus. We developed a novel 'estrogen response element (ERE) Chip' microarray bearing 297 genes including both known estrogen target genes and genes identified by searching the mouse genome database to have EREs, AP-1 sites, and Sp1 sites, all targets of estrogen receptor (ER) regulation. 400-500 bp PCR products of these 297 genes were printed onto nylon membranes creating the 'ERE Chip' microarray. This microarray is unique because it is the first estrogen-responsive gene-specific microarray to identify changes in uterine gene expression in young versus old mice. Using this ERE microarray we identified 10 uterine genes whose expression was up-regulated in old mice, e.g. beta-actin, calcium binding protein 45a, Sp1, and COUP-TFII. In contrast, the expression of only 4 uterine genes, i.e., complement C3, lactoferrin, Muc-1, and 17-beta-hydroxysteroid dehydrogenase 8 (H2-Ke6) was down-regulated in old mice. These changes may reflect an increase in stromal and a decrease in glandular epithelial gene expression, and may be associated with age-related changes in these tissue compartments within the uterus, possibly leading to the decline in reproductive function in C57Bl/6 mice.

The two primary sources of nutrition for infants are human milk and infant formula. Both contain an array of endogenous and exogenous chemicals that may act through many separate hormonal mechanisms. The safety of infant nutrition sources has been questioned based on the possibility that exogenous chemicals may exert adverse effects on nursing or formula-fed infants through estrogen-mediated mechanisms. In response to these and other concerns, the National Research Council recommended assessing the estrogenic potency of natural and anthropogenic hormonally active agents. Furthermore, the Endocrine Disruptor Screening and Testing Advisory Committee of the U.S. Environmental Protection Agency specifically recommended testing chemicals present in human milk as a representative mixture to which large segments of the population are exposed. To date, no clinical or epidemiologic evidence demonstrates that levels of chemicals currently found in human milk or infant formulas cause adverse effects in infants. Nonetheless, the question is sufficiently important to warrant a consideration of how best to evaluate potential estrogenic risks. We reviewed the types of data available for measuring estrogenic potency as well as methods for estimating health risks from mixtures of chemicals in infant nutrition sources that act via estrogenic mechanisms. We conclude that the science is insufficiently developed at this time to allow a credible assessment of health risks to infants based on estimates of estrogenic potency or on an understanding of toxicologic effects mediated by estrogenic mechanisms. However, clinical and epidemiologic data for infant nutrition sources may provide insights about risks of such substances in human milk and infant formulas.

Soy foods may have various health benefits, but little is known about the patterns and correlates of soy consumption among postmenopausal women in the United States.

We assessed the reliability and validity of a soy food-frequency questionnaire (FFQ) and examined demographic, lifestyle, and dietary correlates of plasma isoflavone concentrations in postmenopausal women.

In this cross-sectional study, soy isoflavone intake and plasma isoflavone concentration were analyzed in 96 postmenopausal women aged 50-79 y; the data were obtained at 2 visits that were 1 wk apart. Intake was determined with a 20-item soy FFQ and a comprehensive FFQ that included questions about tofu and soymilk. Fasting plasma daidzein and genistein concentrations were determined with liquid chromatography-mass spectrometry.

Intraclass correlations between week 1 and week 2 values were >0.98 for both the soy and comprehensive FFQs. Median reported isoflavone intake was <2 mg/d. Pearson's product-moment correlation coefficients relating isoflavone intakes with plasma isoflavone concentrations ranged from 0.35 to 0.43. Plasma isoflavone concentrations were positively associated with age, fiber consumption, servings of fruit and vegetables, and dietary supplement use and were inversely associated with caffeine consumption; no associations with body mass index, education, dietary beliefs, activity level, alcohol intake, or fat intake were observed.

Within a population with low soy consumption, the soy FFQ and comprehensive FFQ showed good reliability and moderate validity. Associations of plasma isoflavone concentrations with other dietary behaviors suggest that these compounds may serve as biomarkers of health behaviors in populations with low soy consumption.

The estrogenic responses of several phytoestrogens including genistein, daidzein, coumestrol, alpha-zearalanol, zearalenone, naringenin, taxifolin and biochanin A were compared over a wide dose range using an in vitro assay that measures transcriptional activation of the estrogen receptor (ER) and an in vivo immature mouse uterotrophic assay consisting of measuring uterine wet weight increase plus sensitive morphological and biochemical endpoints in the uterus. The transcriptional activation assay showed activation of the ER by all compounds tested except taxifolin with varying magnitudes of response as compared to estradiol or diethylstilbestrol. Results from the uterotropic bioassay showed that genistein, coumestrol, zearalanol, and zearalenone caused an increase in uterine wet weight, while naringenin, taxifolin, daidzein and biochanin A failed to do so over the dose range tested. However, sensitive morphological and biochemical parameters such as uterine epithelial cell height increase, uterine gland number increase, and induction of the estrogen-responsive protein lactoferrin demonstrated that all compounds tested in this study gave some measure of estrogenicity although a wide range of estrogenic responses across compounds was shown. Use of multiple in vitro and in vivo estrogenic endpoints as described in this paper will be useful in developing estrogenic profiles for individual compounds and ultimately mixtures of compounds. Furthermore, having an estrogenic "fingerprint" for each phytochemical is an essential first step in determining potential adverse effects of exposure to phytoestrogens.

Approximately 30% of women afflicted with migraine have menstrually associated attacks. These migraines are often refractory to treatment. Evidence suggests estrogen and progestin fluctuations may influence menstrual migraine. Phytoestrogens have demonstrated estrogenic effects in some tissues, but are without stimulation of the endometrium, suggesting decreased risk with long-term use. This study was undertaken to assess the efficacy of a phytoestrogen combination in the prophylactic treatment of menstrual migraine. Forty-nine patients were randomized to receive either placebo, or a daily combination of 60 mg soy isoflavones, 100 mg dong quai, and 50 mg black cohosh, with each component standardized to its primary alkaloid. Patients received study medication for 24 weeks. Average frequency of menstrually associated migraine attacks during weeks 9-24 was reduced from 10.3 +/- 2.4 (mean +/- s.e.m.) in placebo treated patients to 4.7 +/- 1.8 (P < 0.01) in patients treated with the phytoestrogen preparation.

Despite strong observational evidence for a beneficial role of oestrogen in cardiovascular disease, recent trial results suggest that hormone replacement therapy (HRT) may have adverse effects in menopausal women with established coronary heart disease. Isoflavones are oestrogen analogues found in plants with oestrogen-like properties and, because of a favourable side-effect profile, may be ideal alternatives to HRT with respect to cardiovascular benefits. Endothelial function is a marker of cardiovascular health. We aimed to determine the effect of isoflavones on endothelial function using the brachial artery reactivity test.

Twenty-nine healthy menopausal women underwent entry and exit brachial artery reactivity testing following randomization to 2 weeks of an oral soy isoflavone concentrate containing 80 mg of soy isoflavones (Archer Daniel Midland Inc., IL, USA) or placebo.

At study exit, there was no difference between placebo and isoflavone groups with respect to flow-mediated dilation (%FMD(max)), change (entry to exit) in %FMD(max) or response to nitroglycerine (%TNG). Subgroup analyses assessing lipid and oestrogen effects did not produce any significant results.

These results suggest that short-term oral isoflavone supplements do not improve endothelial function in healthy menopausal women.

Environmental oestrogens have been implicated in the pathogenesis of hormonally treated cancers (such as breast and prostate cancer), male infertility, and abnormalities of the male and female reproductive tracts. They may be derived from plants (phytoestrogens), pharmaceuticals, or other synthetic compounds not originally intended to have oestrogenic activity (including soy based infant formulas). This review will discuss the evidence from both animal studies and humans for an effect of these ubiquitous compounds on the development of the human female genital tract, in addition to prolonging the menstrual cycle, alleviating symptoms of the menopause, and protecting against the development of endometrial carcinoma.

Modern molecular methods for precancer diagnosis have expanded the range of detectable disease to a preclinical level and provided material for histopathological correlation. The precancer scenario begins with sporadic acquisition of rare PTEN mutation bearing glands, which are morphologically unremarkable, and progresses to discrete foci of cytologically altered glands, readily visible on routinely stained sections. Clinical outcome studies of women with endometrial lesions have established threshold diagnostic features that confer increased cancer risk. This class of high risk lesions has been designated endometrial intraepithelial neoplasia (EIN). EIN is diagnosed by presence of cytological demarcation, crowded gland architecture, minimum size of 1mm, and careful exclusion of mimics. Most EIN lesions have been diagnosed as atypical endometrial hyperplasias in the World Health Organisation system. Specialised molecular and morphometric analyses have been extremely useful in redefining clinically relevant premalignant endometrial disease, but translation to improved patient care requires the informed participation of pathologists.

Consumption of phytoestrogens and mycoestrogens in food products or as dietary supplements is of interest because of both the potential beneficial and adverse effects of these compounds in estrogen-responsive target tissues. Although the hazards of exposure to potent estrogens such as diethylstilbestrol in developing male and female reproductive tracts are well characterized, less is known about the effects of weaker estrogens including phytoestrogens. With some exceptions, ligand binding to the estrogen receptor (ER) predicts uterotrophic activity. Using a well-established and rigorously validated ER-ligand binding assay, we assessed the relative binding affinity (RBA) for 46 chemicals from several chemical structure classes of potential phytoestrogens and mycoestrogens. Although none of the test compounds bound to ER with the affinity of the standard, 17beta-estradiol (E(2)), ER binding was found among all classes of chemical structures (flavones, isoflavones, flavanones, coumarins, chalcones and mycoestrogens). Estrogen receptor relative binding affinities were distributed across a wide range (from approximately 43 to 0.00008; E(2) = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.

Controversy has arisen concerning the likelihood of adverse health effects due to exposure to hormonally active agents or endocrine modulators such as environmental estrogens. With the aim to improve the basis for their toxicological evaluation, several chemicals of anthropogenic (bisphenol A, octylphenol, o,p'-DDT) and of natural origin (daidzein, genistein) were investigated with regard to their mode of hormonal action and potency as well as toxicokinetics. Experimental toxicodynamic and toxicokinetic data illustrate important points in a comparative assessment of environmental estrogens. A novel concept, the Hygiene-Based Margine of Safety (HBMOS), has been suggested to characterize the relative impact of these potential endocrine modulators on human health: It integrates exposure scenarios (i.a. those generated within the European Existing Chemicals Programme) and in vivo rodent potency data for xenoestrogens and for dietary phytoestrogens. On the basis of these informations, HBMOS values calculated for the alkylphenol and bisphenol A appear sufficiently high to ensure the absence of a practical risk to human health under the present exposure conditions. For slowly accumulating compounds (e.g. DDT) with much longer half-lifes than isoflavones, such comparison should be based on comparative blood levels rather than on scenarios of daily exposures.

To assess the effects of a red clover-derived isoflavone extract on the Ki-67 proliferative marker of endometrial biopsies in 45-to 50-year-old perimenopausal women. We hypothesized that we would be able to detect a decrease in the Ki-67 proliferative index during the late follicular phase after a 3-month course of approximately 50 mg red clover isoflavones. Isoflavones have been found to have some antiestrogenic effects, and an antiproliferative effect during the perimenopausal period may be especially useful owing to the excessive endometrial proliferation often characteristic of this period.

In a double-blind, randomized, controlled study, 30 women between the ages of 45 and 50 years consented to an endometrial biopsy before and after a 3-month course of either placebo or active isoflavone extract. The biopsies were timed as close as possible to days 7-11 of the menstrual cycle, and simultaneous measurements of transvaginal endometrial thickness, uterine artery Doppler, hormone profiles, lipids, and bone markers were performed.

Of 30 women, 2 did not return for a second biopsy, and a third had an unsuccessful second biopsy. Four subjects were excluded from the Intention to Treat analysis because they did not have a menstrual bleed within the time frame of the study (3 subjects) or were tested on day 13 instead of between days 7 and 11 of the cycle (1 subject). There was no change in the Ki-67 proliferation index after treatment in either group. Eight subjects in the placebo group and eight in the P-07 group had proliferative endometrial biopsies that were synchronized with estradiol levels at baseline and post-treatment, and analysis of these subjects revealed no detectable change in the relationship between estradiol levels and Ki-67 with treatment in either group. There was no change in fasting lipids, bone markers, uterine Doppler resistance, or pulsatility index.

In this small pilot study, we did not find, using immunohistochemical quantification of the Ki-67 antigen, that red clover isoflavones had an antiproliferative effect in the endometrium. Small sample size, examination of a relatively short interval in the menstrual cycle, and isoflavone formulation may have contributed to our lack of findings; however, we believe that the issue of isoflavones and their possible antiproliferative effect is deserving of further study. A simpler physiological model with less hormonal variability, such as healthy, recently menopausal women on predetermined doses of estrogen, may prove to be more informative.

The phytoestrogen genistein was studied in normal and malignant experimental uterine models in vivo. The action of genistein on the uterus and vagina of ovariectomized DA/Han rats after 3 day oral administration (25, 50 or 100 mg/kg/BW/d) was compared to ethinyl oestradiol (0.1 mg/kg/BW/d). Effects on uterine and vaginal morphology, uterine growth and uterine gene expression were studied. A dose dependent increase of the uterine wet weight and the uterine and vaginal epithelial height, a dose dependent up-regulation of complement C3, down-regulation of clusterin mRNA expression and a stimulation of the vaginal cornification was observed after administration of genistein. Uterine gene expression and vaginal epithelium respond to genistein at doses where no significant effects on uterine wet weight were detectable. In general the vagina was more sensitive to genistein than the uterus. To analyse the action of genistein in malignant uterine tissue, the impact of a 28 d treatment with 50 mg/kg/d of genistein on the in-vivo tumour growth of RUCA I endometrial adenocarcinoma cells, following subcutaneous inoculation into syngeneic DA/Han rats, was assessed. In contrast to ethinyl oestradiol (0.1 mg/kg/BW/d), a dose of 50 mg/kg/BW/d of genistein did not affect tumour growth. Nevertheless C3 and TRPM2 mRNA expression in the tumour were both significantly stimulated by ethinyl oestradiol and genistein. In comparison to ovariectomized animals genistein up-regulated uterine wet weight and uterine dependent gene expression in tumour bearing animals. In conclusion, four independent uterine and vaginal parameters indicate genistein is a weak oestrogen receptor agonist in the uterus and vagina of female DA/Han rats, and evidence is provided for a selective oestrogen receptor modulator (SERM)-like action of genistein in normal and malignant uterine tissue.

Three practical diets were formulated to contain 0, 500, or 1000 ppm genistein. The three diets were distributed for 1 year to groups of rainbow trout undergoing their first gametogenesis and until spawning. Growth performance of rainbow trout was not affected by dietary treatments. Plasma cholesterol levels were equivalent between groups. In males, a slight but constant induction of vitellogenin (VTG) synthesis and a decrease in testosterone levels were observed. A slight decrease in plasma levels of betaFSH and betaLH was noticed at the end of spermatogenesis in the male fish fed a diet with 500 ppm (genistein) (from 2.16 +/- 0.39 to 1.47 +/- 0.23 for betaFSH and from 0.44 +/- 0.09 to 0.31 +/- 0.09 for betaLH). There was a significantly reduced 17alpha,20beta(OH)(2)-progesterone (from 10.93 +/- 0.88 in control to 5.46 +/- 0.92 in males and from 251.22 +/- 21.40 to 183.22 +/- 13.48 in females). Testicular development was accelerated in genistein-fed fish, and sperm motility and concentration were decreased in a dose-dependent manner at spawning. In females, a significant increase in plasma VTG occurred only at the beginning and at the end of oogenesis. Testosterone levels were decreased at the beginning of oogenesis. Both betaFSH and betaLH were decreased by genistein (from 6.38 +/- 1.55 to 3.44 +/- 0.82 for betaFSH and from 15.18 +/- 3.00 to 6.93 +/- 0.99 for betaLH in females), whereas spawning was delayed only in females fed the diet with 500 ppm of genistein. Gamete quality was impaired only in this group, as underlined by a lower percentage of ovulating females (from 100 to 79% at the end of the trial), a lower fertilization rate, and a lower viability of fry. These results may be explained by the agonistic/antagonistic effect of genistein on estrogen function related to the tissue ratio between endogenous estrogens/genistein.

The present study investigated the effect of genistein, daidzein and estradiol on in vitro rat uterine responsiveness to oxytocin (OT) and PGF(2)alpha or luprostiol (L). In a first experiment, animals were either sham-operated (SH; n=5), or ovariectomized (OVX; n=20) and orally treated for three months with either genistein (G; n=5; 10 microg/g BW/d) or daidzein (D; n=5; 10 microg/g BW/d) or 17 alpha-ethinylestradiol (E; n=5; 23 microg/kg BW/d) or untreated (OVX; n=5). At necropsy, the basal uterine tension was lower in OVX, G and D than in SH, the highest value being measured in E. Oxytocin (10(-12); 10(-11) M) or PGF(2)alpha (10(-12); 10(-9) M) induced an increase in SH, but not in OVX, E and G. In D, only the highest doses were efficient. In a second experiment, 20 intact animals were s.c. injected with either genistein (G; n=5; 10 microg/g BW) or daidzein (D; n=5; 10 microg/g BW) or estradiol benzoate (E; n=5; 23 microg/kg BW) or vehicle (C: controls; n=5), and killed 24 h later. In C and E, OT (10(-15) to 10(-10) M) or L (10(-12) to 10(-7) M) stimulated uterine contractile activity in a dose-dependent manner until a maximal level. On the opposite, in G and D, contractile agents (except the highest luprostiol doses) did not stimulate myometrium contractions. Moreover, radioligand binding assays showed that genistein or daidzein inhibited the specific binding of [(3)H] estradiol to the calf uterus estrogen receptor (ER). Therefore, it could be postulated that both genistein and daidzein might bind to the rat uterus ER, inducing either anti-estrogenic or very weak estrogenic effects (depending on the experimental conditions) on in vitro uterine responsiveness to OT and PGF(2)alpha or luprostiol.

Two Spectroscopic Data-Activity Relationship (SDAR) models based on (13)C nuclear magnetic resonance (NMR) and electron ionization mass spectra (EI MS) data were developed for 108 compounds whose relative binding affinities (RBA) to the estrogen receptor are known. The (13)C NMR and EI MS data were used as spectrometric digital fingerprints to reflect the electronic and structural characteristics of the compounds. Both SDAR models segregated the 108 compounds into 20 strong, 15 medium, and 73 weak relative binding classifications. The first SDAR model, based on (13)C NMR data alone, gave a leave-one-out (LOO) cross-validation of 75.0%. The second SDAR model, based on a composite of (13)C NMR and EI MS data, gave a LOO cross-validation of 82.4%. Many of the misidentifications from the cross-validations were between medium and weak classifications, where there were fewer specific spectrometric characteristics to identify the relationship of spectra to estrogen receptor binding. Real and predicted (13)C NMR chemical shifts were used to test the predictive behavior of both SDAR models. The ease of use and speed of SDAR modeling may facilitate their use with other toxicological endpoints.

Postmenopausal women have sought nonestrogen alternatives to hormone replacement in order to avoid possible risks and side effects of the therapy. Selective estrogen receptor modulators have been developed to tailor therapy to a specific risk/benefit profile that will best fit the patient. More women have looked to phytoestrogens, such as the isoflavones found in the soy plant, to tailor their menopausal therapy in a "natural" way. This review examines the evidence regarding the risks and benefits of isoflavones as hormone replacement therapy. Controlled trials have shown a reduction in postmenopausal hot flashes when subjects' diets were supplemented with soy. There is less evidence for a benefit in vaginal dryness symptoms. Furthermore, dietary supplementation also appears to lower total and low-density lipoprotein cholesterol in hypercholesterolemic subjects. A synthetic isoflavone, ipriflavone, has been shown in controlled trials to prevent postmenopausal bone loss, though there is much less evidence that soy isoflavones will accomplish this goal. Finally, although unopposed estrogen replacement may promote breast and endometrial cancer, there is no evidence that phytoestrogens will do the same. In contrast, great interest has been taken in the potential cancer-protective effects of phytoestrogens, though prospective evidence in postmenopausal women is not available. Although data regarding the use of isoflavone extracts are incomplete, dietary supplementation with soy foods appears to be a safe and possibly beneficial option for postmenopausal women.

The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>>>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured RUCA-I cells represent a valuable endometrial derived model system to assess the relative potencies and the molecular mode of action of environmental estrogens in vitro. Our results further show that no intimate correlation exists between the relative binding affinity and the biological response of these compounds. Therefore, data obtained from single-parametric analyses may result in misleading conclusions. On the other hand, the presented in vitro data will provide us with tools to study the activity of xenoestrogens in vivo and thus carry risk assessment one step further.