Coumestrol induces oxidative stress and impairs migration and embryonic growth.

In brief: Healthy development of the placenta is dependent on trophoblast cell migration and reduced oxidative stress presence. This article describes how a phytoestrogen found in spinach and soy causes impaired placental development during pregnancy. Abstract: Although vegetarianism has grown in popularity, especially among pregnant women, the effects of phytoestrogens in placentation lack understanding. Factors such as cellular oxidative stress and hypoxia and external factors including cigarette smoke, phytoestrogens, and dietary supplements can regulate placental development. The isoflavone phytoestrogen coumestrol was identified in spinach and soy and was found to not cross the fetal-placental barrier. Since coumestrol could be a valuable supplement or potent toxin during pregnancy, we sought to examine its role in trophoblast cell function and placentation in murine pregnancy. After treating trophoblast cells (HTR8/SVneo) with coumestrol and performing an RNA microarray, we determined 3079 genes were significantly changed with the top differentially changed pathways related to the oxidative stress response, cell cycle regulation, cell migration, and angiogenesis. Upon treatment with coumestrol, trophoblast cells exhibited reduced migration and proliferation. Additionally, we observed increased reactive oxygen species accumulation with coumestrol administration. We then examined the role of coumestrol within an in vivo pregnancy by treating wildtype pregnant mice with coumestrol or vehicle from day 0 to 12.5 of gestation. Upon euthanasia, fetal and placental weights were significantly decreased in coumestrol-treated animals with the placenta exhibiting a proportional decrease with no obvious changes in morphology. Therefore, we conclude that coumestrol impairs trophoblast cell migration and proliferation, causes accumulation of reactive oxygen species, and reduces fetal and placental weights in murine pregnancy.

Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells.

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion.

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.

The binding of lignans, flavonoids and coumestrol to CYP450 aromatase: a molecular modelling study.

Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Effect of in utero-administered coumestrol, equol, and organic selenium on biomarkers for phase 2 enzyme capacity and redox status.

The aim of the present study was to investigate the effect of in utero administration of coumestrol, equol, and selenium-enriched yeast on selected hepatic phase 2 enzymes, plasma hormone levels, and markers for redox status in plasma and red blood cells (RBCs). The test compounds were administered via the diet to pregnant Sprague-Dawley rats throughout gestation. Within 24 h following delivery dams and offspring were sacrificed, and blood, liver, and reproductive organs were sampled. Coumestrol, equol, and selenium-enriched yeast did not significantly affect hepatic glutathione S-transferase (GST), quinone reductase (QR), or RBC glutathione peroxidase (GPx) in the offspring, whereas significant increases in GST, QR, and GPx activities in dams were observed following administration of selenium-enriched yeast. The level of 17beta-estradiol in offspring from coumestrol-exposed dams was significantly increased compared with the control. The present results indicate that selenium-enriched yeast, coumestrol, and equol affect selected hepatic phase 2 enzymes and GPx in RBC in dams, whereas the offspring in general were refractive to the employed treatments. Further studies are warranted to investigate whether the observed in utero effects imposed by the selected plant compounds confer permanent alterations on the health status of the animal resulting in an altered resistance to cancer.

Detection of phytoestrogens in samples of second trimester human amniotic fluid.

There is widespread concern that fetal exposure to hormonally active chemicals may adversely affect development of the reproductive tract. Therefore, the present study was performed to develop the necessary analytical methods and test the hypothesis that dietary phytoestrogens can be quantified in second trimester human amniotic fluid. Amniotic fluid samples (n=59) from women (n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (+/- standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44 +/- 1.34 and 1.69 +/- 1.48 ng/ml with maximum levels of 5.52 and 6.54 ng/ml, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure.

Differential estrogen receptor binding of estrogenic substances: a species comparison.

The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.

Fitoestrógenos: una nueva preocupación en la alimentación infantil / Phytoestrogens: a new preoccupation in paediatric nutrition

Los fitoestrógenos son sustancias ambientales naturales, producidas por plantas, que a pesar de su estructura química distinta de los estrógenos, actúan como tales. Estudios en adultos sugieren que tendrían efectos protectores para cánceres hormonodependientes (de próstata y mama), dislipidemias y de la mineralización ósea. Se clasifican en isoflavonas, cumestanos y lignanos, y se encuentran principalmente en legumbres y poroto de soya, brotes de poroto, forraje y granos, y en cereales de grano entero y semillas, respectivamente. Estudios recientes han demostrado que los alimentos infantiles, incluyendo algunas fórmulas lácteas, yogur y alimentos de soya, contienen cantidades considerables de fitoestrógenos. Los efectos de estos sobre la salud infantil no han sido del todo aclarados. Existen evidencias epidemiológicas y clínicas de que al actuar como estrógenos débiles prodrían determinar adelanto de los eventos puberales y telarquia en la niña y ginecomastia en el varón. Se hace una revisión del tema y se plantea la necesidad de realizar estudios destinados a aclarar los efectos de los estrógenos ambientales sobre la salud infantil

Characterization of estrogenicity of phytoestrogens in an endometrial-derived experimental model.

Severe developmental and reproductive disorders in wild animals have been linked to high exposure to persistent environmental chemicals with hormonal activity. These adverse effects of environmental estrogens have raised considerable concern and have received increasing attention. Although numerous chemicals with the capacity to interfere with the estrogen receptor (ER) have been identified, information on their molecular mechanism of action and their relative potency is rather limited. For the endometrium, the lack of information is due to the lack of a suitable experimental model. We investigated the functions of phytoestrogens in an endometrial-derived model, RUCA-I rat endometrial adenocarcinoma cells. The cells were cultured on a reconstituted basement membrane to preserve their functional differentiation and estrogen responsiveness. We assessed the relative binding affinity to the estrogen receptor of the selected phytoestrogens coumestrol, genistein, daidzein, and the putative phytoestrogen mangostin compared to estradiol by a competitive Scatchard analysis. The following affinity ranking was measured: 17beta-estradiol >>> coumestrol > genistein > daidzein >>> mangostin. In addition, we investigated the capacity of these compounds to promote the increased production of complement C3, a well-known estradiol-regulated protein of the rat endometrium. All substances tested increased the production of complement C3, although different concentrations were necessary to achieve equivalent levels of induction compared to estradiol. Mechanistically we were able to demonstrate that the increase of complement C3 production was mediated by primarily increasing its steady-state mRNA level. These findings indicate that RUCA-I cells represent a sensitive model system to elucidate relative potencies and functions of environmental estrogens in an endometrium-derived model.

Binding of phyto-oestrogen and oestradiol-17 beta by cytoplasmic receptors in the pituitary gland and hypothalamus of the ewe.

The binding of the phyto-oestrogens, genistein and coumestrol by oestrogen receptors in pituitary and hypothalamic cytosol of the ewe was studied in relation to the binding of [3H]-oestradiol-17 beta. It was determined that the interaction of these compounds was competitive. The apparent dissociation constant for oestradiol-17 beta in ewe pituitary cytosol was determined to be 0 x 26 +/- 0 x 12 (S.E.M.) nmol/l. Apparent inhibition constants for coumestrol and genistein were determined to be 60 and 170 nmol/l respectively. These results suggested that phyto-oestrogens can interfere with the normal oestrogen feedback mechanisms with respect to release of gonadotrophine in the ewe.