Isoflavones stimulate estrogen receptor-mediated core histone acetylation

The isoflavones genistein and daidzein and the daidzein metabolite equol have been reported to interact with estrogen receptors (ERs). Some studies indicate that they behave clinically like estrogen in some estrogen-deficiency diseases. However, the detailed molecular mechanism used by these compounds to create beneficial effects in patients with estrogen-related diseases has not been clarified. Using histone acetyltransferase (HAT) assay, we found that equol, genistein, and AglyMax had significant effects on ERalpha-mediated histone acetylation. Although 17beta-estradiol (E2)-dependent HAT activity of steroid receptor coactivators 2 (SRC2) and p300 mediated by ERbeta could be detected, it was weaker than that mediated by ERalpha. Equol, genistein, AglyMax, and daidzein all markedly stimulated ERbeta-mediated histone acetylation. On the other hand, anti-estrogenic compounds ICI 182,780 (ICI) and tamoxifen (TA) did not have an effect on HAT activity mediated by either ERalpha or ERbeta. Our data indicate that estrogenic ligands exert their effects by elevating histone acetylation and coactivator activity of ER, and suggest that the risk of estrogen-related diseases might be reduced by a sufficient amount of genistein or AglyMax supplements.     

Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback

Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4-7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5alpha-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) beta, and no affinity for ERalpha. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

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.     

Three isoflav-3-enes and a 2-arylbenzofuran from the root bark of Erythrina burttii

From the root bark of Erythrina burttii three isoflav-3-enes, 7,4'-dihydroxy-2'-methoxy-6-(1",1"-dimethylallyl)isoflav-3-ene (trivial name, burttinol-A), 4'-hydroxy-2'-methoxy-2",2"-dimethylpyrano[5",6":8,7]isoflav-3-ene (trivial name, burttinol-B), 7,4'-dihydroxy-2'-methoxy-8-(3",3"-dimethylallyl)isoflav-3-ene (trivial name, burttinol-C), and 2-arylbenzofuran, 6,4'-dihydroxy-2'-methoxy-5-(1",1"-dimethylallyl)-2-arylbenzofuran (trivial name, burttinol-D) were isolated. In addition, the known compounds, abyssinone V-4'-methyl ether, bidwillol A, calopocarpin, erybraedin A, erythrabyssin II, isobavachalcone, phaseollidin and phaseollin were identified. The structures were determined on the basis of spectroscopic evidence.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

In vitro evaluation of estrogenic, estrogen antagonistic and progestagenic effects of a steroidal drug (Org OD-14) and its metabolites on human endometrium

The human endometrial model for in vitro evaluation of estrogenic, estrogen antagonistic, and progestagenic effects of endogenous steroids, natural products or synthetic drugs was applied to the study of Org OD-14, an analog of norethynodrel developed by Organon International, Oss, The Netherlands, and some of its metabolites. Estrogen antagonistic actions of Org OD-14 and its 4-ene isomer were evident from their ability to suppress the enhancement of PGF2 alpha output elicited by estradiol on fragments of secretory endometrium and to decrease the rate of output of the prostaglandin by proliferative tissue, already stimulated by endogenous estrogens. These inhibitory effects were similar to those obtained with progesterone and do not appear to involve competition for the estrogen receptor since the antiestrogen 4-hydroxyamoxifen was not active in parallel incubations of proliferative endometrium. The progestagenic effects of Org OD-14 and its 4-ene isomer were also evident from their capability to enhance estradiol 17 beta-dehydrogenase activity and glycogen accumulation in specimens of proliferative endometrium. Estrogenic effects of the 3 alpha- and 3 beta-hydroxy metabolites of Org OD-14 were demonstrated by their stimulatory actions on PGF2 alpha output during incubations of secretory endometrium. The estrogenic and progestagenic actions of these compounds are in general agreement with their relative affinity for binding to the estradiol and progesterone receptors, although their actions may be influenced by intracellular metabolism in the endometrial tissue. For instance, the similarity in progestagenic activity of Org OD-14 and the 4-ene isomer, contrasting with their different affinities for the progesterone receptor, may result from in situ isomerization of Org OD-14 to the 4-ene metabolite.     

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.     

Absolute configurations of isoflavans

The absolute configurations of isoflavans and isoflavanquinones isolated from Cyclolobium, Dalbergia and Machaerium species were established by comparison of their ORD curves with that of (3S)-5,7,3′,4′-tetra-methoxyisoflavan and (3S)-7,4′-dimethoxyisoflavan-2′,5′-quinone, respectively. The assignments were checked by the ozonolysis of the isoflavan (?)-duartin to (R)-paraconic acid and the oxidation of isoflavans to isoflavanquinones. The PMR spectra of the dihydropyran ring of the isoflavans are discussed in terms of the preferred conformation of this ring.     

The phytoestrogen equol increases nitric oxide availability by inhibiting superoxide production: an antioxidant mechanism for cell-mediated LDL modification

Estrogen replacement therapy (ERT) is reported to lower the incidence of cardiovascular disease in postmenopausal women. ERT also lowers the levels of oxidatively modified low-density lipoprotein (LDL). Because modified LDL can mediate the development of atherosclerosis by inflammatory processes, ERT may exert its LDL protective effect through enhanced antioxidant activity in vascular tissues. Plant sources of estrogenic compounds have been used as alternatives for ERT because they avoid a number of negative health effects produced by estrogen. In this study, the antioxidant properties of the soy isoflavone metabolite, equol (an estrogenic metabolite of daidzein) were studied. Equol has a greater antioxidant activity than the parent isoflavone compounds genistein and daidzein, found in high concentration in soy. Equol inhibits LDL oxidation in vitro and LDL oxidative modification by J774 monocyte/macrophages to LDL(-), an electronegative modified LDL found in human plasma. An antioxidant effect of equol was found to be mediated by inhibition of superoxide radical (O(2)(-*)) production and manifested through enhanced levels of free nitric oxide (NO) that prevents LDL modification. Thus, when NO levels were increased by donor agents, generators, or compounds that facilitate nitric oxide synthase activity, LDL(-) formation by J774 cells was strongly inhibited. Conversely, inhibition of NO production enhanced LDL(-) formation, and the combination of reduced NO and increased O(2)(-*) production yielded maximum LDL(-) formation. Pretreatment of cells with equol inhibited production of O(2)(-*) by J774 cells apparently via the inactivation of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex. Decreased O(2)(-*) production resulted in increased free NO levels (but not total NO production) indicating that decreased reactions between O(2)(-*) and NO are an outcome of equol's antioxidant activity in cell culture.     

Equol,an Isoflavone Metabolite,Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER−) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.     

Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17β-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.     

Homology modeling, active site prediction, and targeting the anti hypertension activity through molecular docking on endothelin - B receptor domain

In cardiovascular system, activation of Endothelin receptors causes vasoconstriction which leads to Pulmonary Arterial Hypertension (PAH). Endothelin receptor antagonism has emerged as an important therapeutic strategy in pulmonary arterial hypertension. Bosentan is intended to affect vasoconstriction, hypertrophic and fibrotic effects by blocking the actions of receptors ET(A) and ET(B). In this study we identified the action of Bosentan on endothelin B receptor using docking studies with homology modeled endothelin B receptor. Through the modeled protein, the flexible Docking study was performed with Bosentan and its derivatives with theoretically predicted active sites. The results indicated that amino acid ARG82, ARG84 and HIS197 present in endothelin B receptor are core important for binding activities and these residues are having strong hydrogen bond interactions with Bosentan. We have investigated the Bosentan and its derivatives interactions and scoring parameters using gold docking package. Among the docked compounds, one of the Bosentan derivatives BD(6) shows better interaction than Bosentan with endothelin B receptor. Our results may be helpful for further investigations in both in vivo and in vitro conditions.     

Systemic administration of diarylpropionitrile (DPN) or phytoestrogens does not affect anxiety-related behaviors in gonadally intact male rats

The development of highly selective agonists for the two major subforms of the estrogen receptor (ERα and ERβ) has produced new experimental methodologies for delineating the distinct functional role each plays in neurobehavioral biology. It has also been suggested that these compounds might have the potential to treat estrogen influenced behavioral disorders, such as anxiety and depression. Prior work has established that the ERβ agonist, diarylpropionitrile (DPN) is anxiolytic in gonadectomized animals of both sexes, but whether or not this effect persists in gonadally intact individuals is unknown. Isoflavone phytoestrogens, also potent but less selective ERβ agonists, have also been shown to influence anxiety in multiple species and are becoming more readily available to humans as health supplements. Here we determined the effects of 0.5, 1 or 2 mg/kg DPN, 1 mg/kg of the ERα agonist propyl-pyrazole-triol (PPT), 3 or 20 mg/kg of the isoflavone equol (EQ) and 3 or 20 mg/kg of the isoflavone polyphenol resveratrol (RES) on anxiety behavior in the gonadally intact male rat using the light/dark box and the elevated plus maze. We first determined that DPN can be successfully administered either orally or by subcutaneous injection, although plasma DPN levels are significantly lower if given orally. Once injected, plasma levels peak rapidly and then decline to baseline levels within 3 h of administration. For the behavioral studies, all compounds were injected and the animals were tested within 3 h of treatment. None of the compounds, at any of the doses, significantly altered anxiety-related behavior. Plasma testosterone levels were also not significantly altered suggesting that these compounds do not interfere with endogenous androgen levels. The results suggest that the efficacy of ERβ agonists may depend on gonadal status. Therefore the therapeutic potential of ERβ selective agonists to treat mood disorders may be limited.     

Isoflavan phytoalexins from Anthyllis,lotus and Tetragonolobus

Two previously unreported phytoalexins, 7,4′dihydroxy-2′-methoxy- and 7,2′,4′-trihydroxyisoflavan, have been isolated from the fungus-inoculated leaves of Anthyllis vulneraria and 5 Tetragonolobus species. Examination of Lotus corniculatus revealed the co-occurrence of the latter with the known isoflavans, vestitol and sativan. Only 7,2′4′-trihydroxyisoflavan and vestitol were produced by the closely related L. uliginosus.     

The isoflavone Equol mediates rapid vascular relaxation: Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells

We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nM) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nM, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER)alpha and ERbeta. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.     

Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women

Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E(1)) relative to 16alpha-hydroxyestrone (16alpha-OH E(1)) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E(1), 16alpha-OH E(1), and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E(1) and 16alpha-OH E(1) were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48h apart in excretion of 2-OH E(1) or 16alpha-OH E(1) (P=0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16alpha-OH E(1) (r=0.32, P=0.02), but was not correlated with 2-OH E(1) or 2-OH E(1):16alpha-OH E(1) ratio (r=0.21, P=0.14, and r=-0.05, P=0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E(1):16alpha-OH E(1) ratio (r=0.38, P=0.005), but was not correlated with 2-OH E(1) or 16alpha-OH E(1) (r=0.15, P=0.29, and r=-0.17, P=0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E(1):16alpha-OH E(1) ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk.     

Identification of phytoestrogens in the urine of male dogs

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, less than 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.     

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.     

Effects of dietary equol on the pituitary of the ovariectomized rats.

The aim of our study was to evaluate the effects of dietary equol, metabolite of a phytoestrogen daidzein, on the secretion of prolactin (PRL) and lutenizing hormone (LH), as well as the expression of estrogen receptors (ERalpha, ERbeta and truncated estrogen receptor-1 (TERP-1) in the pituitary gland of ovariectomized (ovx) female Sprague-Dawley rats. Two doses of equol (50 mg/kg of chow and 400 mg/kg of chow) were used and the results were compared with the effects of estradiol 3-benzoate (E2B), also given at two doses (4.3 mg/kg of chow and 17.3 mg/kg of chow). Treatment period was 3 months. Dietary equol administration at the high dose increased significantly serum PRL levels. This effect was also observed in the E2B group but this difference did not reach statistical significance. Surprisingly, high dose dietary equol treatment also significantly increased serum LH levels, which was in contrast to E2B treatment where serum LH levels were significantly decreased at both doses. Serum LH levels in the equol low group were unaffected. Equol treatment had no effects on pituitary ERalpha or ERbeta gene expression. In contrast, high dose E2B treatment increased significantly pituitary ERalpha mRNA levels but decreased those of ERbeta. Both doses of E2B also increased significantly pituitary TERP-1 mRNA levels. This effect was also observed in the equol high group but at a much smaller magnitude. In conclusion, high dose dietary equol administration to ovx rats exerts estrogenic like effects on the lactotropes and anti-estrogenic on the gonadotropes.     

Estrogen, nitric oxide, and hypertension differentially modulate agonist-induced contractile responses in female transgenic (mRen2)27 hypertensive rats

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17beta-estradiol (E(2)) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E(2) or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E(2) treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with N(omega)-nitro-L-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E(2)-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.