Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17beta-estradiol (E(2)). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E(2) to generate 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), respectively. Studies suggest that 2-OHE(2) and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE(2) is carcinogenic in rodent models. Thus, agents that increase the metabolism of E(2) by Cyp1A1 to produce 2-OHE(2) may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50muM concentrations for time points ranging from 3 to 48h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 microM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE(2) by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE(2) by Cyp1A1 is increased.     

Genotoxicity of phytoestrogens

Plant extracts containing phytohormones are very popular as 'alternative' medicine for many kinds of diseases. They are especially favored by women who enter menopause and are concerned about the side effects of hormone replacement therapy. However, adverse health effects of phytoestrogens have often been ignored. This review examines the literature on genotoxicity and apoptotic effects of phytohormones. Genistein, coumestrol, quercetin, zearalenone, and resveratrol exerted genotoxic effects in in vitro test systems. Other phytoestrogens such as lignans, the isoflavones daidzein and glycetein, anthocyanidins, and the flavonol fisetin exhibited only weak or no effects in vitro. However, some metabolites of daidzein showed a genotoxic activity in vitro. Practically all of the phytoestrogens exhibit pro-apoptotic effects in some cell systems. Further investigations regarding dose-response-relationships and other aspects relevant for extrapolation to human exposure seem necessary. Until then, care may be advised in taking concentrated phytohormones. Nevertheless, the intake of substantial amounts of plant-food in a normal diet constitutes an important, individual contribution to cancer prevention.     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

Synergistic chemoprotective mechanisms of dietary phytoestrogens in a select combination against prostate cancer

Combination of dietary phytoestrogens with diverse molecular mechanisms may enhance their anticancer efficacy at physiological concentrations, as evidenced in epidemiological studies. A select combination of three dietary phytoestrogens containing 8.33 μM each of genistein (G), quercetin (Q) and biochanin A (B) was found to be more potent in inhibiting the growth of androgen-responsive prostate cancer cells (LNCaP) as well as DU-145 and PC-3 prostate cancer cells in vitro than either 25 μM of G, B or Q or 12.5+12.5 μM of G+Q, Q+B or G+B. Subsequent mechanistic studies in PC-3 cells indicated that the action of phytoestrogens was mediated both through estrogen receptor (ER)-dependent and ER-independent pathways as potent estrogen antagonist ICI-182780 (ICI, 5 μM) could not completely mask the synergistic anticancer effects, which were sustained appreciably in presence of ICI. G+Q+B combination was significantly more effective than individual compounds or their double combinations in increasing ER-β, bax (mRNA expression); phospho-JNK, bax (protein levels); and in decreasing bcl-2, cyclin E, c-myc (mRNA expression); phospho-AKT, phospho-ERK, bcl-2, proliferating cell nuclear antigen (protein levels) in PC-3 cells. Phytoestrogens also synergistically stimulated caspase-3 activity. Our findings suggest that selectively combining anticancer phytoestrogens could significantly increase the efficacy of individual components resulting in improved efficacy at physiologically achievable concentrations. The combination mechanism of multiple anticancer phytochemicals may be indicative of the potential of some vegetarian diet components to elicit chemopreventive effects against prostate cancer at their physiologically achievable concentrations, in vivo.     

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.     

Phytoestrogens and their low dose combinations inhibit mRNA expression and activity of aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens inhibit aromatase, the enzyme that converts androgens to oestrogens. Kinetic studies in cell-free preparations show that they may inhibit aromatase by competitive binding to the enzyme, but there is a paucity of studies investigating longer-term effects of phytoestrogens on the expression of steroidogenic enzymes. This study tested the hypothesis that phytoestrogens could reduce aromatase activity by down-regulation of its expression. Experiments were carried out on primary cultures of human granulosa-luteal (GL) cells after they had been exposed to phytoestrogens for 48 h. Aromatase activity was measured by the ability of cells to convert testosterone to estradiol over a 4 h period and aromatase mRNA expression (mRNA_arom) was subsequently measured from the same cells using quantitative real-time PCR. The compounds investigated were the flavones, apigenin and quercetin, and the isoflavones, genistein, biochanin A and daidzein at doses of 10 μM and 100 nM. Combinations of these compounds at the lower dose were also investigated. All compounds tested dose-dependently reduced mean mRNA_arom compared with controls. Apigenin was the most potent inhibitor with significant inhibition of mRNA_arom seen at both 10 μM and 100 nM, whilst other flavonoids (except biochanin A) only induced significant inhibition (p ≤ 0.05) at the higher dose. Low dose (100 nM) mixtures of the compounds were ineffective except for a combination of the three isoflavones that induced a significant inhibition of mRNA_arom. The changes in aromatase activity paralleled the mRNA_arom results and additional studies showed that the reduction in aromatase activity was significantly delayed in time compared with the reduction in mRNA_arom. This is the first study to compare the action of various phytoestrogens, either singly or in low-dose combination, on the expression and activity of aromatase in human cells and it suggests that chronic dietary exposure and tissue accumulation of low-dose mixtures of phytoestrogens could have important consequences for aromatase activity and the production of oestrogens.     

Co-administration of quercetin and catechin in rats alters their absorption but not their metabolism

Quercetin and catechin are among the major flavonoids in plant foods and their intake has been associated to a risk reduction in several degenerative diseases. The aim of the present study was to bring data on the bioavailability of quercetin and catechin when administered simultaneously. The study was performed on rats adapted to diets containing (i) 0.25% quercetin, or (ii) 0.25% catechin, or (iii) 0.25% quercetin+0.25% catechin. Quercetin, catechin and their metabolites were determined in plasma, urine and liver by HPLC with UV or coulometric detection. When quercetin and catechin were fed in association, their respective plasma concentration significantly decreased (-35% and -28% respectively), whereas the urinary and hepatic concentrations were only affected for quercetin (-36%). These data may be explained by a competitive interaction between quercetin and catechin at the digestive level, leading to a reduction of the intestinal absorption of quercetin and a possible delaying of catechin absorption over time. The simultaneous administration of quercetin and catechin had no effect on the formation of their glucurono and sulfo conjugates, indicating the absence of competition between quercetin and catechin for the corresponding conjugative enzymes.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons

Epidemiological data from retrospective and case-control studies have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer's disease. In addition, ERT has been found to promote cellular correlates of memory and to promote neuronal survival both in vivo and in vitro. Phytoestrogens have been proposed as potential alternatives to ERT. To determine whether phytoestrogens exert estrogen agonist effect in neural tissue, investigations of neuroprotective and neurotrophic efficacy of phytoestrogens were conducted. Six phytoestrogens, genistein, genistin, daidzein, daidzin, formononetin, and equol, were tested for their neuroprotective efficacy against two toxic insults, glutamate excitotoxicity and beta-amyloid(25-35). Neuronal membrane damage was quantitatively measured by lactate dehydrogenase (LDH) release, and neuronal mitochondrial viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid (MTT) assay. Results of these studies demonstrated that all phytoestrogens induced a modest but significant reduction in LDH release following exposure to glutamate and beta-amyloid(25-35). In contrast, none of phytoestrogens induced a significant increase in reduced MTT levels, which occurred in the presence of a full estrogen agonist, 17beta-estradiol. Analysis of the neurotrophic potential of genistein and daidzein, two phytoestrogens that exerted a significant reduction in LDH release, demonstrated that neither of these molecules promoted hippocampal neuron process outgrowth. Results of these analyses indicate that although phytoestrogens exert a neuroprotective effect at the plasma membrane, they do not sustain neuron mitochondrial viability nor do they induce cellular correlates of memory as neurite outgrowth and synaptogenesis are putative mechanisms of memory. Data derived from these investigations would predict that phytoestrogens could exert some neuroprotective effects analogous to that of antioxidants, but that these molecules are not functional equivalents to endogenously active 17beta-estradiol or to estrogen replacement formulations and, therefore, would raise the concern that they may not reduce the risk of Alzheimer's disease or sustain memory function in postmenopausal women.     

Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassays (TR-FIA), with europium labeled phytoestrogens as tracers, were developed for the quantitative measurement of genistein, daidzein and enterolactone in plasma and urine for the purpose of screening large populations and studies on possible correlation between the values in biological fluids and the risk of western diseases. The mean values of the three phytoestrogens in plasma as determined by TR-FIA were similar to those obtained by gas chromatography-mass spectrometry (GC-MS). The urinary excretion levels of total individual phytoestrogens were higher than those obtained by GC-MS, with the exception of the daidzein values. However, comparing the assay results obtained by the present method and those obtained by GC-MS, a strong correlation was evident (r = 0.87 - 0.99, p < 0.001). We measured plasma levels of genistein, daidzein and enterolactone in 111 healthy Japanese women The mean and median levels of genistein were 406.8 and 306.3 nmol/l, respectively, and those of daidzein were 118.4 and 76.8 nmol/l, respectively. These levels are higher than those reported for Americans and Western Europeans. Isoflavone intake as calculated from dietary records (genistein: mean, 86.5 mircomol/day and daidzein: mean, 57.4 micromol/day) was correlated with the plasma concentrations observed (genistein: r = 0.287, p < 0.01 and daidzein: r = 0.313, p < 0.01). Plasma enterolactone levels were low in Japanese women (mean, about 10 nmol/l). The levels of urinary excretions of genistein, daidzein were also measured and it was found that, in the majority, the levels ranged between 5-25 and 5-50 micromol/24 h, respectively. In contrast, healthy Finnish women showed very low values of isoflavones (below 10 nmol/l in plasma (n = 87) and below 0.6 micromol/24 h in urine (n = 126) for both compounds) and high levels of enterolactone in both plasma and urine (plasma: mean, 25 nmol/l and urine: majority range, 1-7 micromol/24 h).     

The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells

A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) alpha and ERbeta and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ERalpha-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17beta-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ERalpha as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a Gbetagamma-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression.