Identification of the Potent Phytoestrogen Glycinol in Elicited Soybean (Glycine )max

Stephen M. Boué, Syreeta L. Tilghman, Steven Elliott, M. Carla Zimmerman, K. Y. Williams, Florastina Payton-Stewart, Allen P. Miraflor, Melanie H. Howell, Betty Y. Shih, Carol H. Carter-Wientjes, Chris Segar, Barbara S. Beckman, Thomas E. Wiese, Thomas E. Cleveland, John A. McLachlan and Matthew E. Burow

Endocrinology 2009 150:2446-2453 originally published online Dec 30, 2008; , doi: 10.1210/en.2008-1235

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Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online REPRODUCTION-DEVELOPMENT

Identification of the Potent Phytoestrogen Glycinol in Elicited Soybean (Glycine max)

Stephen M. Boue´,* Syreeta L. Tilghman,* Steven Elliott, M. Carla Zimmerman, K. Y. Williams, Florastina Payton-Stewart, Allen P. Miraflor, Melanie H. Howell, Betty Y. Shih, Carol H. Carter-Wientjes, Chris Segar, Barbara S. Beckman, Thomas E. Wiese, Thomas E. Cleveland, John A. McLachlan, and Matthew E. Burow

United States Department of Agriculture (S.M.B., B.Y.S., C.H.C.-W., T.E.C.), Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana 70179; Departments of Medicine, Section of Hematology and Medical Oncology (S.L.T., S.E., F.P.-S., M.E.B.), Pulmonary Diseases, Critical Care, and Environmental Medicine (S.L.T.), Surgery (M.E.B.), and Pharmacology (M.C.Z., A.P.M., M.H.H., B.S.B., J.A.M.), The Tulane Cancer Center (S.L.T., F.P.-S., J.A.M., M.E.B.), and The Center for Bioenvironmental Research (S.L.T., S.E., K.Y.W., B.S.B., T.E.W., J.A.M., M.E.B.), Tulane University Health Science Center, New Orleans, Louisiana 70112; and Xavier University School of Pharmacy (C.S., T.E.W.), New Orleans, Louisiana 70125

The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent es- trogen antagonists in vitro and in vivo. The discovery of the glyceollins’ ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 ␮M, which correlated with comparable colony formation of MCF-7 cells at 10 ␮M. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor- ␣ ⌱ ϭ 1). Competitive binding assays revealed a high affinity of glycinol for both ER ( C50 13.8 nM) and ␤ ⌱ ϭ ER ( C50 9.1 nM). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ER␣ and ER␤. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods. (Endocrinology 150: 2446–2453, 2009)

pidemiologic studies support the view that consumption of form of isoflavones are formed from microorganism-induced Esoy prevents certain hormonally induced cancers and other fermentation and hydrolysis. diseases associated with estrogen deficiency (1–6). Asian women Isoflavones are considered phytoalexins, low-molecular- who consume a traditional low-fat soy diet have a 4- to 6-fold weight antimicrobial compounds and are synthesized de novo, lower risk of developing breast cancer when compared with accumulating in different plant tissues in response to stress, women living in the industrialized Western world (1–6). Many physical stimuli, or infectious agents (12–17). In soybean, of the health benefits of soybean have been attributed, in part, to several significant changes in isoflavone concentration occur the presence of isoflavones (3, 6–10). The primary isoflavones in in response to stress or elicitor treatment. The predominant soybean are genistein, daidzein, glycitein, and their respective phytoalexins produced in soybean are glyceollins I, II, and III, ␤-glucosides. In most soy foods, isoflavones are present primar- and these compounds demonstrate antifungal activity against ily as ␤-glucosides, esterified with malonic or acetic acid (11). several plant pathogens (13–18). Recent research in our lab- However, in fermented soy foods, higher levels of the aglycone oratory focused on the antiestrogenic activity of the glyceol-

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: CS, Charcoal stripped; DMSO, dimethylsulfoxide; E2, estradiol; ER, estrogen Printed in U.S.A. receptor; ERE, estrogen response element; FBS, fetal bovine serum; PgR, progesterone Copyright © 2009 by The Endocrine Society receptor; RBA, relative binding affinity; SDF, stromal-cell-derived factor. doi: 10.1210/en.2008-1235 Received August 20, 2008. Accepted December 19, 2008. First Published Online December 30, 2008 * S.M.B. and S.L.T. contributed equally to this work and both should be considered as first authors of this manuscript.

2446 endo.endojournals.org Endocrinology, May 2009, 150(5):2446–2453 Endocrinology, May 2009, 150(5):2446–2453 endo.endojournals.org 2447

lins in vitro (19) and in vivo (20, 21). Our results have led to glycinol has potent estrogenic activity and may prove beneficial the examination of other soybean phytoalexins for estrogenic for the use of postmenopausal women requiring hormone re- and antiestrogenic activities. placement therapy. Several other phytoalexins that are structurally similar to the glyceollins, occur at low concentrations including glyceollidin (22, 23), glyceocarpin (22–24), and glycinol (22–27) shown in Materials and Methods Fig. 1. Glycinol, a precursor in the biosynthetic pathway of the glyceollins, was first isolated by Lyne and Mulheirn (25). In a Chemicals and plasmids study by Weinstein et al. (24), glycinol inhibited the growth of all ICI 182,780 (Fulvestrant) was purchased from Tocris Bioscience six bacteria examined and the three fungi Phytophthora me- (Ellisville, MO). 17␤-Estradiol was purchased from Sigma (St. Louis, gasperma f. sp glycinea (race 1), Saccharomyces cerevisae, and MO). The solvents acetonitrile (HPLC grade), methanol, and ethanol were purchased from Aldrich Chemical Co. (St. Louis, MO). H O Cladosporium cucumerinium. These results suggested that gly- 2 treated with a Millipore system (Bedford, MA) was used during sample cinol possessed antimicrobial and antifungal activity; however, preparation procedures and HPLC analyses. no other biological activity was examined. Because of the struc- ER␤ cDNA was generously provided by Jan-Åke Gustafsson (Karolinska tural similarity to daidzein and coumestrol in addition to various Institute, Stockholm, Sweden) in pBluescript. ER␣ and ER␤ expression ␣ ␤ other environmental estrogens studied (28, 29), it was hypoth- vectors were constructed by inserting the ER and ER cDNA respec- tively into pcDNA 3.1 vector (Invitrogen, Carlsbad, CA). ER␣ cDNA esized that glycinol would have estrogenic activity. (2090 bp) was cleaved from plasmid (pBluescript) with BamHI/EcoRI In this study, we examined the estrogenic activity of the in- and then ligated into the pcDNA3.1. ER␤ cDNA (1460 bp) was duced soybean isoflavone glycinol using several in vitro assays. cleaved from Plasmid (pBluescript) with HindIII/BamHI and then Glycinol’s effect on estrogen receptor (ER) activity was analyzed ligated into the pcDNA3.1. Each construct was verified by detailed using an ER-positive MCF-7 human breast carcinoma cell line restriction mapping. HEK 293 cells were cultured in 5% charcoal-stripped (CS) DMEM transfected with an estrogen response element (ERE)-luciferase and seeded into a 24-well plate at a density of 50,000 cells/well and reporter. We also examined glycinol’s effect on expression levels allowed to attach overnight. Cells were transfected with 0.2 ␮g ER(2)-luc of two estrogen-responsive genes by semiquantitative real time plasmid (Panomics, Fremont, CA), 0.1 ␮g pcDNA, 0.1 ␮g pcDNA3.1B- RT-PCR: stromal-cell-derived factor (SDF)-1 and progesterone ER␤,or0.1␮g pcDNA3.1B-ER␣ plasmids the next day using Effectene receptor (PgR). Furthermore, viability and proliferative proper- transfection reagent (QIAGEN, Valencia, CA) according to the manu- facturer’s protocol. After a 6-h transfection, cells were treated with com- ties exerted by MCF-7 cells was explored using a colony forma- pounds [dimethylsulfoxide (DMSO), estradiol (E ), glycinol, or fulves- ␣ ␤ 2 tion assay. ER and ER binding assays were conducted to de- trant] overnight. On the following day, the cells were lysed with 150 ␮l termine the binding affinities of glycinol to both ER subtypes. In of the M-Per mammalian extraction reagent (Pierce, Rockford, IL). After addition, ligand-receptor docking studies using computer mod- 18 h cell lysates were measured for luciferase activity. One hundred eling were performed to analyze the interaction of glycinol with microliters of the cell extract were assayed using the Bright-glo luciferase assay substrate (Promega, Madison, WI) and determined in a AutoLumat the ER␣ and ER␤ ligand binding domains. These in vitro studies Plus lunimometer (Berthold, Bad Wildbad, Germany). support our hypothesis and demonstrate for the first time that Isolation of glycinol 2 Glycinol was isolated using a procedure developed by Qi et al. (27). Seeds from the soybean [Glycine max (L.)] cultivar Asgrow 5902 were OH O obtained from Helena Chemical Co. (Thibodaux, LA). Seeds were sur- OH face sterilized for 3 min in 70% EtOH followed by a quick deionized H2O 1.6 O rinse and two 2-min rinses in deionized H2O. Seeds were presoaked in OH sterile deionized H O for 4–5 h before placement into treatment cham- Glycinol 2 Daidzin bers (10 g/chamber). Each chamber consisted of a petri dish (100 ϫ 15 mm, four compartments), each compartment lined with two autoclaved

1.2 Glycinol filter papers (Whatman, Middlesex, UK) moistened with 0.5 ml distilled

H2O. Seeds were cut and treated with a solution of 0.01 mol/liter silver nitrate. All chambers were stored at 25 C in the dark for 3 d and then AU transferred to Ϫ70 C. Soy extracts were extracted from 10 g finely

0.8 ground seeds in 20 ml methanol. Genistin HPLC analyses were performed on a Waters 2695 combined with a Waters UV-visible 2996 photodiode array detector (Waters Associated,

Milford, MA). Isoflavones were separated using a Luna II C18 reverse-

Malonyldaidzin ϫ ␮

Malonylgenistin phase column (4.6 250 mm; 5 m; Phenomenex, Torrance, CA). A 0.4 Glyceollins guard column containing the same packing was used to protect the an- ␮ Glycitin alytical column. The injection volume of sample was 20 l with a flow rate of 1.0 ml/min with the following solvent system: A ϭ 0.1% acetic acid/water, B ϭ acetonitrile; 15% B for 8 min, then to 58% B in 50 min, 0 0 10203040506070 then to 90% B in 10 min followed by holding at 90% B for 10 min. The Retention Time (min) spectra were collected between 220 and 400 nm by photodiode array FIG. 1. HPLC chromatogram of soybean seeds after treatment with silver nitrate. detector. Predominant constitutive isoflavones are daidzin, genistin, malonyldaidzin, and Glycinol was isolated using semipreparative HPLC using a Whatman malonylgenistin. Induced isoflavone phytoalexins are glycinol and glyceollins (I, II, ODS-2 10 mm ϫ 500 mm column at a flow rate of 3.0 ml/min with the and III). AU, Absorbance units. following solvent system: A ϭ acetonitrile, B ϭ water; 5% A for 15 min, 2448 Boue´ et al. Glycinol: A Novel Phytoestrogen Endocrinology, May 2009, 150(5):2446–2453

then 5% A to 90% A in 40 min followed by holding at 90% A for 20 min. ER␣ and ER␤ binding assays Glycinol was confirmed by UV-visible spectrophotometry, mass spec- Receptor binding determinations of glycinol were achieved using the trometry, and nuclear magnetic resonance analyses. The solvents aceto- method of Bolger (30). In this method, recombinant ER is in equilibrium nitrile (HPLC grade) and methanol were purchased from Aldrich. Water with a fluorescent estrogen ligand (ES2; Panvera, Madison, WI) and a was obtained using a Millipore system and used during sample prepa- concentration of the competitor (glycinol). The relative displacement of ration procedures and HPLC analyses. the ES2 is measured as a change in polarization anisotropy. Serial dilu- tions of competitors (glycinol and estradiol) were prepared from DMSO stock solutions in screening buffer at the desired concentrations. The ER Cell culture and ES2 were combined with each competitor aliquot to a final concen- MCF-7 breast cancer cells and human embryonic kidney, HEK 293, tration of 2 nM ER and 3 nM ES2, respectively. In addition, both a cells were cultured in 150-cm2 culture flasks in DMEM supplemented no-binding control (ER ϩ ES2 only, equivalent to 0% competitor inhi- with 10% fetal bovine serum (FBS) (Life Technologies, Inc.-BRL, Gaith- bition) and a 100% binding control (only free ES2, no ER, equivalent to ersburg, MD), basal medium Eagle (BME) and MEM amino acids, L- 100% competitor inhibition) were prepared. All competitor and controls glutamine, sodium pyruvate and penicillin-streptomycin, and porcine were prepared in duplicate within a binding experiment. After a 2-h Ϫ8 insulin (10 M) (Sigma). The culture flasks were maintained in 5% CO2 incubation at room temperature, the anisotropy value for each sample at 37 C. and control were measured using the Beacon 2000 (Invitrogen, Carlsbad, CA). Anisotropy values were converted to percent inhibition using the ϭ Ϫ Ϫ ϫ following formula: I% (A0 A)/(A0 A100) 100, where I% is the Luciferase assays percent inhibition, A0 is 0% inhibition, A100 is 100% inhibition, and A MCF-7 breast cancer cells were cultured in 5% charcoal-stripped represents the observed value. This conversion to percent inhibition media overnight. Cells were plated in 24-well plates in 5% charcoal- makes the data more intuitive and normalizes the day-to-day differences stripped phenol red-free media overnight. Cells were transfected with 0.3 of multiple experiments. The percent inhibition vs. competitor concen- ␮g of ER(2)-luc plasmid (Panomics) for 6 h according to the manufac- tration curves was analyzed by nonlinear least-squares curve fitting turer’s protocol using Effectene (QIAGEN) and treated with vehicle (Prism 5.0a; GraphPad Software, San Diego CA, www.graphpad. (DMSO) or glycinol (0.01–10 ␮M) overnight. Media were removed and com) to yield IC50 values (the concentration of competitor needed to cells were lysed with reporter lysis buffer. Relative light units were mea- displace half of the bound ligand). To compare binding affinities of the sured in an Opticomp II luminometer (MGM Laboratories, Hamden, test compounds to those reported in the literature, IC50 values were CT) using luciferase reagent (Promega). converted to relative binding affinities (RBAs) using E2 as a standard. The ϭ ϫ HEK 293 cells were cultured in 5% FBS-DMEM and seeded into a E2 RBA was set equal to 100 RBA (IC50/IC50 of E2) 100. 24-well plate at a density of 50,000 cells/well and allowed to attach overnight in 5% CS-FBS. Cells were transfected with 0.2 ␮g ER(2)-luc Colony formation assay plasmid (Panomics), 0.1 ␮g pcDNA, 0.1 ␮g pcDNA3.1B-ER␤,or0.1␮g Cells were cultured in 5% FBS-DMEM and media were changed to pcDNA3.1B-ER␣ plasmids the next day using Effectene transfection phenol red-free 5% CS-DMEM 2 d before assay. Cells were seeded at a reagent (QIAGEN) according to the manufacturer’s protocol. After a 6-h density of 3000 cells/well in a six-well plate. The cells were allowed to transfection, cells were treated with compounds (DMSO, E2, glycinol, or attach overnight and treated on the following day with DMSO vehicle or ϩ fulvestrant) overnight. On the following day, the cells were lysed with 1nM E2, glycinol, or glycinol fulvestrant. Media were replaced every 150 ␮l of the M-Per mammalian extraction reagent (Pierce). After 18 h 7 d and treated with appropriate drug for 3 wk. After 3 wk the media were cell lysates were measured for luciferase activity. One hundred microli- removed and the cells were fixed with formaldehyde and dried overnight. ters of the cell extract were assayed using the Bright-glo luciferase assay The cells were then washed and stained with crystal violet and dried. The substrate (Promega) and determined in a Berthold AutoLumat Plus colonies were counted. lunimometer. Docking models of glycinol to ER␣ and ER␤ RNA extraction and semiquantitative real-time RT-PCR The glycinol used in this study possessed two chiral centers within its structure, resulting in four possible enantiomers of the compound. Of ϫ 6 2 MCF-7 cells were seeded at a density of 2 10 cells per 25 cm these, configurations found in nature were selected for ligand-receptor culture flask in phenol red containing 5% FBS-DMEM. On the following modeling (docking) studies. The SS configuration or (Ϫ)-glycinol struc- day, cells were washed in PBS and media were changed to phenol red-free ture was converted to a unique SMILE string with ChemDraw (Cam- media supplemented with 5% CS-DMEM and grown to 50–80% con- bridgeSoft, cambridgesoft.com) and then converted to a three-dimen- fluency for 48 h before treatment with DMSO vehicle, E2, glycinol, or sional structure using CONCORD (R. S. Pearlman, distributed by Tripos fulvestrant. RNA was extracted using QiaShredders (QIAGEN) and pu- International, St. Louis, MO). The initial three-dimensional model was rified on RNeasy columns (QIAGEN) according to the manufacturer’s then optimized in Sybyl 8.0 (Tripos International) using the MMFF94 protocol. RNA quality and concentration were determined by absor- force field and the conjugated gradient method with a termination of bance at 260 and 280 nm. Total RNA was reverse transcribed using the 0.005 kcal/mol. After optimization, the glycinol structure was assigned iScript kit (Bio-Rad Laboratories, Hercules, CA). The levels of ER␣, AM1 charges using MOPAC 6.0 (Colorado Springs, CO) distributed SDF-1, and PgR transcripts were determined using real-time semiquan- with Sybyl (Tripos International, St. Louis, MO). titative PCR. The primer sequences for PgR, SDF-1 and ER␣ are (sense Docking and scoring of glycinol was performed using Surflex-Dock in and antisense, respectively): PgR, 5Ј-TACCCGCCCTATCTCAAC- Sybyl 8.0. Crystal structure from the Protein Data Bank (pdb) of the Ј Ј Ј Ј ␣ TACC-3 ,5-TGCTTCATCCCCACAG-ATTAAACA-3 ; SDF-1, 5 - human ER ligand-binding domain in complex with E2 (pdb:1ERE) Ј Ј ␤ ϫ AGTCAGGTGGTGGCTTAACAG-3 ,5-AGAGGAGGTGAAGGC- and human ER ligand-binding domain in complex with E2 (pdb: 2 7R) AGTGG-3Ј; and ER␣,5Ј-GGCATGGTGGAGATCTTCGA-3Ј,5Ј-CC- were used for the docking studies. For this study, only the A chain of each TCTCCCTGCAGATTCATCA-3Ј. PCR mix contained optimal crystal structure was retained for docking. Docking preparation of the concentrations of primers, cDNA, and SYBR Green PCR master mix receptor included extraction of E2 from each of the crystal structures, (Bio-Rad). ␤-Actin, PgR, SDF-1, and ER␣ genes were amplified in trip- addition of hydrogens to the protein, and the generation of a protomol licate. Quantification and relative gene expression was calculated by surface of the binding cavity. The glycinol model was then docked into comparing PgR, SDF-1, and ER␣ relative gene expression to internal the crystal structure protein model with Surflex-Dock (default settings ␤-actin control. The ratio between these values obtained provided the with reign flexibility sampled) and the resulting 10 poses best were sorted Ϫ relative gene expression levels. by the Surflex-Dock scoring function in log10 [Kd (affinity constant)] Endocrinology, May 2009, 150(5):2446–2453 endo.endojournals.org 2449

pcDNA 2500 ERα ERβ c 1400 2000

1200 a

1500 1000 ac b a a 800 1000 600 c a Normalized RLUs

400 Normalized RLUs 500 a 200 b a a 0 0 Control 10 pM 100 nM 10 pM 10 nM 100 nM 1000 nM 1000 nM Control 100 pM E2 1 nM 10 nM 100 nM 1 uM 10 uM E2 ICI E2 + Glycinol Glycinol Glycinol Glycinol Glycinol Glycinol Glycinol Glycinol Glycinol 100 nM + 100 Treatments ICI nM ICI Treatments FIG. 3. MCF-7 ERE-luciferase reporter assay. Cells were cultured in 5% CS- DMEM and seeded into a 24-well plate and allowed to attach overnight. Cells FIG. 2. HEK 293 cells ERE-luciferase reporter assay with glycinol treatments. were transfected with ERE-luciferase plasmid the next day using Effectene Cells were cultured in 5% CS-DMEM and seeded into a 24-well plate at a density (QIAGEN) according to the manufacturer’s protocol. After a 6-h transfection, of 50,000 cells/well and allowed to attach overnight. Cells were transfected with cells were treated with compounds (DMSO or glycinol) and incubated overnight. 0.2 ␮g ERE-luciferase plasmid, and 0.1 ␮g pcDNA, 0.1 ␮gER␤ or 0.1 ␮gER␣ the On the following day, the cells were lysed with 150 ␮l of the M-Per mammalian next day using Effectene (QIAGEN) according to the manufacturer’s protocol. extraction reagent (Pierce). Luciferase activity for 100 ␮l of the cell extract was After a 6-h transfection, cells were treated with compounds (DMSO, E , glycinol 2 assayed using the Bright-glo luciferase assay substrate (Promega) and determined or ICI) and incubated overnight. On the following day, the cells were lysed with in a Berthold AutoLumat Plus lunimometer. Data are represented as relative light 150 ␮l of the M-Per mammalian extraction reagent (Pierce). Luciferase activity for units (RLUs) normalized to untreated vector control (100 Ϯ SEM), and the 100 ␮l of the cell extract was assayed using the Bright-glo luciferase assay values are the means and the SEs of triplicates from a single experiment and substrate (Promega) and determined in a Berthold AutoLumat Plus lunimometer. representative for at least two independent experiments. a, Significant difference Data are represented as relative light units (RLUs) normalized to untreated vector from vector control, P Ͻ 0.05; b, significant difference from vector control, P Ͻ control (100 Ϯ SEM), and the values are the means and the SEs of triplicates from 0.01; c, significant difference from vector control, P Ͻ 0.005, Tukey test. a single experiment and representative for at least two independent experiments. a, Significant difference from vector control, P Ͻ 0.05; b, significant difference from vector control, P Ͻ 0.01; c, significant difference from vector control, P Ͻ 0.005, Tukey test. chose to use HEK 293 cells lacking endogenous ER␣ and ER␤ for this purpose. Estrogen-responsive reporter gene assays were per- units to simulate binding affinities. The best scoring pose of glycinol formed by transiently transfecting the cells with the ERE reporter docked with the ␣- and ␤-forms of the ER were retained for this study. construct in addition to ER␣ or ER␤. Glycinol exhibited potent estrogenic activity (Fig. 2). Treatment with glycinol at 100 nM

displayed moderate activity equivalent to 35% of E2 (1 nM) (Fig. Results 2). However, at 1000 nM, estrogenic activity increased to 90% of E2 (1 nM), which was blocked by the ER down-regulator, ful- Isolation of glycinol vestrant, suggesting an ER-dependent mechanism. When com- Several constitutive isoflavones are responsible for the estro- pared with glycinol-treated HEK 293 cells transfected with ER␣, genic activity observed in soy, including daidzein, genistein, and ER␤-transfected cells exhibited a higher response. We then used glycitein. These isoflavones, as well as coumestrol and other fla- the MCF-7 breast cancer cells to further examine the estroge- vonoids, predominantly act as estrogenic chemicals but may also nicity of glycinol in a system endogenously expressing both ERs. exhibit antiestrogenic activity in a dose-dependent manner (17, ER-positive MCF-7 cells were also used to examine the potential 18, 31). We have shown that under stress conditions or elicitor of glycinol to induce the ER-mediated transcriptional activity. treatment, the concentrations of the constitutive isoflavones in- Glycinol dose-response studies were performed, and doses rang- crease and several isoflavone phytoalexins are produced. Our ing from 100 nM to 10 ␮M enhanced the ER-mediated transcrip- previous results showed that several soy isoflavone phytoalexins tional activity to levels that were 250–750% above the control, are induced with elicitor treatments (19, 32). Coumestrol, the whereas lower doses did not have any effect on activity (Fig. 3). glyceollins, and several other phytoalexins are induced to higher concentrations as a result of secondary metabolism within the Regulation of ER␣, SDF-1, and PgR mRNA levels after plant tissue. These studies resulted in the identification of the glycinol treatment induced pterocarpan glycinol shown in Fig. 1. The isolated com- Three differentially expressed genes were selected for analysis pound was identified as glycinol by comparison with its spec- of expression levels by semiquantitative real-time RT-PCR: ER␣, troscopic data [1H and 13C nuclear magnetic resonance; atmo- SDF-1, and PgR. We chose to examine ER␣ mRNA levels as a spheric pressure chemical ionization (APCI) and electron control. SDF-1 and PgR, both estrogen-responsive genes, which ionization mass spectrometry (EI MS)] with those in the litera- have been shown to have functions that are associated with cell ture (24, 25, 27). proliferation and/or tumorigenesis. SDF-1 has been identified as a novel ER-regulated gene involved in metastasis and a mediator Effect of glycinol on ER transcriptional activation of the mitogenic effects of estrogen in ovarian and breast cancer Based on the similarity of the chemical structure of glycinol to cells (33). In the present study, we examined the effect of glycinol other isoflavones, ERE-based reporter assays were performed to treatment on the mRNA expression of SDF-1, ER␣, and PgR in examine the potential estrogenicity of glycinol. Initially, we MCF-7 cells. 2450 Boue´ et al. Glycinol: A Novel Phytoestrogen Endocrinology, May 2009, 150(5):2446–2453

ER α Glycinol ER-α and ER-β competitive binding assay PgR SDF-1 180 -10 a 160 10 140

120 30

100 50 80

60 70

Relative Gene Expression 40

% Bound inhibitor % Bound Estrogen a 90 Glycinol ER-α 20 Glycinol ER-β a a 0 110 Control 1 nM E2 0.01 uM 0.1 uM 1 uM Glycinol 1 uM Glycinol Glycinol Glycinol + 100 nM ICI -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 Treatments Log [Mol] FIG. 4. ER␣, PgR, and SDF-1 gene expression in MCF-7 breast cancer cells. FIG. 5. Competitive binding curves of glycinol and ER␣/ER␤. Increasing Total R⌵A was isolated from MCF-7 cells, reverse transcribed into cDNA, and concentrations of glycinol were added to ER␣ or ER␤ complex and compared subjected to real-time PCR analysis for quantification. Treatment was as follows: with E . Data points and error bars represent the mean Ϯ SEM of three Ϯ 2 DMSO vehicle control, 1 nM E2, glycinol fulvestrant. The values are the means independent experiments for glycinol and E2 treatment for each concentration and the SEs of triplicates from a single experiment and representative for at least tested. two independent experiments. a, Significant difference from vector control, P Ͻ 0.05; b, significant difference from vector control, P Ͻ 0.01; c, significant difference from vector control, P Ͻ 0.005, Tukey test. Glycinol induces colony formation in MCF-7 cells Upon establishing the ability of glycinol to bind both ER␣ and ER␤ with high affinity, we then examined the biology of glycinol To determine the effect of glycinol on expression of PgR, on cell viability and proliferation using the colony formation SDF-1, and ER␣ genes, MCF-7 cells were treated with glycinol assay (Fig. 6). MCF-7 cells were treated with 1 nM E2, glycinol ␮ ␣ at varying concentrations (0.01–1 M) for 4 h (Fig. 4). ER (0.01–1 ␮M), and 1 ␮M glycinol ϩ 100 nM fulvestrant, a classical mRNA expression remained relatively unchanged with all treat- ER down-regulator. Cells were treated once a week and colonies ments, suggesting that the effects observed by glycinol are not formed after 5 wk of treatment. Upon formation of colonies, cells due to changes in ER␣ mRNA expression. However, estrogen were fixed, stained, and counted. MCF-7 cells treated with ve- induced PgR and SDF-1 gene expression to levels greater than hicle exhibited few colonies; however, treatment with 1 nM E2 100- and 15-fold (respectively) above the control. Glycinol treat- treatment enhanced the number of colonies greater than 600% ments (0.01–1 ␮M) elicited a dose-dependent increase in SDF-1 above the control. Cells treated with glycinol exhibited a dose- expression, which ranged from 6- to 39-fold above the control, dependent increase in colony formation reaching a maximum of whereas PgR gene expression increased from 4- to 18-fold above approximately 300% above control levels. In addition to an the control. As expected, fulvestrant at 100 nM effectively inhib- ited the up-regulation of PgR and SDF-1 expression by glycinol A indicating a direct ER effect. 1000 900 a 800

700

␣ ␤ 600 a Competitive binding of glycinol to ER and ER a 500 Several studies have demonstrated that phytoestrogens exert 400 a their stimulatory effect on the ER by binding to the same site as colonies of Number 300 200 E2 with some differences in ligand binding specificity and trans- 100 ␣ ␤ 0 activation between ER and ER (34–37). Of particular interest DMSO 1 nM E2 10 nM Glycinol 100 nM 1 uM Glycinol 1 uM Glycinol + Glycinol 100 nM ICI was the observation that certain isoflavones may bind with Treatment higher affinity and possess higher agonistic activity toward ER␤ B (35–37). Additionally we found that glycinol induces a greater ER␤ transcriptional response compared with ER␣ in ERE tran- scriptional reporter assays (Fig. 2). Therefore, we chose to assess Control 1 nM Estradiol 10 nM Glycinol the ability of glycinol to bind ER␣ and ER␤ using a competitive binding assay with fluorescent detection. Figure 5 details the results for the competitive binding assay. Glycinol produced a displacement of E bound ER␣ (50%), which occurred at a con- 2 100 nM Glycinol 1 µM Glycinol 1 µM Glycinol ␣ 100 nM ICI centration of 2.46 nM. The IC50 of glycinol for ER was 13.75 FIG. 6. A, Effects of glycinol on colony formation on MCF-7 cells placed in nM. However, the IC of glycinol for ER␤ was 9.05 nM. This 50 phenol red-free DMEM supplemented with 5% dextran-coated charcoal-treated indicated that the ability of glycinol to act as an ER agonist FBS for 48 h before plating. Then 3000 cells/well were plated in six-well plates. occurred through receptor binding, and only slightly greater af- Forty-eight hours later, cells were treated with glycinol or vehicle. B, The picture finity for ER␤ vs. ER␣ was observed, which may account in part is a representative of the best of three experiments. The values are the means ␤ and the SEs of triplicates from a single experiment and representative for at least for the greater glycinol-induced ER transcriptional response in two independent experiments. a, Significant difference from vector control, P Ͻ HEK 293 cells. 0.05; Tukey test. Endocrinology, May 2009, 150(5):2446–2453 endo.endojournals.org 2451

Discussion

Soy is an excellent source of the constitutive isofla- vones daidzein, genistein, and glycitein. Numerous studies have been conducted detailing the estrogenic and antiestrogenic activities of isoflavones and their many health-promoting properties (1–10). Isofla- vones mimic the shape and polarity of the steroid hormone estradiol, and their structural similarity leads to the competitive binding of isoflavones with estradiol for the ERs. Recently after concerns were FIG. 7. Glycinol docked to ER␣ and ER␤. A, Glycinol bound to ER␣ with endogenous estrogen, raised regarding the risks of hormone therapy, there E2. Conserved amino acids arginine (ARG) 394 and glutamate (GLU) 353 (left) with a water molecule hydrogen binds to glycinol, whereas histidine (HIS) 524 (right) hydrogen bonds to a has been increased interest in identifying new natural ␣ hydroxyl of glycinol. E2 is pictured in magenta as found within the crystal structure of ER , plant-derived agents to treat menopausal symptoms. ␤ and glycinol is atom colored. B, Glycinol bound to ER with endogenous estrogen E2. Amino This research has led to the discovery of many new acids arginine 301 and glutamate 260 (left) with a water molecule hydrogen binds to glycinol, estrogenic and antiestrogenic natural compounds whereas histidine 430 (right) hydrogen bonds to a hydroxyl of glycinol. E2 is pictured in magenta as found within the crystal structure of ER␤, and glycinol is atom colored. derived from soy and other plants. The induced soy glyceollins displayed antiestrogenic activity in both in vitro and in vivo experiments using both breast increase in colony formation, there was a difference in the mor- and ovarian cancer cells (19–21). The discovery of an inducible phology, size, and staining intensity of the glycinol-treated col- isoflavone with selective ER modulator activity has led to the onies compared with the estrogen-treated colonies. Glycinol- identification of a new soy phytoalexin, glycinol with estrogenic treated cells stained more intensely and exhibited an increase in activity. Glycinol accumulates in high concentrations in soybean colony size compared with estrogen. Furthermore, the combi- under conditions of stress or elicitor treatment. However, little is nation of glycinol plus fulvestrant led to a marked decrease in known about the hormonal effects of glycinol in mammalian colony formation compared with glycinol alone. systems. Therefore, glycinol was examined in a variety of hor- mone-responsive systems and, in contrast to the glyceollins, dem- Glycinol docks to ER␣ and ER␤ similarly onstrated potent estrogenic effects in each system. Evaluation of the ligand-receptor docking and scoring of gly- Studies with MCF-7 cells showed glycinol-induced gene cinol to both the ER␣ and ER␤ ligand binding domains illustrates transactivation and proliferation when administered in a dose- that glycinol can bind similarly to each of these receptor subtypes dependent manner. Glycinol induced a high level of ER trans- ␮ in a binding interaction that might involve fewer hydrogen bond activation between 100 nM and 10 M, and significant MCF-7 cell proliferation was observed at 1 ␮M. Compared with earlier interactions than E2 (Fig. 7). A Connolly channel, colored blue, which represents the surface of the binding cavity of the ER was results, this observed estrogenic activity is greater than that ob- constructed, and only the three essential amino acids within the served with daidzein and genistein and slightly lower than that binding cavity are presented. In Fig. 7A, these are arginine 394 observed for coumestrol (19). and glutamate 353 (left panel), with histidine 524 [right panel, Consistent with previous results, genistein, coumestrol, and from pdb:1ERE (ER␣)]. At the same time [Fig. 7B, pdb:2ϫ7R daidzein demonstrated a dose-dependent activation of estrogen (ER␤)], the same conserved amino acids are arginine 301 and response in MCF-7 cells (19), with coumestrol showing the glutamate 260 (Fig. 7, left panel), with histidine 430 (Fig. 7, right greatest activity (90% at 100 nM) followed by genistein (110% panel). Within Fig. 7A, it is possible to see glycinol makes the at 1 ␮M) and daidzein (150% at 10 ␮M). same interactions with glutamate 353 and histidine 524 of ER␣ Genistein binds both ER␣ and ER␤; however, it preferentially ␤ ␣ as does E2. However, this simulation suggests that glycinol may binds ER . Its binding affinity to ER is only 4% compared with ␤ not interact with arginine 394 or the water molecule within the E2, whereas the affinity to ER is 87% (38). The relative affin- binding cavity. Figure 7B represents how glycinol can make the ities of glycinol for ER␣ and ER␤ were calculated by dividing the

same interactions with glutamate 260 and histidine 430 of ER␤ IC50 of unlabeled E2 by the IC50 of glycinol and then multi-

as does E2, whereas interactions with arginine 301 and the water plying by 100 (E2 arbitrarily set to 100%). The relative bind- molecule are excluded as observed in the ER␣ docking model. ing affinity of glycinol calculated was 27.2% for ER␤ and The docking simulations suggest that glycinol may bind the ER␣ 17.9% for ER␣. This relative binding affinity is comparable and ER␤ with different ligand conformations. The ER␣ docking with the binding affinity of coumestrol. In earlier work by pose maintains the more flat glycinol conformation, whereas the Nikov et al. (39), the relative binding affinity of coumestrol best ER␤ docking pose takes on the bent conformation. Thus, calculated was 34% for ER␤ and 12% for ER␣. The binding glycinol docking simulations result in a slightly higher interac- affinity of another isoflavone, genistein, has even greater af- tion score for ER␣ binding (5.29) than for ER␤ binding (4.63). finity for ER␤. If realistic, these receptor subtype-specific binding modes may Several phytoalexins containing the pterocarpan structure are contribute to the subtype-specific activity observed in cells. synthesized by soybean tissues when treated with elicitors or 2452 Boue´ et al. Glycinol: A Novel Phytoestrogen Endocrinology, May 2009, 150(5):2446–2453 during stress and insect damage. Glyceollins I, II, and III in ad- Acknowledgments dition to smaller amounts of glyceollin IV, glyceollidin I, and glyceollidin II (glyceocarpin) are used for plant defense. We have Address all correspondence and requests for reprints to: Stephen Boue, Southern Regional Research Center, United States Department of Agri- shown that glyceollins I, II, and III all bind to the ER and display culture, New Orleans, Louisiana 70124. E-mail: sboue@ srrc.ars.usda.gov. antagonist activity using both in vitro and in vivo assays in the This work was supported by the National Institutes of Health (NIH) presence of E2. Further research in our laboratory has deter- Digestive Kidney Disease Research Grant DK 059389 (to M.E.B), Susan mined that glyceollin I is the most active antagonist among the G. Komen Breast Cancer Foundation (BCTR0601198) to M.E.B., NIH Research Supplements to Promote Diversity in Health-Related Research three glyceollin isomers tested. Glycinol also accumulates in elic- (to M.E.B. and S.L.T.), NIH National Research Service Award Training itor-treated soybean and is a precursor in the biosynthetic path- Grant T32-HL-07973-05 (to S.L.T), Office of Naval Research N00014- way of all the glyceollins. Glycinol is derived from daidzein via 06-1-1136 (to J.A.M. and M.E.B.), and United States Department of a pterocarpan by cyclization and 6␣-hydroxylation with re- Agriculture 59-6435-7-188 (to J.A.M. and M.E.B.). Disclosure Summary: The authors have nothing to disclose. tention of configuration (18, 40). For the synthesis of the glyceollins, glycinol is prenylated to produce glyceollidin I and II, followed by cyclization to the corresponding glyceollin (41). The prenylation and cyclization steps are key in con- References verting the estrogenic pterocarpan glycinol into the antiestro- genic pterocarpans glyceollins. These structural changes af- 1. Adlercreutz H 1990 Western diet and Western diseases: some hormonal and biochemical mechanisms and associations. Scand J Clin Lab Invest 50:3–23 fect the binding within the ER pocket, and our modeling data 2. Wu AH, Ziegler RG, Horn-Ross PL, Nomura AM, West DW, Kolonel LN, present ligand receptor models to describe this difference in Rosenthal JF, Hoover RN, Pike MC 1996 Tofu and risk of breast cancer in Asian-Americans. Cancer Epidemiol Biomarkers Prev 5:901–906 activity. 3. Fournier DB, Erdman Jr JW, Gordon GB 1998 Soy, its components, and cancer Other isoflavonoids are inducibly formed and synthesized in prevention: a review of the in vitro, animal and human data. Cancer Epidemiol greater amounts by stressed soy, including daidzein, isofor- Biomarkers Prev 7:1055–1065 4. Baird DD, Umbach DM, Lansdell L, Hughes CL, Setchell KD, Weinberg CR, mononetin, glyceofuran, and coumestrol. Coumestrol is a Haney AF, Wilcox AJ, McLachlan JA 1995 Dietary intervention study to assess coumestan-derived phytoalexin present in several legumes, cer- estrogenicity of dietary soy among postmenopausal women. J Clin Endocrinol tain foods, and forages (31, 42–45). Coumestrol has been well Metab 80:1685–1690 5. Adlercreutz H 1995 Phytoestrogens: epidemiology and a possible role in cancer characterized for its estrogenic activity (46–49) because it in- protection. In: McLachlan JA, Korach KS, eds. Estrogens in the environment, teracts with ERs in vertebrates and acts as an ER agonist (49, 50). III: global health implications. Environ Health Perspect 103(Suppl 7):103–112 ␣ 6. Tham DM, Gardner CD, Haskell WL 1998 Clinical review 97: potential health The IC50 value of coumestrol is 109 nM for ER and 35 nM for benefits of dietary phytoestrogens: a review of the clinical, epidemiological, ␤ ER (39). These IC50 values are higher than those of glycinol: and mechanistic evidence. J Clin Endocrinol Metab 83:2223–2235 7. Murkies AL, Wilcox G, Davis SR 1998 Clinical review 92: phytoestrogens. 13.75 nM for ER␣ and 9.05 nM for ER␤, suggesting glycinol J Clin Endocrinol Metab 83:297–303 binds ER␣ and ER␤ with higher affinity compared with 8. Cline JM, Hughes CL 1998 Phytochemicals for the prevention of breast and coumestrol. endometrial cancer. In: Foon KA, Muss HB, eds. Biological and hormonal therapies of cancer. Boston: Klewer Academic Publishers; 107–134 The ability of glycinol to increase the expression of an ERE- 9. Humfrey CDN 1998 Phytoestrogens and human health effects: weighing up LUC reporter gene in MCF-7 cells suggests that it may exert its the current evidence. Nat Toxins 6:51–59 estrogenic activity, at least in part, via an ERE-dependent mech- 10. Bingham SA, Atkinson C, Liggins J, Bluck L, Coward A 1998 Phyto-estrogens: where are we now? Br J Nutr 79:393–406 anism. In this context, we confirmed that glycinol up-regulates 11. Coward L, Smith M, Kirk M, Barnes S 1998 Chemical modification of isofla- endogenous E2-responsive gene transcription, i.e. SDF-1 and vones in soyfoods during cooking and processing. Am J Clin Nutr 68:1486S– PgR in a dose-dependent manner. Glycinol’s ability to up-regu- 1491S 12. Dakora FD, Phillips DA 1996 Diverse functions of isoflavonoids in legumes late gene transcription was inhibited by the pure ER antagonist transcend anti-microbial definitions of phytoalexins. Physiol Mol Plant fulvestrant and colony assays demonstrated that fulvestrant in- Physiol 49:1–20 13. Darvill AG, Albersheim P 1984 Phytoalexins and their elcitors—a defense hibited the estrogenic effects of glycinol. It was therefore con- against microbial infection in plants. Ann Rev Plant Physiol 35:243–275 cluded that the agonist effects of glycinol were mediated primar- 14. Graham TL, Kim JE, Graham MY 1990 Role of constitutive isoflavone con- ily through ER. jugates in the accumulation of glyceollin in soybean infected with Phytoph- ␣ ␤ thora megasperma. Mol Plant Microbe Interact 3:157–166 When glycinol is docked to ER or ER and compared with 15. Paxton JD 1991 Biosynthesis and accumulation of legume phytoalexins in Mycotoxins and Phytoalexins. In: Sharma RP, Salunkhe DK, eds. Boca Raton, E2, it does fit with the binding cavity and can interact with key residues. However, these models suggest that glycinol does not FL: CRC Press; 485–499 16. Rivera-Vargas LI, Schmitthenner AF, Graham TL 1993 Soybean flavonoid share the same hydrogen bonding network as E2. The docking effects on and metabolism by Phytophthora sojae. Phytochem 32:851–857 simulations also suggest that glycinol may bind ER␣ and ER␤ 17. Graham TL, Graham MY 1991 Glyceollin elicitors induce major but distinctly different shifts in isoflavonoid metabolism in proximal and distal soybean cell with different ligand ring conformations that this differential populations. Mol Plant Microbe Interact 4:60–68 binding mode may be the basis for ER subtype-specific effects 18. Banks SW, Dewick PM 1983 Biosynthesis of glyceollins I, II, and III in soybean. observed in cells. Taken together these results demonstrate for Phytochemistry 22):2729–2733 19. Burow ME, Boue´ SM, Collins-Burow BM, Melnik LI, Duong BN, Carter- the first time the isolation of a novel phytoalexin. We demon- Wientjes CH, Li S, Wiese TE, Cleveland TE, McLachlan JA 2001 Phytochem- strate glycinol to be estrogenic in breast cancer cells and may ical glyceollins, isolated from soy, mediate antihormonal effects through es- trogen receptor ␣ and ␤. J Endocrinol 86:1750–1758 prove beneficial for patients who may require hormone replace- 20. Salvo VA, Boue´ SM, Fonseca JP, Elliott S, Corbitt C, Collins-Burow BM, ment therapy. Curiel TJ, Srivastav SK, Shih BY, Carter-Wientjes C, Wood CE, Erhardt PW, Endocrinology, May 2009, 150(5):2446–2453 endo.endojournals.org 2453

Beckman BS, McLachlan JA, Cleveland TE, Burow ME 2006 Antiestrogenic Gustafsson JA 1997 Comparison of the ligand binding specificity and tran- glyceollins suppress human breast and ovarian carcinoma tumorigenesis. Clin script distribution of estrogen receptors ␣ and ␤. Endocrinology 138:863–870 Cancer Res 12:7159–7164 36. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, Nilsson S 1998 21. Wood CE, Clarkson TB, Appt SE, Franke AA, Boue´SM, Burow ME, McCoy Differential response of estrogen receptor ␣ and estrogen receptor ␤ to partial T, Cline JM 2006 Effects of soybean glyceollins and estradiol on postmeno- estrogen agonists/antagonists. Mol Pharmacol 54:105–112 pausal female monkeys. Nutr Cancer 56:74–81 37. McInerney EM, Weis KE, Sun J, Mosselmam S, Katzenellenbogen BS 1998 22. Moesta P, Hahn MG, Grisebach H 1983 Development of a radioimmunoassay Transcription activation by the human estrogen receptor subtype ␤ (ER␤) for the soybean phytoalexin glyceollin I. Plant Physiol 73:233–237 studied with ER␤ and ER␣ receptor chimeras. Endocrinology 139:4513–4522 23. Garcez WS, Martins D, Garcez FR, Marques MR, Pereira AA, Oliveira LA, 38. Wolters M, Hahn A 2004 Soy isoflavones—a therapy for menopausal symp- Rondon JN, Peruca AD 2000 Effect of spores of saprophytic fungi on phy- toms? Wien Med Wochenschr 154:334–341 toalexin accumulation in seeds of frog-eye leaf spot and stem canker-resistant 39. Nikov G, Hopkins NE, Boue S, Alworth W 2000 Interactions of dietary es- and -susceptible soybean (Glycine max L.) cultivars. J Agric Food Chem 48: trogens with human estrogen receptors and the effect of estrogen receptor- 3662–3665 estrogen response element complex formation. Environ Health Perspect 108: 24. Weinstein LI, Hahn MG, Albersheim P 1981 Host-Pathogen Interactions 867–372 XVIII. Isolation and biological activity of glycinol, a pterocarpan phytoalexin 40. Hagmann ML, Heller W, Grisebach H 1984 Induction of phytoalexin syn- synthesized by soybeans. Plant Physiol 69:358–363 thesis in soybean. Stereospecific 3,9-dihydropterocarpan 6a-hydrolase from 25. Lyne RL, Mulheirn LH 1978 Minor pterocarpinoids of soybean. Tetrahedron elicitor-induced soybean cell cultures. Eur J Biochem 142:127–131 Lett 34:3127–3128 41. Za¨hringer U, Schaller E, Grisebach H 1981 Induction of phytoalexin synthesis 26. Weinstein LI, Albersheim P 1983 Host-Pathogen Interactions XXIII. The in soybean. Structure and reactions of naturally occurring and enzymatically mechanism of the antibacterial action of glycinol, a pterocarpan phytoalexin prepared prenylated pterocarpans from elicitor-treated cotyledons and cell synthesized by soybeans. Plant Physiol 72:557–563 cultures of soybean. Z. Naturforsch 36:234–241 27. Qi Y, Moco S, Boeren S, de Vos C, Bovy A 2005 Isolation and identification 42. Oldfield JE, Fox CW, Bahn AV, Bickoff EM, Kohler GO 1966 Coumestrol in of glycinol form glycine max [L.] Merri. Chin J Chromatogr 23:353–357 alfalfa as a factor in growth and carcass quality in lambs. J Anim Sci 25:167– 28. Burow ME, Tang Y, Collins-Burow BM, Krajewski S, Reed JC, McLachlan 174 JA, Beckman BS 1999 Effects of environmental estrogens on tumor necrosis 43. Wong E, Latch GCM 1971 Coumestans in diseased white clover. Phytochem- factor ␣-mediated apoptosis in MCF-7 cells. Carcinogenesis 20:2057–2061 istry 10:466–468 29. Klotz DM, Beckman BS, Hill SM, McLachlan JA, Walters MR, Arnold SF 44. O’Neil M, Adesnaya SA, Roberts MF, Pantry I 1986 Inducible isoflavonoids 1996 Identification of environmental chemicals with estrogenic activity using from the lima bean, Phaseolus lunatus. Phytochemistry 25:1315–1332 a combination of in vitro assays. Environ Health Perspect 104:84–89 45. Gutendorf B, Westendorf J 2001 Comparison of an array of in vitro assays for 30. Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W 1998 Rapid screening the assessment of the estrogenic potential of natural and synthetic estrogens, of environmental chemicals for estrogen receptor binding capacity. Environ phytoestrogens and xenoestrogens. Toxicology 166:79–89 Health Perspect 106:551–557 46. Bickoff EM, Livingston AL, Hendrickson AP, Booth AN 1962 Relative po- 31. Dewick PM, Barz W, Griesbach H 1970 Biosynthesis of coumestrol in Phaseo- tencies of several estrogen-like compounds found in forages. J Agric Food lus aureus. Phytochemistry 9:775–783 Chem 10:410–412 32. Boue´SM, Carter CH, Ehrlich KC, Cleveland TE 2000 Induction of the soybean 47. Dodge JA, Glaesbbrook AL, Magee DE, Philips DL, Sato M, Short L, Bryant phytoalexins coumestrol and glyceollin by Aspergillus. J Agric Food Chem HU 1996 Environmental estrogens: effect of cholesterol lowering and bone in 48:2167–2172 the ovariectomized rat. J Steroid Biochem Mol Biol 59:155–161 33. Hall JM, Korach KS 2003 Stromal cell-derived factor 1, a novel target of 48. Martin PM, Horwitz KB, Ryan DS, McGuire WL 1978 Phytoestrogen inter- estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian action with estrogen receptors in human breast cancer cells. Endocrinology and breast cancer cells. Mol Endocrinol 17:792–803 103:1860–1867 34. Collins-Burow BM, Burow ME, Duong BN, McLachlan JA 2000 The estro- 49. Whitten PL, Patisaul HB, Young LJ 2002 Neurobehavioral actions of coumes- genic and antiestrogenic activities of flavonoid phytochemicals through estro- trol and related flavonoids in rodents. Neurotoxicol Teratol 24:47–54 gen receptor binding dependent and independent mechanisms. Nutr Cancer 50. Markaverich BM, Webb B, Densmore CL, Gregory RR 1995 Effects of 38:229–244 coumestrol on estrogen receptor function and uterine growth in ovariecto- 35. Kuiper GG, Carlsson B, Grandien K, Enmark E, Ha¨ggblad J, Nilsson S, mized rats. Environ Health Perspect 103:574–581