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Carcinogenesis Advance Access originally published online on January 6, 2006
Carcinogenesis 2006 27(4):856-863; doi:10.1093/carcin/bgi320
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice

Young H. Ju 1, 3, Jodi Fultz 1, Kimberly F. Allred 1, 4, Daniel R. Doerge 2 and William G. Helferich 1, *

1 Department of Food Science and Human Nutrition, University of Illinois, 905 S Goodwin, Room 580 Bevier Hall, Urbana, IL 61801, USA
2 National Center for Toxicological Research, Jefferson, AR 72079, USA
3 Present address: 325 Wallace Hall Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
4 Present address: Department of Physiology, University of Kentucky, Lexington, KY 40536, USA

* To whom correspondence shoule be addressed at: Department of Food Science and Human Nutrition, University of Illinois, 905 S Goodwin, Room 580 Bevier Hall, Urbana, IL 61801, USA. Tel: +1 217 244 5414; Email: helferic{at}uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genistein and daidzein are the main isoflavones in legumes. Equol is an intestinal bacterial metabolite of daidzein. In this study, we evaluated the estrogenic potential of daidzein and synthetic (±)-equol to stimulate growth of estrogen-dependent breast cancer (MCF-7) in vitro and in vivo. We hypothesize that estrogenic effects of daidzein and (±)-equol could modulate the growth of MCF-7 cells both in vitro and also once implanted into ovariectomized athymic mice. At concentrations between 0.001 and 50 µM, daidzein and (±)-equol stimulated the growth of MCF-7 cells with maximal stimulation at 1 µM in vitro. To evaluate their effects on the growth of MCF-7 cells implanted in ovariectomized athymic mice, two dietary dose–response studies [daidzein (125, 250, 500 and 1000 p.p.m.) and (±)-equol (250, 500 and 1000 p.p.m.)] were conducted. Tumor size and body weight were monitored weekly during the study. At completion of the study, we analyzed cellular proliferation of tumors using immunohistochemical staining (ki-67), pS2 expression in tumors using a real time quantitative reverse transcription–polymerase chain reaction (qRT–PCR), and total daidzein and (±)-equol levels in plasma using liquid chromatography–electrospray tandem mass spectrometry (LC–ES/MS/MS). Dietary daidzein had a slight but significant stimulatory effect on MCF-7 tumor growth in mice. No significant induction of pS2 mRNA (an estrogen-responsive marker) in tumors by dietary daidzein was observed. Total plasma daidzein concentrations in plasma were between 0.25 and 1.52 µM. Dietary equol treatment (for 37 weeks) did not stimulate MCF-7 tumor growth. There were no statistical differences in tumor size, proliferation and pS2 expression among any treatment groups. Total equol concentrations in plasma were 2.10–3.21 µM. In conclusion, daidzein and (±)-equol have proliferative effects on MCF-7 cell growth in vitro within the concentration range tested. Dietary daidzein had a slight but significant stimulatory effect on tumor growth, whereas (±)-equol did not stimulate the growth of estrogen-dependent breast tumor growth in athymic mice, increase the cell proliferation in tumors, or induce an estrogen-responsive pS2 expression. Total daidzein or (±)-equol plasma levels in mice fed the isoflavones were in the range that stimulated MCF-7 cell growth in vitro. These results suggest that pharmacokinetic and/or metabolic factors attenuate the estrogenic effects of daidzein and equol in vivo.

Abbreviations: AIN93G, American Institute of Nutrition 93 growth semi-purified diet; BCS, bovine calf serum; CD-BCS, charcoal-dextran stripped BCS; E2, 17ß-estradiol; ER, estrogen receptor; LC-ES/MS–MS, liquid chromatography–electrospray/tandem mass spectrometry; MCF-7, Michigan Cancer Foundation-7; PBS, phosphate buffered saline; MEM, minimal essential media; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; qRT–PCR, real time quantitative reverse transcription polymerase chain reaction; SEM, standard error of mean.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Dietary isoflavones are abundant in legumes, particularly in soy. Genistein and daidzein, the principal soy isoflavones, have attracted attention because of their putative value as natural alternatives to hormone replacement therapy (HRT) for relieving post-menopausal symptoms (1). Although many studies have been conducted to understand the effects of soy isoflavones on breast cancer during various stages of life, the results are inconsistent and the mechanism of action is still unclear. Our previous studies in animals have shown the possibility that genistein stimulates estrogen-dependent human breast tumor growth (2–4) and negates the effectiveness of tamoxifen treatment (5).

Daidzein is the second-most prominent isoflavone in soy products. After ingestion, daidzein is converted by the gut microflora into dihydrodaidzein, S-(–)-equol (70%) and O-desmethylangolensin (5–20%) (6–10). Equol was first isolated from the urine of pregnant mares in 1932 (11) and was subsequently identified in the plasma of sheep (derived from formononetin in red clover) (12) and in the urine of human (from daidzein) (13). Equol is consistently observed in high amounts in blood from experimental animals, including rodents and monkeys. However, equol is observed in only ~35% of humans, presumably because of differences in the composition of gut microflora (14–18). It has been shown that the amount of urinary equol excretion is correlated to the consumption of soy products (14,19,20) and a reduced risk of breast cancer (21–23).

Equol is a chiral molecule that can exist as the R-(+)- and S-(–)-enantiomers and previous work suggested that both enantiomers were more estrogenic in vitro than daidzein (24–26). Daidzein acts as an agonist for both ER{alpha} and ERß and induces ER-mediated estrogenic responses both in vitro (27) and in vivo (28–32). Daidzein has much lower affinity than genistein for binding to and transactivation through ER{alpha} and ERß (26). In mice, genistein and equol are uterotrophic, but daidzein is not (33–35). The use of soy products and isoflavone supplements by women with breast cancer is increasing to treat cancer-related symptoms as well as to relieve menopausal symptoms, and to enhance general well-being based on their estrogenic properties (36). Isoflavone levels in dietary supplements vary and can contain over 100 mg per serving. Major isoflavone components in dietary supplements are biochanin A, genistin, and genistein, and formononetin, daidzin and daidzein (17). The dietary effect of equol has not been studied apart from the other isoflavones, principally because there is no commercial source for sufficient quantities. The significant binding affinity and effective transactivation activity of synthetic racemic (±)-equol on the ER subtypes and the association of the excretion of equol with effects on breast cancer risk prompted the current study. In this study, we tested the hypothesis that daidzein and equol have biological effects on the growth of estrogen-dependent human breast cancer cells in vitro and after implantation into athymic mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Daidzein was purchased from Indofine (Somerville, NJ) and Plantech (Reading, UK) supplied dihydrodaidzein. Racemic equol (purity ~98% as determined by 1H-NMR and LC/MS, data not shown) was prepared as described by Muthyala et al. (37). Minimal Essential Medium (MEM, without gentamicin, with glutamine) and phenol red-free MEM was purchased from the Media Facility at University of Illinois at Urbana-Champaign. Bovine calf serum (BCS) was purchased from Hyclone (Logan, UT). Penicillin/streptomycin and trypsin/EDTA were purchased from Invitrogen (Houston, TX). Laboratory animal diet and dietary components were purchased from Dyets (Bethlehem, PA). Reagents for qRT–PCR were purchased from PE Applied Biosystems (Foster City, CA), Synthegen (Houston, TX) and Invitrogen (Carlsbad, CA).

Human breast cancer cell maintenance
MCF-7 cells are estrogen-dependent tumor cells isolated from a post-menopausal woman (38). MCF-7 cells were maintained in MEM supplemented with 5% BCS, 1% penicillin (100 U/ml)/streptomycin (100 µg/ml) and 1 nM 17ß-estradiol (E2). MCF-7 cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air as a monolayer culture in plastic culture plates (100 mm diameter). One week before the cell proliferation assay or the injection of MCF-7 cells into athymic mice, the medium was switched to phenol red-free MEM containing 5% charcoal dextran stripped (CD)-BCS (2) and 1% penicillin/strepomycin.

Cell proliferation
To compare the effects of physiological and phamacological dosage of isoflavones on MCF-7 cell proliferation, a modified colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (39) was used. Mitochondrial dehydrogenase activity was measured by MTT. MCF-7 cells were grown in estrogen-free culture media (phenol red-free MEM containing 5% CD–BCS and 1% penicillin/streptomycin) for 2 weeks prior to the treatment. For the growth experiments, MCF-7 cells (1.5 x 104) were inoculated in triplicate in 1 ml of estrogen-free culture media in a 24-well polystyrene culture plate. After 24 h, the MCF-7 cells in each well were washed with 1 ml of phosphate-buffered saline (150 mM PBS, pH 7.4, without Ca2+ and Mg2+) and treated with various concentrations (0.001, 0.01, 0.1, 1, 5, 10 and 50 µM) of daidzein or (±)-equol every 48 h. After the 5 day treatment, the cells were treated with MTT for 5 h and treated with 10% SDS (in 0.01 N HCl) for 12–18 h. Optical density (OD) was measured at 570 nm. OD values were normalized to number of cells based on a standard curve. Daidzein and (±)-equol were also evaluated in the presence of ICI 182 780 (100 nM).

Athymic nude mice
Female athymic BALB/c (nude) mice were purchased from Charles River Laboratories (Wilmington, MA) and acclimated for a week. The xenograft model used in this study, ovariectomized athymic mice implanted with MCF-7 cells, is an appropriate model of post-menopausal women with estrogen-dependent breast cancer. Mice were ovariectomized at 21 -day of age by the vendor and the allowed to recover for 7 day. During the study, the mice were maintained under the standard light/dark cycle (12 h light, 12 h dark).

E2 pellet preparation
An E2 pellet contained 2 mg of E2 and 18 mg of cholesterol. An E2 pellet was placed subcutaneously under the skin of each mouse before MCF-7 cells were injected into the mice (40).

Diet formulation
American Institute of Nutrition 93 growth diet (AIN93G) semi-purified diet (Dyets, Bethlehem, PA) was selected as a base diet for control mice as it has been established as meeting all of the nutritional requirements of mice (41). Soy oil was removed from all diets and corn oil added in order to eliminate any additional components of soy being added to the diets. Treatment animals were fed AIN93G diet plus different dosages of daidzein (125, 250, 500 or 1000 p.p.m.) or (±)-equol (250, 500 or 1000 p.p.m.).

Tumor growth analysis
Study I: daidzein
One week after the E2 pellets were inserted into mice, MCF-7 cells [1 x 105 cells/40 µl of Matrigel® (Collaborative Biomedical Products, Bedford, MA] were injected at 40 µl/site into each of the four sites on the backs of the athymic mice. All mice were on AIN93G diet. Tumors were grown until their average cross-sectional area reached 35 mm2. Mice were divided into six treatment groups: MCF-7 control, E2 control, 125, 250, 500 and 1000 p.p.m. of daidzein (10–11 mice/group). E2 pellets were removed from all mice, except the E2 group, and dietary daidzein treatment was started. Tumor growth and body weight were measured weekly for 21 week and cross-sectional area was determined using the formula [length/2 x width/2 x {pi}] (42). Owing to a skin rash in all treatment as the attending veterinarian clinician recommended the study be terminated. All mice were killed at week 21 and tumors and blood were collected for analysis. Food intake was measured throughout the study. At the end of the study, uterine weight was measured. Tumors and blood samples were collected for analysis.

Study II: equol
When the mean tumor surface area reached 37 mm2, mice were divided into five treatment groups: MCF-7 control, E2 control, 250, 500 and 1000 p.p.m. (±)-equol (11–12 mice/group). Tumor growth was monitored for 37 weeks. All the other experimental steps were the same as described for Study I.

Ki-67 Expression (a measure of proliferation)
Cell proliferation in tumors was determined using immunohistochemical analysis. Tumors in 10% formalin were embedded in paraffin blocks, cut into 5 µm sections and placed on microscope slides. Slides were deparaffinized by immersing in xylene twice for 12 min each and hydrated by immersing in a series of 100% ethanol, 95% ethanol and three times in dH2O for 5 min each. To block endogenous peroxidase, slides were immersed in 0.3% H2O2 for 20 min and then washed with dH2O. Slides were placed in citrate buffer (pH 6), microwaved for 20 min, and then cooled at 24°C. Then, slides were washed in PBS (pH 7.4) for 5 min. Anti-human Ki-67 antibody (Pharmingen, San Diego, CA) (1 : 3000 dilution in 1% BSA/PBS) was added to the slides, which were then incubated for 1 h at 24°C in a humidity chamber. Slides were washed in PBS before the addition of biotinilated anti-mouse secondary antibody (VECTASTAIN Elite ABS reagent, Vector Laboratories, Inc. Burlingame, CA) was added to slides and incubated for 30 min at 24°C. Slides were then washed in PBS. One drop of 3,3'-diaminobenzidine tetrahydrachloride (DAB) prepared right before used was added to each slide. Slides were then washed twice in dH2O and PBS and counterstained with 20% hematoxlin for 1 min. The slides were then dehydrated by submerging them in 80% ethanol for 5 min, 95% ethanol for 5 min and 100% ethanol for 5 min, followed by submerging slides in xylene four times for 5 min each. Slides were mounted and analyzed using a light microscope. Both positive and background stained cells were counted in a given area of tissue. A total of 25 fields from 5 tumors per treatment group were evaluated. The data were then presented as a percentage of cells proliferating in a given area of tumor.

RNA preparation and analysis of changes in gene expression using qRT–PCR
Expression of pS2 mRNA was analyzed using qRT–PCR. Tumors with areas similar to the mean tumor surface area of the each treatment group were used for mRNA analysis. RNA from frozen tumor (≤200 mg) was prepared as described by Ju et al. (5). cDNA were generated using 10 ng of RNA and TaqMan Reverse Transcription Reagents (PE Applied Biosystems, Foster City, CA). The pS2 primers and fluorescence (6-FAM)-labeled probes were designed using Primer and Probe Design Express (PE Applied Biosystems) (39). The human GAPDH primers and a fluorescent (6-FAM/TAMRA)-labeled probe (User Bulletin #2, PE Applied Biosystems) were used as a control. PCR and analysis of PCR products were performed using an ABI PRISM 7700 Sequence Detector (PE Applied Biosystems). Data were analyzed using a comparative threshold cycle (Ct) method (User Bulletin, PE Applied Biosystems). Amplicons were run as triplicates in separate tubes to permit quantification of target genes normalized to a control, GAPDH.

Plasma analysis
Blood samples were collected by cardiac puncture at sacrifice, placed into EDTA-containing tubes and centrifuged at 500 g for 5 min. Plasma samples were stored at –20°C until analyzed by liquid chromatography and isotope dilution electrospray tandem mass spectrometry (LC–ES/MS/MS) using a method previously validated (43). Levels of total daidzein, dihydrodaidzein and equol were determined in 25–50 µl plasma after treatment with ß-glucuronidase/sulfatase for deconjugation and addition of deuterated daidzein (d3) and equol (d4) as internal standards. Deuterated daidzein was used as the internal standard for dihydrodaidzein. Five to eight plasma samples per treatment group were analyzed. The limits of quantification for the three analytes were ~0.01 µM, method accuracy was 88–98%, and the method precision was 3–13% (43).

Statistics
Data from tumor area at final week, cell proliferation, qRT–PCR and plasma analysis were analyzed using one-way or repeated-measures analysis of variance according to the characteristics of the dataset using the SAS program. If the overall treatment F-ratio was significant (P < 0.05), the differences between treatment means were tested with Fisher's least significant difference (LSD) test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of daidzein and (±)-equol on the growth of MCF-7 cells in vitro
MCF-7 cellular proliferation is expressed as mean number of MCF-7 cells ± SEM. At concentrations of daidzein and (±)-equol ≤1 µM, MCF-7 cell proliferation increased in a concentration-dependent manner to a maximum of 1 µM, which was 2.5-fold higher than the MCF-7 control for daidzein (gray bars, Figure 1A) and 2.3-fold higher for (±)-equol (gray bars, Figure 1B). The daidzein- and (±)-equol-induced MCF-7 cell growth was blocked by ICI 182,780 (100 nM), further confirming that the isoflavone-induced cell growth is ER-mediated (white bars, Figure 1A and 1B). At all concentrations of daidzein and (±)-equol, there were significant differences between isoflavone alone and isoflavone + ICI 182,780. At higher concentrations of daidzein and equol (5–50 µM), MCF-7 cell proliferation decreased from the maximum level.


Figure 1
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  		Fig. 1. Effects of daidzein and equol on the growth of MCF-7 cells. Daidzein (A) and (±)-equol (B) at concentrations between 1 nM and 50 µM in the presence of (white bars) and in the absence of ICI 182,780 (100 nM) (gray bars) were evaluated for their effects on MCF-7 cell growth using MTT assay. Values are means ± SEM (n = 9). Bars with different letters differ, P < 0.05.

 
Effect of daidzein and (±)-equol on pS2 mRNA expression in MCF-7 cells
MCF-7 cells were treated with daidzein or equol at a concentration of 1 µM, which induced highest cell proliferation for 24 h, and pS2 mRNA expression in MCF-7 cells was evaluated. Daidzein (1 µM) increased pS2 mRNA level by 4.18 ± 0.54 fold, and equol increased by 3.79 ± 0.20 fold over the MCF-7 control (Figure 2) (P < 0.05). Genistein induced similar levels of pS2 expression.


Figure 2
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  		Fig. 2. Estrogen-responsive pS2 gene expression in tumors. MCF-7 cells were treated with 1 µM daidzein, equol or genistein for 24 h and analyzed using qRT–PCR. Numbers on the y-axis represent the relative mRNA level; bars ± SEM. Data points were obtained from three replicas. GAPDH was used as a standard. Bars with different letter are significantly different, P < 0.05.

 
Tumor growth
Study I: daidzein
Mean tumor surface area in the E2 positive control group reached 138.0 mm2 3 weeks after the study started and mice were terminated because of a high tumor burden. Tumors in the rest of the treatment groups were monitored for 21 weeks (Figure 3). After removal of the E2 pellets, tumors in the MCF-7 and daidzein treatment groups regressed. Tumors in the daidzein treatment groups regressed slower than ones in the MCF-7 control group. Tumors in the 125 and 250 p.p.m. daidzein groups showed steady regression during 21-weeks dietary treatment, but tumors in the 500 and 1000 p.p.m. groups regressed for 18 weeks and then appeared to grow slowly. At week 21, the mean tumor surface areas were 4.8 ± 0.7 mm2, 14.3 ± 2.2 mm2 (by 3.0x larger than tumors in the MCF-7 control group), 12.0 ± 1.5 mm2 (by 2.5x), 20.8 ± 2.6 mm2 (by 4.3x) and 30.9 ± 3.9 mm2 (by 6.4x), for the MCF-7, 125, 250, 500 and 1000 p.p.m. daidzein groups, respectively. There were statistically significant differences in tumor area between the MCF-7 control and all the daidzein treatment groups (P < 0.05) (Figure 3). The average tumor size of the 1000 p.p.m. daidzein group was significantly than other daidzein treatment group (P < 0.05) (Figure 3). No statistically significant difference was observed between the 125 and 250 p.p.m. group (P = 0.3857) or between 250 and 500 p.p.m. (P = 0.0652).


Figure 3
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  		Fig. 3. Effect of dietary daidzein on MCF-7 tumor growth in athymic mice. Mice were assigned into six treatment groups: MCF-7 control (12 mice; 48 tumors at K 0 and 29 tumors at week 21), E2 control (9 mice; 34 tumors at week 0 and 34 tumors at week 3), 125 p.p.m. daidzein (8 mice; 31 tumors at week 0 and 23 tumors at 21), 250 p.p.m. daidzein (9 mice; 35 week at week 0 and 26 tumors at week 0), 500 p.p.m. daidzein (9 mice; 35 tumors at week 0 and 27 tumors at week 21) and 1000 p.p.m. daidzein (9 mice; 34 tumors at week 0 and 2 tumors at week 21). Week 0 was the first day that animals were started on the experimental diets. Tumors were then measured weekly for 21 weeks. Data are expressed as average cross sectional tumor area (mm2 ± SEM) for all tumors in each treatment.

 
Study II: equol
Tumors in the E2 group reached 123.3 mm2 3 weeks after the study started and mice were terminated because of a high tumor burden. Tumors in the rest of the treatment groups were monitored for 37 weeks (Figure 4). After removal of the E2 pellets, tumors in all equol treatment groups regressed. At week 37, the mean tumor surface areas were 6.9 ± 1.1, 9.1 ± 3.1, 12.1 ± 2.4 mm2, and 10.1 ± 2.4 mm2, for MCF-7 control, 250, 500, and 1000 p.p.m. equol groups, respectively. No statistically significant differences were observed between the MCF-7 and equol treatment groups (Figure 4).


Figure 4
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  		Fig. 4. Effect of dietary (±)-equol on MCF-7 tumor growth in athymic mice. Mice were assigned into six treatment groups: MCF-7 control (9 mice; 34 tumors at week 0 and 20 tumors at week 37), E2 control (10 mice; 38 tumors at week 0 and 38 tumors at week 3), 250 p.p.m. equol (10 mice; 36 tumors at weeks 0 and 22 tumors at week 37), 500 p.p.m. equol (9 mice; 34 tumors at week 0 and 25 tumors at week 37) and 1000 p.p.m. equol (9 mice; 34 tumors at week 0 and 21 tumors at week 37). Week 0 was the first day that animals were started on the experimental diets. Tumors were then measured weekly for 37 weeks. Data are expressed as average cross sectional tumor area (mm2 ± SEM) for all tumors in each treatment.

 
Cell proliferation in tumors
Percentages of Ki-67 expression were 2.4% for the MCF-7 the control, 3.6% for the 125 p.p.m. daidzein, 5.6% for the 250 p.p.m. daidzein, 5.9% for the 500 p.p.m. daidzein, 7.7% for the 1000 p.p.m. daidzein and 55.1% for the E2 control group, respectively (Figure 5). There was significant difference between the MCF-7 and 1000 p.p.m. daidzein groups (P < 0.05) and between the MCF-7 and E2 groups (P < 0.05) (Figure 5A). Ki-67 expression was unchanged for all equol groups but significantly increased for the E2 group (P < 0.05) (Figure 5B).


Figure 5
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  		Fig. 5. Effect of dietary daidzein and (±)-equol on the cellular proliferation on MCF-7 tumors. Ki-67 expression was evaluated as a measure of proliferation. A total of 25 fields from 5 tumors per treatment group were evaluated. y-axis (cell proliferation) is presented as the percentage of cells actively proliferating in a given area of tissue (± SEM). (A) Cell proliferation induced by Daidzein. (B) Cell proliferation induced by (±)-equol. Bars with different letters differ, P < 0.05.

 
Body weight, food intake, and uterine wet weight
Body weights were monitored weekly and no significant difference was observed among the treated and control groups (data not shown). No significant differences were observed in food intake among any of the treatment groups (data not shown). No significant difference was observed in uterine weight between the MCF-7 and daidzein/equol groups (data not shown).

pS2 expression in tumors
To evaluate the capability of daidzein and equol to modulate the expression of an estrogen-responsive gene, pS2, mRNA in tumors was analyzed using qRT–PCR (Figure 6). Expression of pS2 is presented as relative pS2 mRNA expression ± SEM. In the daidzein groups, pS2 expression was significantly increased by E2 (15.0x), by 500 p.p.m. daidzein (3.1x) and by 1000 p.p.m. daidzein (4.1x), compared with the MCF-7 group (P < 0.05). In the equol groups, E2 significantly enhanced pS2 expression over the MCF-7 group (P < 0.05). There were no statistically significant differences among the equol treatment groups.


Figure 6
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  		Fig. 6. Estrogen-responsive pS2 gene expression in tumors. Six to seven tumors per treatment group were analyzed using qRT–PCR. Numbers on the y-axis represent the relative mRNA level; bars ± SEM. GAPDH was used as a standard. (A) Dietary daidzein induced pS2 expression. (B) Dietary (±)-equol induced pS2 expression. There was no statistical difference among any treatment groups. Bars with different letter are significantly different, P < 0.05.

 
Isoflavone levels in plasma
While no daidzein or equol was detected in plasma from mice in the MCF-7 and E2 groups (<0.01 µM), the concentration of total daidzein in plasma from daidzein-fed animals increased regularly as the dietary dose increased (125–1000 p.p.m.) with levels that ranged from 0.25 to 1.54 µM (Figure 7A, white bars). Plasma concentrations of daidzein measured in the 1000 p.p.m. daidzein group was significantly higher than that in 125 p.p.m. daidzein group (P < 0.05). Formation of equol in daidzein-fed mice was highly variable; in the 125 p.p.m. group, only two of the six mice formed equol, in the 250 p.p.m. group, one of the eight did, in the 500 p.p.m. group, one of the seven did, and in the 1000 p.p.m. group, none did. Equol levels in these mice ranged from <LOD to 1.01 µM (Figure 7A, gray bars). In addition, dihydrodaidzein was observed in some but not all mice (Figure 7A, black bars); in the 125 p.p.m. group, only three of the six mice formed equol, in the 250 p.p.m. group, four of the eight did, in the 500 p.p.m. group, 4 of 7 did, and in the 1000 p.p.m. group, 1 of 8 did. No statistically significant correlations between the total equol or total dihydrodaidzein levels and daidzein dietary dosages were observed. Total equol in plasma from equol-fed mice ranged from 2.10 to 3.21 µM (Figure 7B) and there were no statistically significant differences between groups.


Figure 7
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  		Fig. 7. (A) Total daidzein, equol and dihydrodaidzein (aglycone + conjugated) plasma levels in dietary daidzein dose–response study (Study 1). Total aglycone level on the y-axis represents µM ± SEM (total daidzein, white bars; total equol, gray bars; dihydrodaidzein, diagonal-patterned bar). (B) Total equol plasma levels in dietary equol dose-response study (Study 2). Total aglycone level on the y-axis represents µM ± SEM (total equol; gray bars). Bars with different letter are significantly different P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Based on in vitro studies (44–46) and computational models for ER binding affinities (47), it seemed likely that other dietary isoflavones and metabolites could produce effects similar to those observed for genistein in vitro. Genistein, daidzein and equol are agonists for both ER{alpha} and ERß, with preferential binding to and transactivation through ERß (37). However, the data presented here for daidzein and equol point out several critical differences in vivo. Similar to genistein, daidzein and equol showed proliferative effects on MCF-7 cell growth in vitro (Figure 1). Daidzein (0.001–50 µM) and (±)-equol (0.001–20 µM) stimulated growth with the maximal stimulation at 1 µM. The concentration dependence for effects by genistein (48), daidzein and equol (Figure 1) on MCF-7 cell growth in vitro are similar. Our previous studies have demonstrated that genistein stimulates MCF-7 cell growth in vitro at concentrations from 0.1 to 20 µM, with maximal stimulation at 1 µM, and induces expression of the estrogen-responsive gene pS2 (48,49). In addition, there is evidence for an anti-proliferative effect on cell growth apparent at concentrations above 10 µM (48,50,51). In this study, we also observed that daidzein and equol (at 1 µM) enhanced pS2 mRNA expression in MCF-7 cells in vitro (Figure 2), suggesting that the cell stimulatory effect observed in Figure 1 may involve an ER-regulated mechanism. Daidzein- and equol-stimulated cell growth was blocked by ICI 182 780 (Figure 1), confirming that the stimulatory effect on cell growth involves an ER-mediated activity. Administration of genistein to athymic mice in the diet between 125 and 1000 p.p.m. produced circulating total genistein concentrations of 0.4–3.4 µM, increased pS2 expression and markers of proliferation and stimulated growth of implanted MCF-7 tumors (2). Soy protein isolates containing increasing concentrations of genistein stimulated the growth of MCF-7 tumors in a dose-dependent manner (3). Consumption of genistein in pure or highly enriched forms (as in soy protein isolates) has a greater stimulatory effect on MCF-7 tumor growth than the same content of dietary genistein in soy flour. These results suggest that the matrix in soy flour alters the effects of dietary genistein on estrogen-dependent tumor growth (52). These estrogen agonistic actions of genistein, mediated through ERs (53), occur at circulating levels of total genistein that are quite similar to those observed in humans consuming soy foods (54–59) and nutritional supplements (60). Unlike genistein, equol, administered through the diet at levels comparable with those used previously for genistein (2), did not enhance growth of implanted MCF-7 tumors in vivo (Figure 4). And, dietary daidzein produced a modest, but significant, stimulatory effect (Figure 3). We observed a weak estrogenic effect of dietary daidzein at the concentration range tested (Figure 3), although it was not as potent as that of genistein (2). This mild estrogenic effect is likely due to enhanced cellular proliferation (Figure 5). Furthermore, evidence for ER activation (i.e. elevated pS2 mRNA expression) was not observed in mice fed equol and was significant but weak in mice fed daidzein at 250 ppm and above (Figure 6).

The absence of estrogenic effects in mice fed daidzein or equol is notable because the 'steady-state' plasma levels of total daidzein or equol (Figure 7) were quite similar to those previously measured for total genistein (2) and comparable with those that have estrogenic effects in vitro (Figure 1). These results are consistent with the conclusions of our recent study (61) that pharmacokinetic factors beyond bioavailability, possibly reflecting differences in the Phase II metabolism and distribution of active estrogenic isoflavone aglycones in the target tissue, are possible explanations for the observed in vitro–in vivo dichotomy between genistein and equol.

Isoflavone supplements have been consumed by post-menopausal women to relieve menopausal symptoms despite equivocal clinical evidence for efficacy (62,63). Furthermore, there is considerable controversy regarding whether isoflavones and their metabolites can produce adverse effects, including possibly stimulating the growth of estrogen-dependent breast tumors in women. Epidemiological evidence suggests that consumption of soy-rich diets can reduce breast cancer risk, particularly during the pre-pubertal period of life (64), and it is hypothesized that equol may be a contributor to the beneficial effects of soy (65). However, other epidemiological investigations show an increased odds ratio for breast cancer incidence for women with higher plasma and urinary phytoestrogen levels (66). Long-term dietary studies of equol, free from the confounding presence of other isoflavones, have not been possible in laboratory animal models until recently because synthetic material in sufficient quantities has not been available (37). The present study, which used a pre-clinical model for post-menopausal estrogen-dependent breast cancer, suggests that dietary daidzein and its metabolite equol have only minimal estrogenic effects. The clear difference in pharmacodynamic effects from genistein (61) suggests that it, and not daidzein or equol, is the active soy isoflavone that affects breast cancer growth in the athymic mouse-MCF-7 xenograft model. This conclusion is consistent with previous research conducted using a pre-pubertal rat model of breast cancer (67). Lamartiniere et al. (67) reported that DMBA-induced mammary tumor multiplicity was not affected in rats fed daidzein-containing diets (250–1000 p.p.m.), despite the observation of high circulating levels of total daidzein (0.98–3.8 µM) and equol (0.16–1.0 µM). These internal exposures to daidzein and equol also failed to stimulate mammary gland differentiation. Those results were opposite to those previously observed by the same research group from identical pre-pubertal treatment with genistein, which produced significant reductions in tumor multiplicity and increased mammary gland differentiation (68). Ward et al. (69) reported that 200 p.p.m. daidzein intake for two weeks produced 0.18–0.36 µM daidzein and did not change uterine weight in four mouse strains [two inbred strains (C57BL/6 and C3H) and two outbred strains (CD-1 and Swiss–Webster)].

In summary, dietary daidzein and (±)-equol produce concentrations of total isoflavones in mouse plasma that are similar to those observed in humans. However, these internal exposures to equol did not stimulate growth of estrogen-dependent human breast tumor (MCF-7) growth or induce estrogen-responsive pS2 expression in tumors in vivo. Similarly, dietary daidzein had a very modest stimulatory effect on MCF-7 tumor growth and did not increase pS2 gene expression. Findings from this study and our previous studies (52,61) suggest that genistein is the major active estrogenic component from dietary soy isoflavone consumption and is likely responsible for the estrogenic effects on mammary and other estrogen-responsive tissues.


   Acknowledgments
 
Supported by CA77355 to W.G.H. from National Cancer Institute; by P01-AG024387 to W.G.H., Y.H.J. and D.R.D. from National Institute on Aging with additional support from the National Institute for Complementary and Alternative Medicine, Office of Dietary Supplements, and the Women's Health Initiative; and partially supported by the National Institutes of Environment, Health and Science Training Program Grant PHS T32 ES07326 to Y.H.J. from National Institute of Health. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Adlercreutz,H., Mousavi,Y., Clark,J., Hockerstedt,K., Hamalainen,E., Wahala,K., Makela,T. and Hase,T. (1992) Dietary phytoestrogens and cancer: in vitro and in vivo studies. J. Steroid Biochem. Mol. Biol., 41, 331–337.[CrossRef][ISI][Medline]
  2. Ju,Y.H., Allred,C.D., Allred,K.F., Karko,K.L., Doerge,D.R. and Helferich,W.G. (2001) Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice. J. Nutr., 131, 2957–2962.[Abstract/Free Full Text]
  3. Allred,C.D., Allred,K.F., Ju,Y.H., Virant,S.M. and Helferich,W.G. (2001) Soy diets containing varying amounts of genistein stimulate growth of estrogen-dependent (MCF-7) tumors in a dose-dependent manner. Cancer Res., 61, 5045–5050.[Abstract/Free Full Text]
  4. Allred,C.D., Allred,K.F., Ju,Y.H., Clausen,L.M., Doerge,D.R., Schantz,S.L., Korol,D.L., Wallig,M.A. and Helferich,W.G. (2004) Dietary genistein results in larger MNU-induced, estrogen-dependent mammary tumors following ovariectomy of Sprague-Dawley rats. Carcinogenesis, 25, 211–218.[Abstract/Free Full Text]
  5. Ju,Y.H., Doerge,D.R., Allred,K.F., Allred,C.D. and Helferich,W.G. (2002) Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice. Cancer Res., 62, 2474–2477.[Abstract/Free Full Text]
  6. Bannwart,C., Fotsis,T., Heikkinen,R. and Adlercreutz,H. (1984) Identification of the isoflavonic phytoestrogen daidzein in human urine. Clin. Chim. Acta., 136, 165–172.[CrossRef][Medline]
  7. Chang,Y.C., Nair,M.G. and Nitiss,J.L. (1995) Metabolites of daidzein and genistein and their biological activities. J. Nat. Prod., 58, 1901–1905.[CrossRef][Medline]
  8. Rafii,F., Hotchkiss,C., Heinze,T.M. and Park,M. (2004) Metabolism of daidzein by intestinal bacteria from rhesus monkeys (Macaca mulatta). Comp. Med., 54, 165–169.[Medline]
  9. Joannou,G.E., Kelly,G.E., Reeder,A.Y., Waring,M. and Nelson,C. (1995) A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids. J. Steroid Biochem. Mol. Biol., 54, 167–184.[CrossRef][ISI][Medline]
  10. Wang,X.-L., Hur,H.-G., Lee,J.H., Kim,K.T. and Kim,S.-I. (2005) Enantioselective Synthesis of S-Equol from Dihydrodaidzein by a Newly Isolated Anaerobic Human Intestinal Bacterium. Appl. Environ. Microbiol., 71, 214–219.[Abstract/Free Full Text]
  11. Marrian,G.F. and Haslewood,G.A.D. (1932) Equol, a new inactive phenol isolated from the ketohydroxyoestrin fraction of mares urine. Biochem. J., 26, 1227–1232.
  12. Shutt,D.A. and Braden,A.W.H. (1968) The significance of equol in relation to the oestrogenic responses in sheep engesting clover with a high formononetin content. Aust. J. Agric. Res., 19, 545–553.[CrossRef]
  13. Axelson,M., Kirk,D.N., Farrant,R.D., Cooley,G., Lawson,A.M. and Setchell,K.D. (1982) The identification of the weak oestrogen equol [7-hydroxy-3-(4'-hydroxyphenyl)chroman] in human urine. Biochem. J., 201, 353–357.[ISI][Medline]
  14. Rowland,I.R., Wiseman,H., Sanders,T.A., Adlercreutz,H. and Bowey,E.A. (2000) Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora. Nutr. Cancer, 36, 27–32.[CrossRef][ISI][Medline]
  15. Xu,X., Harris,K.S., Wang,H.J., Murphy,P.A. and Hendrich,S. (1995) Bioavailability of soybean isoflavones depends upon gut microflora in women. J. Nutr., 125, 2307–2315.[Abstract/Free Full Text]
  16. Lampe,J.W., Karr,S.C., Hutchins,A.M. and Slavin,J.L. (1998) Urinary equol excretion with a soy challenge: influence of habitual diet. Proc. Soc. Exp. Biol. Med., 217, 335–339.[Abstract]
  17. Setchell,K.D., Brown,N.M., Desai,P., Zimmer-Nechemias,L., Wolfe,B.E., Brashear,W.T., Kirschner,A.S., Cassidy,A. and Heubi,J.E. (2001) Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J. Nutr., 131, 1362S–1375S.[Abstract/Free Full Text]
  18. Bayer,T., Colnot,T. and Dekant,W. (2001) Disposition and biotransformation of the estrogenic isoflavone daidzein in rats. Toxicol. Sci., 62, 205–211.[Abstract/Free Full Text]
  19. Lampe,J.W. (2003) Isoflavonoid and lignan phytoestrogens as dietary biomarkers. J. Nutr., 133 (Suppl 3), 956S–964S.[Abstract/Free Full Text]
  20. Chen,Z., Zheng,W., Custer,L.J., Dai,Q., Shu,X.O., Jin,F. and Franke,A.A. (1999) Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai. Nutr. Cancer, 33, 82–87.[ISI][Medline]
  21. Duncan,A.M., Merz-Demlow,B.E., Xu,X., Phipps,W.R. and Kurzer,M.S. (2000) Premenopausal equol excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epidemiol Biomarkers Prev., 9, 581–586.[Abstract/Free Full Text]
  22. Ingram,D., Sanders,K., Kolybaba,M. and Lopez,D. (1997) Case-control study of phyto-oestrogens and breast cancer. Lancet, 350, 990–994.[CrossRef][ISI][Medline]
  23. Zheng,W., Dai,Q., Custer,L.J., Shu,X.O., Wen,W.Q., Jin,F. and Franke,A.A. (1999) Urinary excretion of isoflavonoids and the risk of breast cancer. Cancer Epidemiol Biomarkers Prev., 8, 35–40.[Abstract/Free Full Text]
  24. Chang,Y.C. and Nair,M.G. (1995) Metabolism of daidzein and genistein by intestinal bacteria. J. Nat. Prod., 58, 1892–1896.[CrossRef][Medline]
  25. Morito,K., Hirose,T., Kinjo,J., Hirakawa,T., Okawa,M., Nohara,T., Ogawa,S., Inoue,S., Muramatsu,M. and Masamune,Y. (2001) Interaction of phytoestrogens with estrogen receptors alpha and beta. Biol. Pharm. Bull, 24, 351–356.[CrossRef][ISI][Medline]
  26. Sathyamoorthy,N. and Wang,T.T. (1997) Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells. Eur. J. Cancer, 33, 2384–2389.[CrossRef][ISI][Medline]
  27. Kaldas,R.S. and Hughes,C.L.,Jr (1989) Reproductive and general metabolic effects of phytoestrogens in mammals. Reprod Toxicol., 3, 81–89.[CrossRef][ISI][Medline]
  28. Baird,D.D., Umbach,D.M., Lansdell,L., Hughes,C.L., Setchell,K.D., Weinberg,C.R., Haney,A.F., Wilcox,A.J. and McLachlan,J.A. (1995) Dietary intervention study to assess estrogenicity of dietary soy among postmenopausal women. J. Clin. Endocrinol. Metab., 80, 1685–1690.[Abstract/Free Full Text]
  29. Cassidy,A., Bingham,S. and Setchel,K.D. (1994) Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. American J. Clinical Nutrition, 60, 333–340.
  30. Hargreaves,D.F., Potten,C.S., Harding,C., Shaw,L.E., Morton,M.S., Roberts,S.A., Howell,A. and Bundred,N.J. (1999) Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast. J. Clin. Endocrinol. Metab., 84, 4017–4024.[Abstract/Free Full Text]
  31. Setchell,K.D., Gosselin,S.J., Welsh,M.B., Johnston,J.O., Balistreri,W.F., Kramer,L.W., Dresser,B.L. and Tarr,M.J. (1987) Dietary estrogens–a probable cause of infertility and liver disease in captive cheetahs. Gastroenterology, 93, 225–233.[ISI][Medline]
  32. Setchell,K.D., Brown,N.M. and Lydeking-Olsen,E. (2002) The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. J. Nutr., 132, 3577–3584.[Abstract/Free Full Text]
  33. Jefferson,W.N., Padilla-Banks,E., Clark,G. and Newbold,R.R. (2002) Assessing estrogenic activity of phytochemicals using transcriptional activation and immature mouse uterotrophic responses. J. Chromatography B, 777, 179–189.[CrossRef]
  34. Selvaraj,V., Zakroczymski,M.A., Naaz,A., Mukai,M., Ju,Y.H., Doerge,D.R., Katzenellenbogen,J.A., Helferich,W.G. and Cooke,P.S. (2004) Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice. Biol. Reprod., 71, 966–972.[Abstract/Free Full Text]
  35. Tang,B.Y. and Adams,N.R. (1980) Effect of equol on oestrogen receptors and on synthesis of DNA and protein in the immature rat uterus. J. Endocrinol., 85, 291–297.[Abstract]
  36. Patterson,R.E., Neuhouser,M.L., Hedderson,M.M., Schwartz,S.M., Standish,L.J., Bowen,D.J. and Marshall,L.M. (2002) Types of alternative medicine used by patients with breast, colon, or prostate cancer: predictors, motives, and costs. J. Altern. Complement Med., 8, 477–485.[CrossRef][ISI][Medline]
  37. Muthyala,R.S., Ju,Y.H., Sheng,S., Williams,L.D., Doerge,D.R., Katzenellenbogen,B.S., Helferich,W.G. and Katzenellenbogen,J.A. (2004) Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta. Bioorg. Med. Chem., 12, 1559–1567.[CrossRef][Medline]
  38. Soule,H.D., Vazguez,J., Long,A., Albert,S. and Brennan,M. (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl Cancer Inst., 51, 1409–1416.[ISI][Medline]
  39. Ju,Y.H., Clausen,L.M., Allred,K.F., Almada,A.L. and Helferich,W.G. (2004) beta-Sitosterol, beta-Sitosterol Glucoside, and a Mixture of beta-Sitosterol and beta-Sitosterol Glucoside Modulate the Growth of Estrogen-Responsive Breast Cancer Cells In Vitro and in Ovariectomized Athymic Mice. J. Nutr., 134, 1145–1151.[Abstract/Free Full Text]
  40. McManus,M.J. and Welsch,C.W. (1981) Hormone-induced ductal DNA synthesis of human breast tissues maintained in the athymic nude mouse. Cancer Res., 41, 3300–3305.[ISI][Medline]
  41. Reeves,P.G. (1997) Components of the AIN-93 diets as improvements in the AIN-76A diet. J. Nutr., 127, 838S–841S.
  42. Gottardis,M.M., Wagner,R.J., Borden,E.C. and Jordan,V.C. (1989) Differential ability of antiestrogens to stimulate breast cancer cell (MCF-7) growth in vivo and in vitro. Cancer Res., 49, 4765–4769.[Abstract/Free Full Text]
  43. Chang,H.C., Churchwell,M.I., Delclos,K.B., Newbold,R.R. and Doerge,D.R. (2000) Mass spectrometric determination of Genistein tissue distribution in diet-exposed Sprague-Dawley rats. J. Nutr., 130, 1963–1970.[Abstract/Free Full Text]
  44. Schmidt,S., Michna,H. and Diel,P. (2005) Combinatory effects of phytoestrogens and 17[beta]-estradiol on proliferation and apoptosis in MCF-7 breast cancer cells. J Steroid Biochem Mol Biol, 94, 445–449.[CrossRef][ISI][Medline]
  45. Verma,S.P. and Goldin,B.R. (1998) Effect of soy-derived isoflavonoids on the induced growth of MCF-7 cells by estrogenic environmental chemicals. Nutr. Cancer, 30, 232–9.[ISI][Medline]
  46. Willard,S.T. and Frawley,L.S. (1998) Phytoestrogens have agonistic and combinatorial effects on estrogen-responsive gene expression in MCF-7 human breast cancer cells. Endocrine, 8, 117–121.[Medline]
  47. Fang,H., Tong,W. Shi,L.M. et al (2001) Structure-Activity Relationships for a Large Diverse Set of Natural, Synthetic, and Environmental Estrogens. Chem. Res. Toxicol., 14, 280–294.[CrossRef][ISI][Medline]
  48. Hsieh,C.Y., Santell,R.C., Haslam,S.Z. and Helferich,W.G. (1998) Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res., 58, 3833–3838.[Abstract/Free Full Text]
  49. Wang,T.T., Sathyamoorthy,N. and Phang,J.M. (1996) Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis, 17, 271–275.[Abstract/Free Full Text]
  50. Martin,P.M., Horwitz,K.B., Ryan,D.S. and McGuire,W.L. (1978) Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology, 103, 1860–1867.[Abstract]
  51. Wang,C. and Kurzer,M.S. (1997) Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells. Nutr. Cancer, 28, 236–247.[ISI][Medline]
  52. Allred,C.D., Allred,K.F., Ju,Y.H., Goeppinger,T.S., Doerge,D.R. and Helferich,W.G. (2004) Soy processing influences growth of estrogen-dependent breast cancer tumors. Carcinogenesis, 25, 1649–1657.[Abstract/Free Full Text]
  53. Pettersson,K. and Gustafsson,J.Å. (2001) Role of Estrogen Receptor Beta in Estrogen Action. Annu. Rev. Physiol., 63, 165–192.[CrossRef][ISI][Medline]
  54. Xu,X., Wang,H.J., Murphy,P.A., Cook,L. and Hendrich,S. (1994) Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J. Nutr., 124, 825–832.[Abstract/Free Full Text]
  55. Adlercreutz,H., Markkanen,H. and Watanabe,S. (1993) Plasma concentrations of phyto-oestrogens in Japanese men. Lancet, 342, 1209–1210.[CrossRef][ISI][Medline]
  56. Lu,L.J., Lin,S.N., Grady,J.J., Nagamani,M. and Anderson,K.E. (1996) Altered kinetics and extent of urinary daidzein and genistein excretion in women during chronic soya exposure. Nutr. Cancer, 26, 289–302.[ISI][Medline]
  57. Izumi,T., Piskula,M.K., Osawa,S., Obata,A., Tobe,K., Saito,M., Kataoka,S., Kubota,Y. and Kikuchi,M. (2000) Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr., 130, 1695–1699.[Abstract/Free Full Text]
  58. Setchell,K.D., Brzezinski,A., Brown,N.M., Desai,P.B., Melhem,M., Meredith,T., Zimmer-Nechimias,L., Wolfe,B., Cohen,Y. and Blatt,Y. (2005) Pharmacokinetics of a slow-release formulation of soybean isoflavones in healthy postmenopausal women. J. Agric Food Chem., 53, 1938–1944.[CrossRef][Medline]
  59. Busby,M.G., Jeffcoat,A.R. and Bloedon,L.T. et al. (2002) Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men. Am. J. Clin. Nutr., 75, 126–136.[Abstract/Free Full Text]
  60. Doerge,D.R., Chang,H.C., Churchwell,M.I. and Holder,C.L. (2000) Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab Dispos., 28, 298–307.[Abstract/Free Full Text]
  61. Allred,C.D., Twaddle,N.C., Allred,K.F., Churchwell,M.I., Ju,Y.H., Helferich,W.G. and Doerge,D.R. (2005) Soy processing affects metabolism and disposition of dietary isoflavones in ovariectomized Balb/c mice. J. Agric. Food Chem., 53, 8542–8550.[CrossRef][ISI][Medline]
  62. Tice,J.A., Ettinger,B., Ensrud,K., Wallace,R., Blackwell,T. and Cummings,S.R. (2003) Phytoestrogen supplements for the treatment of hot flashes: the Isoflavone Clover Extract (ICE) Study: a randomized controlled trial. Jama, 290, 207–214.[Abstract/Free Full Text]
  63. Wuttke,W., Jarry,H., Becker,T., Schultens,A., Christoffel,V., Gorkow,C. and Seidlova-Wuttke,D. (2003) Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?. Maturitas, 44 (Suppl 1), S9–20.
  64. Wu,A.H., Wan,P., Hankin,J., Tseng,C.C., Yu,M.C. and Pike,M.C. (2002) Adolescent and adult soy intake and risk of breast cancer in Asian-Americans. Carcinogenesis, 23, 1491–1496.[Abstract/Free Full Text]
  65. Setchell,K.D.R., Clerici,C., Lephart,E.D. et al. (2005) S-Equol, a potent ligand for estrogen receptor {beta}, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am. J. Clin. Nutr., 81, 1072–1079.[Abstract/Free Full Text]
  66. Committee on Toxicity of Chemicals in Food/Consumer Products and the Environment (2003), The Food Standards Agency, London The Food Standards Agency, In COT Report – Phytoestrogens and Health
  67. Lamartiniere,C.A., Wang,J., Smith-Johnson,M. and Eltoum,I.E. (2002) Daidzein: bioavailability, potential for reproductive toxicity, and breast cancer chemoprevention in female rats. Toxicol. Sci., 65, 228–238.[Abstract/Free Full Text]
  68. Fritz,W.A., Coward,L., Wang,J. and Lamartiniere,C.A. (1998) Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis, 19, 2151–2158.[Abstract/Free Full Text]
  69. Ward,W.E., Kim,S., Chan,D. and Fonseca,D. (2005) Serum equol, bone mineral density and biomechanical bone strength differ among four mouse strains. 16, 743–749.
Received September 10, 2005; revised September 24, 2005; accepted December 17, 2005.


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