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BOR - Papers in Press, published online ahead of print May 24, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.052142
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BIOLOGY OF REPRODUCTION 75, 477–486 (2006)
DOI: 10.1095/biolreprod.106.052142
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys1

Charles E. Wood 2 3, Susan E. Appt 3, Thomas B. Clarkson 3, Adrian A. Franke 4, Cynthia J. Lees 3, Daniel R. Doerge 5, and J. Mark Cline 3

Department of Pathology/Section on Comparative Medicine,3 Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1040 Cancer Research Center of Hawaii,4 Honolulu, Hawaii 96813 Division of Biochemical Toxicology,5 National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, Arkansas 72079

ABSTRACT

Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.

estradiol, estrogen receptor, female reproductive tract, mammary glands, uterus

INTRODUCTION

Soy isoflavonoids are natural dietary compounds widely marketed and consumed for their potential health benefits [1]. The physiologic effects of soy isoflavonoids have been studied extensively in recent years [2–3], although public health recommendations regarding isoflavonoid intake remain controversial. Much of this uncertainty has focused on potential adverse effects of isoflavonoids in estrogen-sensitive tissues. Soy isoflavonoids have structural similarities to mammalian estrogens (Fig. 1), bind and transactivate estrogen receptors [4], induce proliferation in estrogen-sensitive endometrial and breast tumor cells in culture [5–7], and elicit clear estrogenic effects in rodent models, particularly when given as purified supplements [7–10]. Naturally, this evidence has raised concern that high levels of soy isoflavonoid intake may promote estrogen-responsive tumors in women.


Figure 1
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  		FIG. 1. Chemical structures of the isoflavonoids genistein, daidzein, and equol compared with estradiol. Genistein (4',5,7-trihydroxyisoflavone) and daidzein (4',7-dihydroxyisoflavone) are the primary isoflavones in soy, while equol (4',7-dihydroxyisoflavan) is an isoflavan produced enterically from daidzein

Unopposed estrogen induces endometrial hyperplasia and may increase endometrial cancer risk by at least fivefold in postmenopausal women [11]. Identifying potential estrogen-like uterotrophic effects of isoflavonoids thus has particular public health relevance. Studies investigating soy effects on human reproductive tissues have been largely inconclusive, however. Recently, a large randomized trial of postmenopausal women reported a small but significant increase in endometrial hyperplasia in the group receiving a purified isoflavonoid supplement compared to placebo (3.4% incidence vs. 0% at 5 yr) [12]. Two smaller intervention trials in premenopausal women also concluded that soy may have stimulatory effects in breast tissue [13–14]. In contrast, the majority of other studies in women have found no effect of soy/isoflavonoid intake on reproductive markers [15–21], and population-based studies have generally shown modest protective effects of isoflavonoid intake against both endometrial [22] and breast [23–27] cancer. In our prior work using postmenopausal monkeys, we have yet to identify estrogenic isoflavonoid effects in reproductive tissues, even at doses up to twice those attainable by diet [28–30].

The main serum isoflavonoids resulting from soy intake are genistein, daidzein, and equol [2]. The glycosylated forms of genistein and daidzein (isoflavones) predominate in most soy products, while equol (an isoflavan) is produced from daidzein by enteric bacterial metabolism (Fig. 1). Many of the health claims related to dietary soy have been attributed to genistein, which is the primary serum isoflavonoid in most people consuming soy. The precise effects of individual isoflavonoids are poorly defined, however. In recent years much speculation has focused on the role of equol production in determining soy effects across individuals. Equol is produced in large quantities in all rodents and monkeys but in only about 30% of people who consume soy [31]. Equol binds estrogen receptors with greater affinity than genistein and daidzein [32] and may induce estrogenic effects in cell culture and rodent models [32–35]. Conversely, associative data from studies of women (based on subset analyses of equol producers vs. nonproducers) suggest that equol producers may have lower breast cancer risk [25], reduced mammographic density [36], and lower-risk estrogen profiles [37–38], consistent with an estrogen-sparing effect. Unlike genistein and daidzein, equol has a chiral center and therefore exists as two distinct enantiomeric forms, S-equol and R-equol [39]. The S-enantiomer is the exclusive form produced naturally in the human gut [39], while synthetic equol supplements are composed of a racemic mixture of S- and R-equol. Previous studies have shown that S- and R-equol have distinct biologic properties, particularly with respect to estrogen receptor interactions [32, 39], suggesting that the effects of naturally occurring S-equol may differ from those of the racemic mixture.

In recent years soy isoflavonoids have become increasingly popular as dietary supplements [1], particularly for postmenopausal women seeking a safe natural alternative to traditional hormone therapies [40]. While traditional soy-based Asian diets provide ~20–50 mg of isoflavonoids per day [41], commercially available purified supplements may deliver far greater isoflavonoid amounts (>150 mg per serving), despite little relevant safety data at these doses. The purpose of this study was to evaluate the potential estrogenic effects of high isoflavone and equol doses on markers of uterine and breast cancer risk in a postmenopausal primate model.

MATERIALS AND METHODS

Animal Subjects and Diets

In this study we used 30 adult female surgically menopausal cynomolgus macaques (Macaca fascicularis) with an average age of 18.4 ± 0.5 yr. All animals were originally imported from the Institut Pertanian Bogor in Bogor, Indonesia. Macaques are anthropoid primates with >95% overall genetic coding sequence identity to humans [42], including key genes involved in cancer susceptibility [43]. Female cynomolgus macaques have a 28-day menstrual cycle and reproductive tissues that are similar to women in terms of microanatomy, sex steroid receptor expression, and responses to exogenous estrogens [44–45].

All animals were ovariectomized 4.5 yr before the start of this study and housed since this time in stable social groups of 3–4 animals each. The previous long-term study involving these animals is described elsewhere [28]. Animals were randomized by social group to receive one of three diets containing the baseline control diet supplemented with 1) nothing (control, n = 10), 2) 537 mg/1800 kcal of isoflavones (genistein, daidzein, glycitein) in a concentrate (IF, n = 10), or 3) 1020 mg/1800 kcal of purified racemic equol (EQ, n = 10). Treatments were given for 28 to 33 days. Previous reports in the macaque model [46] indicate that 1 mo is sufficient time for significant estrogenic effects to occur in reproductive tissues.

The IF and EQ supplements were analyzed by the manufacturers using high performance liquid chromatography (HPLC). The isoflavone supplement contained 65.5% genistein, 29.2% daidzein, and 5.3% glycitein (as expressed in aglycone equivalents), while the equol supplement contained a 96.0% pure racemic mixture of S- and R-equol enantiomers. The IF dose was based on analysis of prior rat studies which showed uterotrophic effects at or above doses of ~360 mg/1800 kcal [35, 41]. We gave a total EQ dose (R + S) approximately twice that of the IF group in an attempt to balance the S-equol (considered to be the more active isomer [39]) provided in the EQ diet with total isoflavonoids given in the IF group. Diets provided 0 (Con), 0.297 (IF), and 0.566 mg (EQ) isoflavonoids/kcal and were otherwise identical in macronutrients, cholesterol, calcium, and phosphorus. Animals were fed 120 kcal/kg body weight (BW) (+10% extra to account for waste) once daily. Expressed daily isoflavone and equol doses were scaled to 1800 kcal of diet (the estimated daily intake for a U.S. woman) to approximate equivalent human doses. This type of caloric scaling helps account for differences in metabolic rates between the monkeys and human subjects [47]. Monkeys were thus given approximately 35.7 mg of isoflavones or 68.0 mg of equol per kg BW. The IF supplement used for this study was generously provided by Solae, a division of Dupont (St. Louis, MO). The equol as supplement was provided by Solae and produced by Aldrich (Milwaukee, WI).

Images and data from postmenopausal female macaques treated with estradiol (E2) in previous studies were included as reference controls. Tissue images in Figs. 2–4 labeled E2 were taken from ovariectomized macaques given oral 17beta-estradiol at a physiologic dose equivalent to 1.0 mg/woman/day for 6 mo, as described previously [30]. Corresponding data from these animals is shown with permission in Table 2. For gene expression assays (Fig. 5), archived E2-treated uterine tissues from this same study [30] were used, while E2-treated breast tissues came from a separate group of animals treated with the same 1.0 mg/day dose of oral E2 for 1 mo (unpublished). For gene expression data collection, cDNA from all reference tissues was analyzed simultaneously on the same PCR plate as tissues from the current study. All estradiol treatments were administered within a casein/lactalbumin-based diet similar to that used in the current study.


Figure 2
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  		FIG. 2. High-dose isoflavone (IF) and equol (EQ) effects on reproductive tissues. Uterine weight (A), glandular epithelial area (B), endometrial thickness (C), and uterine area (D) were not significantly different among groups following treatment (ANOVA P > 0.1 for all). Uterine area, as measured by ultrasound, also was not significantly different from baseline for any of the groups (D). On histology, the control, IF, and EQ groups had atrophic uteri with few simple endometrial glands and condensed endometrial stroma (E). Uterine morphology from a reference animal given 1.0 mg/woman/day of estradiol (E2) is shown at right for comparison; note more extensive glands and stromal expansion. Arrow indicates junction between endometrium and myometrium; endometrial lumen is at the top of each image. Bar = 100 µm. n = 7–10 per group


Figure 4
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  		FIG. 4. Mammary gland expression of the proliferation marker MKI67, progesterone receptor (PGR), and estrogen receptor alpha (ESR1). A) Epithelial MKI67 immunostaining in control (Con), isoflavone (IF), and equol (EQ) groups compared with a reference animal given 1.0 mg/woman/day of estradiol (E2). Red nuclei indicate positive cells. Bar = 25 µm. No significant group differences were found for MKI67 (B), PGR (C), or ESR1 (D) (ANOVA P > 0.1 for all). n = 9–10 per group for lobules and 6–8 per group for ducts


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  		TABLE 2. Comparison of reproductive measures in control, isoflavone (IF), and equol (EQ) groups compared to historical control and estradiol-treated (E2) ovariectomized female macaques


Figure 5
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  		FIG. 5. Isoflavone (IF), equol (EQ), and estradiol (E2) effects on estrogen-induced gene expression. Quantitative real-time RT-PCR was used to determine expression of several estrogen-dependent genes in uterine (A–C) and breast tissues (D–F). Asterisks indicate significant differences from the control group (* P < 0.05, ** P <0.01, and *** P <0.0001). n = 10 for Con, IF, and EQ; n = 13 for E2 uteri; n = 9 for E2 breast

All procedures involving these animals were conducted in compliance with state and federal laws, standards of the U.S. Department of Health and Human Services, and guidelines established by the Wake Forest University Animal Care and Use Committee (ACUC). The facilities and laboratory animal program of Wake Forest University are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.

Hysterectomy and Breast Biopsies

At the end of the dietary treatment period, the animals were anesthetized with ketamine, butorphanol, and isoflurane for hysterectomy, breast biopsy, blood collection, vaginal cytology, and body weight measurement. Surgical procedures were performed by an experienced veterinary surgeon (C.J.L.). For the hysterectomy, the ventral abdomen was given an aseptic prep and the uterus was removed via laparotomy and weighed. For the breast biopsy, a 1.5-cm incision was made in a preselected breast quadrant and a small (~0.5 gram) sample of mammary gland was removed. The animals were monitored and given analgesia during recovery following ACUC-approved clinical procedures. A portion of the uterine and mammary gland samples were flash-frozen in liquid nitrogen for mRNA analysis, while the remaining tissue was fixed at 4°C in 4% paraformaldehyde for 24 h, transferred to 70% ethanol, and then processed for histology using standard procedures.

Serum Isoflavonoids

Serum concentrations of the major soy isoflavonoids (genistein, daidzein, equol) were determined by liquid chromatographic-photodiode array mass spectrometric analysis at the Cancer Research Center of Hawaii, using techniques described previously [48]. Measurements were performed on serum samples collected 4 h after feeding. Isoflavonoid concentrations were measured before and after treatment of the samples with deconjugating enzymes to determine aglycone (unconjugated) and total concentrations. Conjugated isoflavonoids were calculated by subtracting the aglycone fraction from the total. Equol enantiomers were also measured in a randomly selected subset of serum samples (n = 6 IF, n = 6 EQ). For these, chiral analysis was performed at the National Center for Toxicological Research using HPLC separation and electrospray tandem mass spectrometry (ES-MS/MS) with isotope dilution quantification [32].

Serum Estrone

Serum estrone concentrations were measured on 24-h fasted serum samples collected at the time of biopsy. Estrone was quantitated by radioimmunoassay (RIA) using a commercially available kit and protocol from Diagnostic Systems Laboratories (E1, DSL-8700; Webster, TX). Assays were performed in the Clinical Pathology Laboratory at the Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine.

Morphometry and Histology

Uterine area was measured at baseline and after treatment by transabdominal ultrasound using a SonoSite 180 portable ultrasound machine with a 5.0 MHz linear transducer (SonoSite, Bothell, WA). Uterine thickness and maximal transverse cross-sectional area were then measured on a static representative digital image. Fixed tissue sections were stained with hematoxylin and eosin (H&E) and digitized using a Hitachi VK-C370 camera and video capture board (Scion LG-3; Scion, Inc., Frederick, MD). Two transverse uterine sections were made for each animal, one immediately caudal to the uterotubal junction and the other 3 mm caudal to the first section. Endometrial thickness and glandular area were quantified by histomorphometry, using techniques described previously [29, 45]. Endometrial thickness was measured in triplicate at sites of maximal perpendicular depth on both uterine sections; for statistics, the average of these six measurements was used for each animal. For glandular area measurements, three microscopic fields were randomly selected on each uterine section and examined at a magnification of 20x. Glandular area was determined by manual tracing of glandular endometrial units and expressed as a percentage of the total area examined. All morphology measurements were taken using public domain software (NIH ImageJ 1.33, available at http://rsbweb.nih.gov/ij/). H&E-stained uteri were also evaluated qualitatively for histologic changes by a board-certified veterinary pathologist (J.M.C.).

Immunohistochemistry

Immunostaining procedures were performed on fixed, paraffin-embedded uterine and mammary gland tissues using commercially available primary monoclonal antibodies for the proliferation antigen MKI67 (antigen identified by monoclonal antibody Ki-67, clone MIB1, Dako, Carpinteria, CA), progesterone receptor (PGR) (NCL-PGR, Novocastra, Newcastle–upon–Tyne, U.K.), and estrogen receptor alpha (ESR1, NCL-ER-6F11, Novocastra). Antibodies were diluted 1:50 for MKI67, 1:10 (uterus) or 1:100 (breast) for PGR, and 1:100 for ESR1. Staining methods included antigen retrieval with citrate buffer (pH 6.0), biotinylated rabbit anti-mouse Fc antibody as a linking reagent, alkaline phosphatase-conjugated streptavidin as the label, and Vector Red as the chromogen (Vector Laboratories, Burlingame, CA). Cell staining was quantified by a computer-assisted counting technique, using a grid filter to select cells for counting and our modified procedure of cell selection, described previously [45]. Numbers of positively stained cells were measured as a percentage of the total number examined (100 cells per slide). For mammary gland, a single section was counted; for uterus, two slides were counted. Adjacent tissue sections from each animal were used for histomorphometry and immunostaining.

Quantitative Real-Time RT-PCR

Uterine and mammary gland expression of chemokine (C-X-C motif) ligand 12 (CXCL12, also known as SDF1), progesterone receptor (PGR), trefoil factor 1 (TFF1, also known as pS2), and estrogen receptor alpha (ESR1) were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). CXCL12, PGR, and TFF1 are upregulated through ESR1-mediated pathways and thus serve as markers of ligand-dependent estrogen receptor activation within breast and/or uterine tissue [49–51]. Macaque-specific qRT-PCR primer-probe sets were generated for TFF1, ESR1, and the internal control genes GAPDH and ACTB (beta actin). Primer/probe sequences for GAPDH (GenBank Accession number: DQ464111) and TFF1 (GenBank Accession number: DQ464113) have been published previously [27]. Sequences for ACTB and ESR1 were as follows: ACTB-forward 5'-ACCCCAAGGCCAACCG-3', ACTB-reverse 5'-CCTGGATGGCCACGTACATG-3', ACTB-probe 5'-AAGATGACCCAGATCATG-3' (GenBank Accession number: DQ464112); and ESR1-forward 5'-TCACATGATCAACTGGGCAAAGA-3', ESR1-reverse 5'-AGAAGGTGGACCTGATCATGGA-3', ESR1-probe 5'-CACAAAGCCTGGCACCC-3' (GenBank Accession number: DQ469336). Human ABI Taqman primer-probe sets were used for CXCL12 (Assay ID: Hs00171022_m1) and PGR (Assay ID: Hs00172183_m1). All probe sequences covered exon boundaries to eliminate any genomic DNA amplification. RNA was extracted from frozen uterine and mammary tissues using TRI Reagent (Molecular Research Center, Cincinnati, OH), quantitated, screened for intactness using gel electrophoresis, and reverse-transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City CA). Real-time PCR reactions (20 µl volume) were performed on an Applied Biosystems ABI PRISM 7000 Sequence Detection System using Taqman Universal Master Mix and associated reagents. The thermocycling protocol involved initial incubations of 2 min at 50° and 10 min at 95°, followed by 40 PCR cycles of 95° for 15 sec and 60° for 1 min. Gene expression is expressed relative to internal control genes (GAPDH, ACTB) and stock breast tissue (as an external calibrator) using the formula 2–{Delta}{Delta}Ct, as described in the Applied Biosystems User Bulletin #2 (available online at http://www.ukl.uni-freiburg.de/core-facility/taqman/user_bulletin_2.pdf). The average Ct value of GAPDH and ACTB was used for relative calibration. Calculations were performed using ABI Relative Quantification SDS Software v1.1.

Vaginal Maturation

Vaginal keratinocytes were collected from the anterior vagina with a cotton swab, rolled onto a glass slide, and fixed using a commercial fixative (Spray-Cyte, Surgipath Medical Industries, Richmond, IL). Slides were stained using a modified Papanicolau method. Maturation value (MV) was calculated using the following formula: MV = (0.2 x % parabasal cells) + (0.6 x % intermediate cells) + (% of superficial cells).

Statistical Analysis

A general linear model was used to determine group means and test for significant group differences following treatment. All measurements were made blinded to treatment group. For uterine measures, endometrial thickness was unmeasurable (no defined lumen) in four samples (3 Con, 1 EQ) and uterine area in 3 samples (1 Con, 1 IF, 1 EQ). For breast measures, lobular epithelium was absent in one sample (Con) and ductal epithelium in nine samples (4 Con, 3 IF, 2 EQ), reducing sample size for these particular endpoints. All variables were evaluated for their distribution and equality of variances between groups, and log10 transformations were performed where appropriate to improve normality and homogeneity of variance. For log-transformed data, reported values were retransformed to the original scale using the inverse log. Data are reported either as mean (± standard error) for untransformed data (Figs. 2–4) or mean (90% confidence interval) for retransformed data (Fig. 5, Tables 1–2). Data were analyzed using the SAS statistical package (version 8; SAS Institute, Cary, NC). A two-tailed significance level of 0.05 was chosen for all comparisons.


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  		TABLE 1. Serum isoflavonoid concentrations 4 h after feeding control, isoflavone (IF), and equol (EQ) diets

RESULTS

Body Weight

Dietary intake was similar among the three treatment groups. Mean body weights were 3.68 ± 0.20, 3.72 ± 0.20, and 3.65 ± 0.20 kg at baseline (ANOVA P = 0.96) and 3.65 ± 0.20, 3.67 ± 0.20, and 3.60 ± 0.20 kg at posttreatment (ANOVA P = 0.96) for the control, IF, and EQ groups, respectively. Average change in body weight over the 1-mo treatment period was –0.03 ± 0.04, –0.05 ± 0.04, and –0.05 ± 0.04 kg for control, IF, and EQ groups (ANOVA P = 0.89), none of which were different from zero change (P > 0.1 for all).

Serum Estrone

Serum estrone concentrations at the end of the study were 52.4 (48.8–56.4) pg/ml in the control group, 44.2 (41.1–47.5) pg/ml in the IF group, and 43.13 (40.1–46.4) pg/ml in the EQ group (ANOVA P = 0.14). These values are in the reported range for postmenopausal women [52].

Serum Isoflavonoids

Total serum isoflavonoid concentrations were significantly higher in both the IF and EQ groups compared to the control group (P < 0.0001). Four hours after feeding, mean total serum isoflavonoids were below detectable limits (<20 nmol/L) in the control group, 2570.7 nmol/L in the IF group, and 6944.8 nmol/L in the EQ group (P = 0.01 vs. IF). Total isoflavonoid concentrations ranged from 641.4 to 6748.2 nmol/L (median, 3045.8 nmol/L) in the IF group and from 3239.2 to 12386.9 nmol/L (median, 6548.3 nmol/L) in the EQ group. Average serum concentrations were at least 3 times higher in the IF group and at least 8 times higher in the EQ group than nonfasted values reported for women consuming a soy-based Asian diet, which range from about 300 [53] to 800 nmol/L [54]. Despite these high circulating levels, greater than 99% of total serum isoflavonoids were in a conjugated form in both the IF (99.1%) and EQ (99.5%) groups (Table 1). Among isoflavonoids, the fraction of unconjugated genistein and daidzein (1%–2%) was generally higher than that of equol (≤0.5%).

Equol was the predominant circulating isoflavonoid, comprising 72.5% of total isoflavonoids in the IF group and 99.7% in the EQ group. Serum equol resulting from the IF diet was composed exclusively of the S enantiomer (P > 0.0001 for S vs. R), while serum equol in the EQ group contained roughly equivalent amounts of the S and R enantiomers (S/R = 1.15, P = 0.73 for S vs. R) (Table 1). Total equol concentrations from chiral analysis (R + S) were highly correlated with concentrations measured by photodiode array mass spectrometry (r = 0.99, P < 10–8).

Uterine Morphology and Proliferation

The primary endpoint in this study was endometrial stimulation, as determined by morphologic measures and endometrial gland expression of the proliferation antigen MKI67. No significant differences in uterine weight, endometrial thickness, or epithelial area were present among treatment groups (ANOVA P > 0.1 for all) (Fig. 2, A–C). Uterine area did not differ among groups before (ANOVA P = 0.23) or after (ANOVA P = 0.44) treatment or between baseline and treatment within each group (P > 0.1 for all) (Fig. 2D). Morphologic values for all groups were consistent with those of previous ovariectomized control animals and significantly lower than historical values following estrogen treatment (Table 2). Histologic evaluation of uteri revealed diffuse atrophy in all groups, characterized by simple, well-spaced glands, cuboidal epithelium, and densely packed stromal cells (Fig. 2E). Typical estrogenic effects would include a thickened endometrium, more columnar epithelial cells, increased glandular complexity, and stromal edema. No such changes were seen, and no hyperplastic lesions were noted in any of the groups.

MKI67 expression is an important marker of hormone-associated risk in our model [45, 55], while progesterone receptor (PGR) is a reliable marker of estrogen exposure [45, 50]. Immunostaining of endometrium for MKI67 and PGR was not significantly different among treatment diets in either superficial or basal epithelium (ANOVA P > 0.1 for all) (Fig. 3). Epithelial MKI67 labeling was also similar to historic control animals and significantly lower than reference values for estrogen-treated animals (Table 2). Within the endometrial stroma, a slight but significant increase in basal gland MKI67 expression was found in the IF group (P = 0.01 vs. control group), while no group differences in stromal PGR were noted (ANOVA P = 0.51) (Fig. 3, B and C).


Figure 3
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  		FIG. 3. Endometrial expression of markers for cellular proliferation (MKI67 antigen) and estrogen exposure (progesterone receptor, PGR). A) Immunostaining for MKI67 in glandular epithelium in control (Con), isoflavone (IF), and equol (EQ) groups compared with a reference animal given 1.0 mg/woman/day of estradiol (E2). Red nuclei indicate positive cells. Bar = 25 µm. Expression of MKI67 (B) and PGR (C) were not significantly different among groups in either superficial or basal epithelium. Within stroma, MKI67 was low but significantly greater in the basal compartment in the IF group compared to control. n = 10 per group. Asterisks indicate significant differences from the control group (* P < 0.05)

Breast Proliferation and Estrogen Exposure

Breast epithelial expression of MKI67, PGR, and ESR1 did not differ significantly among treatment groups for either lobules or ducts (ANOVA P > 0.1 for all) (Fig. 4, A–D). As in the uterus, epithelial proliferation was comparable to historic control animals and lower than reference values for estrogen-treated animals (Table 2). On histology, all breast biopsies exhibited diffuse glandular atrophy, and no proliferative lesions were noted.

Uterine and Breast Gene Expression

To evaluate high-dose IF and EQ effects on gene markers of estrogen receptor activity, we next measured intratissue expression of estrogen receptor alpha (ESR1) and several ESR1-driven genes: TFF1, CXCL12, and PGR. Tissues from historical estradiol-treated animals were analyzed concurrently for reference. Within uterine tissues, gene expression for CXCL12 and PGR was significantly higher in both IF and E2 groups, while ESR1 expression was higher only in the IF group (P < 0.05 vs. control group) (Fig. 5, A–C). Within breast tissue, expression of TFF1 and PGR was significantly higher in the E2 group (P < 0.05 for both) but not in the IF or EQ groups (P > 0.1 vs. control) (Fig. 5, D–F). Incidentally, TFF1 expression was below detectable limits within uterine tissues, while CXCL12 was constitutively expressed in breast tissues (data not shown).

Vaginal Maturation

Vaginal keratinocyte maturation provides one of the most sensitive markers of reproductive tract estrogen exposure [28]. Maturation index values were 47.7 ± 4.0, 41.2 ± 4.0, and 38.1 ± 4.0 at baseline and 43.8 ± 4.1, 40.2 ± 4.1, and 34.0 ± 4.1 after treatment in the control, IF, and EQ groups, respectively. No differences were found between baseline and treatment maturation values for any of the groups (ANOVA P > 0.1 at both timepoints).

DISCUSSION

In this study we used a postmenopausal primate model to evaluate the short-term effects of high-dose soy isoflavonoid supplements on reproductive tissues. We gave the more bioactive aglycone forms and administered them in isolation (i.e., outside the native soy protein matrix), excluding potential moderating effects of other soy components [56]. Despite serum isoflavonoid concentrations up to 10 times greater than human values after a high-soy meal, we found little evidence of adverse stimulatory effects in uterine or mammary gland tissues. The only estrogenic effects noted were a slight increase in stromal proliferation and an increase in two estrogen-driven genes in the uteri of the isoflavone group; uterine and mammary gland histology, morphometry, and epithelial proliferation remained unaffected by both isoflavone and equol treatment. These findings suggest that comparable soy isoflavonoid exposure would have minimal estrogenic effects in human reproductive tissues and would not likely contribute to increased uterine or breast cancer risk.

Our findings are consistent with the majority of previous soy/soy isoflavonoid trials in women, which have generally used isoflavonoid doses ≤150 mg per day and found no endometrial or breast effects [15–21]. In the only previous study in women to report uterotrophic effects of soy isoflavones [12], serum estrogens, serum isoflavonoid profile, body mass index, and other relevant factors were not analyzed, raising uncertainty about the causative role of isoflavonoid intake. The two studies reporting estrogen-like effects of soy in human breast tissue are also less than definitive, as neither reported an increase in breast proliferation and each study was confounded by either repeated sampling effects [13] or varying concurrent breast diseases [14]. However, we should make clear that the estrogenic properties of soy formulations in reproductive tissues may vary depending on isoflavonoid composition and bioavailability. Equol was the major serum isoflavonoid in both IF and EQ groups in this study, while genistein is the major serum isoflavonoid in most people consuming soy/isoflavonoid supplements. Several recent studies suggest that dietary equol may have less estrogenic potential in vivo than genistein or daidzein [35, 57, 58], an idea supported in part by our data showing increased uterine PGR and CXCL12 gene expression in the IF but not EQ group. It remains uncertain whether equol production (as seen in our model) may preclude or mask any estrogenic effects of nonequol isoflavonoids.

The lack of estrogenic proliferative effects from higher-dose isoflavonoids contrasts with certain data from in vitro and rodent models [7–10]. This discrepancy may relate to several factors, including isoflavonoid dose, route of administration, formulation, the amount of background estrogen, and estrogen sensitivity among the different models. In vitro studies have shown clear estrogen-like effects of isoflavonoids at doses ranging from <1 to 10 µM [4–6], similar to serum concentrations in this study. Such dose effects in culture may overestimate isoflavonoid exposure, however, due to increased conjugation and other metabolic factors found only in vivo. Cells in culture are directly exposed to aglycone isoflavones, while data in this study show that >99% of serum isoflavonoids from dietary intake remain in an essentially inactive conjugated state. This distinction may be particularly important for daidzein and equol, as shown by a recent study of mice in which clear dose-dependent estrogenic effects in vitro were not seen at corresponding serum levels in vivo [57]. (Interestingly, this dichotomy was not seen previously with genistein [7].) While it is possible that aglycone isoflavonoids may accumulate over time within tissues (to greater concentrations than those seen in serum) [59], the dynamics of intratissue isoflavonoid conjugation, bioaccumulation, and metabolism are not well understood.

In studies of mice and rats, uterotrophic isoflavone effects occur at doses far greater than those given in previous studies of monkeys or women. For example, calibrating doses to a caloric scale (to better account for metabolic differences across species) shows that adult rats generally require a dose of at least 360 mg isoflavones per 1800 kcal (or ~40 mg isoflavones per kg body weight) to elicit significant uterotrophic effects [41, 60]. To our knowledge, no rodent studies using dietary soy (as opposed to a purified isoflavone supplement) have achieved this dose or reported uterotrophic isoflavone effects. Many of the relevant studies in rodents have also used purified genistein rather than an isoflavone mixture. As mentioned above, recent studies have found no estrogenic effects of dietary equol on MCF-7 breast cancer cell transplants in nude mice [57] and daidzein on estrogen-related breast differentiation [58], both in contrast to previous reports using genistein [7, 61]. Considered in this context, it seems possible that a high-dose genistein-only supplement may have provided results more consistent with prior rodent studies. Lastly, we have shown previously that isoflavone effects may depend on the amount of background estrogen present [28]. Ovariectomized rodents lack adrenal-derived estrogen precursors [62] and thus have lower serum and tissue estrogens compared to postmenopausal primates. This lack of baseline estrogens in ovariectomized rodents may negate endogenous estrogen interactions and potentially sensitize estrogen-responsive tissues.

Results of this study show that high doses of equol, whether derived naturally as a single isomer from gut microbial production (in the IF group) or given as a purified racemic mixture (in the EQ group), lack estrogenic effects in the primate breast or uterus. This finding is consistent with several longer studies in our model using isoflavone doses ranging from 60 mg to 240 mg per 1800 kcal [28–30]; collectively, these studies raise the question of whether dietary equol is capable of producing estrogen agonist effects at all in primates. Based on current data, equol estrogenicity appears limited to studies administering equol (aglycone) to estrogen sensitive reporter systems or cells in culture [32, 34], injecting equol (aglycone) in ovariectomized rodents [33, 35], and feeding supraphysiologic oral doses of equol to ovariectomized mice (which may still produce only weak effects) [35]. Our data also confirm (in the monkey model) that equol derived from gut metabolism of daidzein is exclusively S-equol and that this pattern is likely due to preferential S-equol production rather than differential bioavailability between R and S enantiomers. Both of these findings are consistent with prior reports in human subjects [39].

Cell culture and rodent models indicate that exposure to soy isoflavonoids may have adverse estrogenic and potentially cancer-promoting effects in reproductive tissues. Nevertheless, the specific isoflavonoid formulation or dose required for estrogenic effects in women has not been identified despite over five decades of study. In this report we used a postmenopausal primate model in an attempt to identify unequivocal proliferative effects of high-dose soy isoflavone or equol supplements. No such effects were seen. These findings suggest that dietary levels of soy isoflavonoid supplements, in particular those containing equol or taken by equol-producers, do not pose a significant risk for promoting uterine or breast cancer. This information may be of value in forming dietary guidelines on soy consumption and in the planning of future clinical trials using soy isoflavonoids.

ACKNOWLEDGMENTS

The authors thank Jean Gardin, Chuck Boyd, Joseph Finley, Hermina Borgerink, Lisa O'Donnell, Maryanne Post, Gerald Perry, and Laurie Custer for their technical contributions. We also thank Dr. Thomas C. Register for generously providing ESR1 primers and probes. Statistical review was provided by Dr. Haiying Chen. Isoflavone and equol supplements were generously provided by Solae, a division of Dupont, St. Louis, MO.

FOOTNOTES

1 Supported by grants from the National Institutes of Health (NCCAM R01-AT00639, NCRR T32 RR 07009, NCI P30-CA71789) and the American Cancer Society (IRG-93-035-09). Back

2 Correspondence: Charles E. Wood, Department of Pathology/Section on Comparative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040. FAX: 336 716 1515; chwood{at}wfubmc.edu Back

Received: 1 March 2006.

First decision: 20 March 2006.

Accepted: 23 May 2006.

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