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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3462-3468
Copyright © 2004 by The Endocrine Society

Breast and Uterine Effects of Soy Isoflavones and Conjugated Equine Estrogens in Postmenopausal Female Monkeys

Charles E. Wood, Thomas C. Register, Mary S. Anthony, Nancy D. Kock and J. Mark Cline

Comparative Medicine Clinical Research Center, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157-1040

Address all correspondence and requests for reprints to: Charles E. Wood, D.V.M., Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1040. E-mail: chwood{at}wfubmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study we evaluated the long-term effects of soy isoflavones on intermediate markers of cancer risk in the normal postmenopausal monkey breast and uterus. Ovariectomized female cynomolgus monkeys were randomized to receive one of three diets for 36 months: 1) isoflavone-depleted soy protein isolate (SPI–) (n = 57); 2) soy protein isolate with the equivalent of 129 mg/d isoflavones (SPI+) (n = 60); or 3) isoflavone-depleted soy protein isolate with conjugated equine estrogens at a dose scaled to approximate 0.625 mg/d in women (n = 62). End points included breast and uterine proliferation markers, sex steroid receptor expression, and serum estrogens. Epithelial proliferation and progesterone receptor expression in the breast and uterus were significantly higher in the conjugated equine estrogen group, compared with SPI+ and SPI– groups, whereas no significant differences were detected between the SPI+ and SPI– groups. SPI+ treatment resulted in significantly lower serum concentrations of estrone (P < 0.01) and estradiol (P < 0.05) vs. SPI–. Within the SPI+ group, serum isoflavone concentrations were inversely correlated with serum estrone and mammary glandular area. These findings suggest that high dietary levels of soy isoflavones do not stimulate breast or uterine proliferation in postmenopausal monkeys and may contribute to an estrogen profile associated with reduced breast cancer risk.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOY ISOFLAVONES ARE phytoestrogenic compounds widely marketed and used as a natural alternative to traditional hormone therapy in postmenopausal women (1). Dietary supplementation with soy isoflavones is particularly common in women at high risk for breast cancer (1, 2) despite controversial evidence on the role of isoflavones in cancer risk. Epidemiologic studies have shown lower rates of breast and endometrial cancer among women consuming higher amounts of soy (3, 4, 5, 6, 7, 8, 9). Soy isoflavones have also been shown to protect against carcinogen-induced mammary tumors in rodents (10, 11) and induce a variety of antiproliferative and proapoptotic responses in cell culture (12, 13, 14, 15, 16, 17). In contrast, several studies have found that the soy isoflavone genistein stimulates growth of MCF-7 breast cancer cells in culture and as xenoplants in nude mice at low micromolar concentrations similar to those found in the serum of women consuming a soy-based diet (18, 19). Several short-term studies have also reported estrogen-like responses in breast tissue (20) or nipple aspirates (21) of women given soy products. These studies have raised concern that isoflavones may stimulate epithelial cell growth in the breast and uterus and promote development of estrogen-responsive tumors. The purpose of this study was to evaluate markers of breast and uterine proliferation in response to long-term treatment with soy isoflavones or conjugated equine estrogens (CEE), a common estrogen replacement therapy in women, in a postmenopausal nonhuman primate model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal subjects

One hundred eighty-nine adult female premenopausal cynomolgus monkeys (Macaca fascicularis) were obtained from the Institute Pertanian Bogor, Bogor, Indonesia. The reproductive physiology of these animals is similar to that of women in several aspects, including a 28-d menstrual cycle (22) and natural ovarian senescence (23). Human and macaque breasts share similar patterns of lobuloalveolar and ductal development and regression (24), cytokeratin phenotype (25), sex steroid receptor expression (26), steroidogenic enzyme distribution (27), and responses to exogenous estrogens (28, 29). Mammary glands and uteri were obtained from 179 of these animals at necropsy. Average age at the time of necropsy was 12.0 yr as estimated from dental examination at the time of import. All procedures involving 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.

Study design

This investigation was part of a large randomized, controlled experiment designed to evaluate multisystem effects of premenopausal oral contraceptives and postmenopausal soy isoflavones and CEE in nonhuman primates. Other aspects of this study have been reported elsewhere (30, 31, 32, 33, 34). The overall study design incorporated premenopausal and postmenopausal treatment phases spanning a total of 5.5 yr, as described previously (30, 31). During the 26-month premenopausal period, half of the animals received, in the diet, a triphasic oral contraceptive (OC) (Triphasil, Wyeth-Ayerst Laboratories, Inc., Philadelphia, PA) containing varying proportions of ethinyl estradiol (0.03–0.04 mg/1800 kcal) and levonorgestrel (0.05–0.125 mg/1800 kcal) for the initial 21 d of each cycle, with a placebo given for the final 7 d, as in women. All monkeys consumed a diet containing 17% of calories from protein, 45% from fat, 38% from carbohydrates, and 0.28 mg cholesterol/kcal.

After the premenopausal phase, all animals were ovariectomized to make them surgically menopausal. The postmenopausal study followed a three-group, parallel arm design and lasted for 36 months. All animals remained in their original social groups. Using a stratified randomization scheme based on premenopausal social group and OC exposure, monkeys were randomly assigned to one of three postmenopausal treatment groups: 1) soy protein isolate depleted of isoflavones (SPI–); 2) soy protein isolate with isoflavones (SPI+); and 3) SPI– diet with CEE. The SPI– diet contained isolated soy protein that had been alcohol washed to remove the isoflavones. The SPI+ treatment group received soy protein isolate containing isoflavones at a dose approximately equivalent to 129 mg/d for women (~91 mg genistein, 31 mg daidzein, and 7 mg glycitein). The CEE group received alcohol-washed soy protein isolate (SPI–) plus CEE (Premarin, Wyeth-Ayerst Laboratories) at a dose comparable with 0.625 mg/d for women. CEE and isoflavone doses were scaled to 1800 kcal of diet (the estimated daily intake for U.S. women) so that monkeys fed 120 kcal of diet per kilogram of body weight (BW) consumed approximately 8.6 mg isoflavones/kg BW or 0.042 mg CEE/kg BW. This caloric adjustment of dose accounts for differences in metabolic rates between the monkeys and human subjects.

The isolated soy proteins used for this study were generously provided by Solae, a division of DuPont (St. Louis, MO). The unextracted soy protein (SUPRO 670-HG) contained, on the average, 1.105 mg genistein, 0.365 mg daidzein, and 0.08 mg glycitein/g soy protein isolate, whereas the alcohol-extracted soy protein (SUPRO 670-IF) contained 0.04 mg genistein, 0.01 mg daidzein, and 0.01 mg glycitein/g isolate (expressed in aglycone units). Soy protein isolate was heat treated before the alcohol extraction to inactivate protease inhibitors. Further details of SPI+ and SPI– diets have been described previously (35). Monkeys were fed 120 kcal/kg BW·d split into two feedings. Diets were formulated to be isocaloric and equivalent for the macronutrients. Serum isoflavone concentrations (genistein, daidzein, equol, dehydrodaidzein, and o-desmethylangolensin) were measured in serum collected 4 h after provision of the morning diet at month 34 of the postmenopausal period. Isoflavone concentrations were determined by HPLC-mass spectrometry in the laboratory of Dr. Stephen Barnes (University of Alabama, Birmingham, AL), as reported previously (31). The mean total serum isoflavone concentration of the SPI+ group was 805 ± 88 nmol/liter aglycone equivalents. Total serum isoflavone concentrations from all SPI– animals were less than 80 nmol/liter.

Necropsy procedures

At the end of the postmenopausal phase, monkeys were sedated with ketamine and euthanized using sodium pentobarbital (100 mg/kg, iv) as recommended by the Panel on Euthanasia of the American Veterinary Medical Association. At necropsy, mammary tissues and uteri were removed and fixed immediately at 4 C in fresh 4% paraformaldehyde solution (Fisher Scientific, Raleigh, NC). After 24 h, tissues were transferred to 70% ethanol. Mammary tissues were sectioned sagitally through the nipple and trimmed to 3 cm in length. Uteri were sectioned transversely at the point of greatest diameter. Fixed tissues were embedded in paraffin and sectioned to 5 µm in thickness for hematoxylin and eosin (H&E) and immunohistochemical staining.

Histomorphometry

Breast and uterine epithelial area and thickness were quantified by histomorphometry, as described previously (28). Briefly, H&E-stained slides were digitized using a Hitachi VK-C370 camera and video capture board (Scion LG-3; Scion, Inc., Frederick, MD), and measurements were taken with public domain software (NIH Image version 1.60; available at http://rsb.info.nih.gov/nih-image/). Three microscopic fields were randomly selected and examined at a magnification of x20; epithelial area was determined by manual tracing of lobuloalveolar (breast) or glandular (endometrium) units and expressed as a percentage of the total area examined. Mammary pad and endometrial thickness were measured at the point of greatest perpendicular depth. H&E-stained mammary glands and uteri were also evaluated qualitatively for histologic changes by two board-certified veterinary pathologists (J.M.C., N.D.K.). All measurements were made blinded to treatment group. Adjacent tissue sections from each animal were used for histomorphometry and immunostaining.

Immunohistochemistry

Immunostaining procedures were performed on fixed, paraffinembedded tissues using commercially available primary monoclonal antibodies for Ki67 (Ki67-MM1, Dako, Carpinteria, CA), progesterone receptor (PR) (NCL-PGR, Novocastra, Newcastle-upon-Tyne, UK), and estrogen receptor-{alpha} (ER{alpha}) (NCL-ER-6F11, Novocastra). Ki67 and PR were used as markers of proliferation and estrogenic stimulation, respectively. Staining methods included antigen retrieval with citrate buffer (pH 6.0), biotinylated rabbit antimouse Fc antibody as a linking reagent, alkaline phosphatase-conjugated streptavidin as the label, and Vector Red as the chromogen. Immunostaining components were obtained as a kit (Vector Laboratories, Burlingame, CA). Staining for Ki67, PR, and ER{alpha} was quantified by a computer-assisted counting technique, using a grid filter to select cells for counting (36) and our modified procedure of cell selection, described previously (26). Numbers of positively stained cells were expressed as a percentage of the total number examined (100 cells).

Hormone assays

Hormone concentrations were measured from serum collected at a preselected time point in month 29 of the postmenopausal period. Estrogen assays were run on a subset of animals based on serum availability at this time point. Blood samples were collected via femoral puncture after the animals entered a squeeze cage and were sedated with ketamine HCl (10 mg/ml, im), 4 h after the morning meal within 1 h of each other. Blood collections were staggered so that all animals in a single pen were sampled on the same day, and all animals were sampled at the same time from the start of treatment. Serum was frozen at –80 C immediately after collection for later analysis. Concentrations of estradiol (E2), estrone (E1), and estrone sulfate (E1-S) were quantitated by RIA using commercially available kits and protocols from Diagnostic Systems Laboratories (E2, DSL-4800 ultrasensitive; E1, DSL-8700; E1-S, DSL-5400; Webster, TX). For E2 assays, serum (0.5 ml) was extracted by adding ethyl ether (4 ml) and vortexing for 5 min. The aqueous layer was frozen in a dry ice/isopropanol bath, and the organic phase was decanted. Extracts were dried and reconstituted with zero standard serum from the RIA kit. For E2 values below the lowest standard in the kit (5.0 pg/ml), a predetermined surrogate value of 4.0 pg/ml was used for statistical analysis. Assays were performed at either the Yerkes Regional Primate Research Center Endocrinology Laboratory (E1) or the Clinical Pathology Laboratory at the Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine (E2, E1-S). Ranges for calibration standards are as follows: E2 (5–750 pg/dl), E1 (7.5–2000 pg/ml), and E1-S (0.05–90 ng/ml). Intraassay coefficients of variation are as follows: E2, 2.70%; E1, less than 10%; and E1-S, 9.76%.

Statistics

All data were subjected to two-way ANOVA using premenopausal treatment (OC, no OC) and postmenopausal treatment (SPI–, SPI+, CEE) as between-subject factors. Body weight was significantly correlated with serum E1 and E1-S and was thus used as a covariate for all measures. All variables were evaluated for their distribution and equality of variances between groups, and log10 transformations were performed for all variables to improve normality and homogeneity of variance. Data in all figures represent retransformed values. A two-tailed significance level of 0.05 was chosen for all comparisons. Analyses were done using the SAS statistical package (version 6.08; SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Age, body weight, and body mass index (BMI)

No significant differences were observed among postmenopausal groups in age, BW, and BMI (BW/trunk length2) during the postmenopausal period (Table 1Go). Mean change in body weight during postmenopausal treatment was +0.51 ± 0.08, +0.54 ± 0.08, and +0.46 ± 0.07 kg for SPI–, SPI+, and CEE groups, respectively (by ANOVA, P = 0.74). Mean change in postmenopausal BMI was +5.30 ± 0.94, +5.13 ± 0.94, and +3.85 ± 0.91 kg/m2 for SPI–, SPI+, and CEE groups, respectively (by ANOVA, P = 0.48).


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  		TABLE 1. Age, body weight, and BMI of postmenopausal groups

 
Mammary gland and uterine measures

Three years of SPI+ treatment did not significantly alter epithelial area, Ki67 expression, or sex steroid receptor expression in the breast or uterus, compared with the SPI– group (Figs. 1–3GoGoGo). The only measure that was significantly different between SPI+ and SPI– groups was mammary thickness, which was lower in the SPI+ animals (P < 0.05). In contrast, CEE treatment resulted in significantly greater mammary gland and endometrial epithelial area, Ki67, and PR, compared with both SPI– and SPI+ groups (Figs. 1–3GoGoGo). ER{alpha} expression in the CEE group was significantly higher in the mammary gland but not endometrium relative to SPI– and SPI+ groups (Figs. 1Go and 2Go). On histology, mild to moderate lobular enlargement was seen in the mammary gland of two of 57 SPI–, none of 60 SPI+, and 31 of 63 CEE-treated animals. Small focal papillary ductal hyperplasias were seen in three of 57 SPI–, none of 60 SPI+, and one of 63 CEE-treated animals. Endometrial hyperplasia of moderate to marked degree was present in none of 57 SPI–, none of 60 SPI+, and 26 of 62 CEE-treated animals. Minimal or mild proliferative changes were seen in three of 57 SPI–, 1 of 60 SPI+, and 32 of 60 CEE-treated animals. All animals in this study had histologic evidence of previous pregnancy as indicated by adventitial expansion and accumulation of extracellular matrix around the myometrial veins (37).



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  		FIG. 1. Mammary gland immunohistochemical measurements of surgically postmenopausal monkeys given SPI– (n = 55), SPI+ (n = 55), or SPI– diet with CEE (n = 60) for 36 months. Data represent retransformed means ± SE. Ki67, MM1 proliferation marker; ER, ER-{alpha}. *, Significant difference from SPI– group (P < 0.01) and SPI+ group (P < 0.05). **, Significant difference from SPI– and SPI+ groups (P < 0.01).

 


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  		FIG. 2. Endometrial immunohistochemical measurements of surgically postmenopausal monkeys given SPI– (n = 55), SPI+ (n = 56), or SPI– diet with CEE (n = 61) for 36 months. Data represent retransformed means ± SE. Ki67, MM1 proliferation marker; ER, ER-{alpha}. **, Significant difference from SPI– and SPI+ groups (P < 0.01).

 


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  		FIG. 3. Mammary gland and endometrial histomorphometric measurements of surgically postmenopausal monkeys given SPI– (n = 55), SPI+ (n = 55), or SPI– diet with CEE (n = 60) for 36 months. Data represent retransformed means ± SE. *, Significant difference from SPI– and CEE groups (P < 0.05). **, Significant difference from SPI– and SPI+ groups (P < 0.01).

 
Substantial interindividual variation was observed in total plasma isoflavone concentrations within the SPI+ group (31). Stratification of SPI+ animals into tertiles based on total plasma isoflavones revealed an inverse association with mammary epithelial area (p for trend = 0.02) (Fig. 4Go); Ki67 and PR expression levels were also lower in the high-isoflavone tertile, although tertile differences were not statistically significant (data not shown). Prior treatment with OCs had no significant carryover effects on mammary gland proliferation at the end of the postmenopausal period; however, postmenopausal uterine weight, endometrial thickness, and endometrial epithelial area were significantly lower in animals previously treated with OCs (Table 2Go).



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  		FIG. 4. Mammary gland epithelial area and serum estrone measurements for the animals fed SPI+ divided into tertiles based on plasma isoflavone concentrations (n = 18–19/group). Data represent retransformed means ± SE. *, Significant difference from low-isoflavone tertile (P < 0.05).

 

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  		TABLE 2. Carryover effects of prior oral contraceptive treatment on postmenopausal mammary gland and uterine proliferation

 
Serum estrogens

SPI+ treatment resulted in significantly lower serum E1 (–24%) and E2 (–20%) concentrations vs. the SPI– group (Table 3Go). E1-S (–14%) was lower in the SPI+ group but not significantly different from SPI–. E2 concentrations were below the lowest standard (<5.0 pg/ml) in 76% of SPI+, 43% of SPI–, and no CEE animals. Total plasma isoflavone levels for SPI+ animals correlated inversely with serum E1 concentrations (r = –0.38, P < 0.005), with markedly lower E1 in the high-isoflavone tertile when compared with the other tertiles (P < 0.01) (Fig. 4Go). Among isoflavone metabolites, the negative correlation with E1 was strongest for equol (r = –0.42; P < 0.001). Body weight was a significant covariate for E1 (P = 0.002) and E1-S (P = 0.04) but not E2 (P = 0.11). Postmenopausal serum estrogen concentrations were not affected by prior treatment with OCs (P > 0.05 for E1, E2, and E1-S).


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  		TABLE 3. Effects of postmenopausal treatment with soy isoflavones or conjugated estrogens on serum estrogens

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first long-term randomized, controlled trial designed to evaluate soy isoflavone effects on breast and endometrial proliferation. In this 3-yr study, high dietary levels of soy isoflavones did not stimulate proliferation or estrogen-responsive markers in the normal postmenopausal breast and uterus of monkeys.

Strengths of this study included the use of a primate model, the extended duration of treatment, relatively large sample size, and prospective parallel-arm design. Limitations include the use of hormonally unstimulated surgically postmenopausal monkeys that have lower endogenous estrogen concentrations, compared with naturally menopausal women (presumably due to less adipose tissue and the lack of ovary-derived C19 estrogen precursors). This relative hypoestrogenemia in our model may have obscured potential antiestrogen effects of soy isoflavones (38) by lowering baseline proliferation. Second, the predominant isoflavone metabolite present in the serum of all isoflavone-treated animals was equol, which comprised over half of the total serum isoflavones (31). Equol is an isoflavone metabolite produced endogenously in only about one third of people consuming soy (39). Whereas the direct effects of equol have not been clearly identified, equol-producing women have previously been reported to have lower circulating estrogens than equol nonproducers (40), consistent with our data.

Results of this study suggest that the soy isoflavones may reduce concentrations of E1 and E2, which are important determinants of breast cancer risk in postmenopausal women (41). This finding is supported by epidemiologic (42) and short-term intervention (43) studies in postmenopausal women as well as in vitro studies showing that genistein may modulate key enzymes involved in estrogen biosynthesis (44, 45, 46, 47). In particular, isoflavones may inhibit 17ß-hydroxysteroid dehydrogenase type I (44, 45) and aromatase (47), which regulate peripheral synthesis of E2. However, we cannot exclude potential estrogen-lowering effects of other lipophilic compounds in soy protein such as sterols and saponins that may have been removed during isoflavone extraction.

In the present study, postmenopausal treatment with CEE increased mammary gland proliferation, ER{alpha} expression, and PR expression. This finding is similar to that of several smaller studies in monkeys (28, 29) as well as observational studies of women (48, 49). Whether a modest increase in breast proliferation as seen in this study translates into higher breast cancer risk in postmenopausal women is unclear. Evidence from several large observational studies in women indicates that current users of estrogen-only therapy have a marginally significant increase in relative risk (from 1.06 to 1.32) vs. nonusers (50, 51, 52), although consensus regarding CEE effects on breast cancer risk is still lacking. Breast cancer risk associated with hormone replacement therapy is higher in lean women with lower endogenous estrogens, compared with those with a higher BMI (53). Given the relative hypoestrogenism of our model, the effects of CEE seen in this study may be greater than those seen in more estrogen-replete postmenopausal women. It should also be noted that in our model CEE with a progestin induced greater breast proliferation than CEE alone (28, 29), consistent with human data showing increased breast cancer risk with combined therapy (51, 52). The marked hyperplastic effect of CEE in the endometrium is consistent with previous monkey data (54) as well as human studies showing increased endometrial proliferation (55) and cancer risk (56) in women taking unopposed CEE.

In this study we observed no significant carryover effects of premenopausal OC treatment on breast proliferation, in support of studies in women showing no long-term effects of prior OC use on breast cancer risk (57). In contrast, several markers of uterine proliferation were significantly lower in OC-treated animals 3 yr into the postmenopausal period. Interestingly, this finding is consistent with studies of women showing a persistent protective effect of combination OCs against endometrial cancer (58).

The role of soy isoflavones in cancer risk has received considerable debate in recent years, and safety concerns remain that soy isoflavones may promote breast and endometrial cell growth through estrogen-like actions. Findings of this study indicate that high dietary levels of soy isoflavones do not stimulate proliferation or induce markers of estrogenicity in the normal postmenopausal breast and uterus of monkeys. Furthermore, higher isoflavone concentrations may contribute to an estrogen profile associated with reduced cancer risk. These findings should be of interest to the many women at high risk of breast cancer who are currently taking supplemental soy products.


   Acknowledgments
 
The authors thank Jean Gardin, Brian McCollough, Kelly Thompson, Dianna Swaim, Tim Vest, and Matt Dwyer for their technical contributions; Professor Thomas B. Clarkson for guidance and assistance; and Professor Stephen Barnes for isoflavone measurements. For hormone measurements, we thank the staff of the Endocrine Core Lab (Yerkes Primate Research Center) and the Clinical Chemistry and Endocrinology Lab (Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine). Soy products were generously provided by Solae (a division of DuPont, St. Louis, MO).


   Footnotes
 
This work was supported by Program Project Grant HL-45666 from the National Institutes of Health/National Heart, Lung, and Blood Institute, Bethesda, Maryland, NIH/National Center for Complementary and Alternative Medicine R01-AT00639, and NIH/National Center for Research Resources T32 RR 07009.

Abbreviations: BMI, Body mass index; BW, body weight; CEE, conjugated equine estrogen; E1, estrone; E2, estradiol; ER{alpha}, estrogen receptor-{alpha}; E1-S, estrone sulfate; H&E, hematoxylin and eosin; OC, oral contraceptive; PR, progesterone receptor; SPI+, diet of soy protein isolate with isoflavones; SPI–, diet of soy protein isolate depleted of isoflavones.

Received December 2, 2003.

Accepted April 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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