American Journal of Primatology 71:722–731 (2009) REVIEW ARTICLE Estrogen/Isoflavone Interactions in Cynomolgus Macaques (Macaca fascicularis) J. MARK CLINE AND CHARLES E. WOOD Wake Forest University Primate Center, Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina Soy isoflavones are phytoestrogenic components of dietary soy, which are widely consumed for their potential health benefits. Soy isoflavones appear to decrease breast and endometrial cancer risk in human observational studies, but paradoxically stimulate growth of breast cancer cells in culture and uterine enlargement in rodents. We have shown that these compounds are not estrogenic in cynomolgus monkeys even at relatively high doses, but that they reduce estrogen-induced proliferative responses of the breast and endometrium. This effect may be mediated through estrogen receptor interactions and/or modulation of endogenous estrogen metabolism. Interindividual variation in isoflavone absorption and metabolism contributes to the degree of estrogen antagonistic effect. Our recent studies have also shown that individual isoflavone metabolites such as glyceollins may have unique selective estrogen receptor modulator-like activity, acting as tissue-specific antagonists without agonist activity. Rodent studies and human epidemiologic data suggest that timing of exposure and dose relative to endogenous estrogen concentrations are important determinants of effect, and studies of dietary soy on breast development and pubertal maturation are under way. Because soy isoflavones are both abundant in standard monkey chow diets and widely available as dietary supplements for human beings, these findings have broad relevance to the health of human and nonhuman primates. Am. J. Primatol. 71:722–731, 2009. r 2009 Wiley-Liss, Inc. Key words: genistein; breast; endometrium; diet INTRODUCTION Soy isoflavones are phytoestrogenic components of dietary soy, which are widely consumed for their potential health benefits. Soy isoflavones in phytoestrogen pharmaceutical and ‘‘nutriceutical’’ products are widely and increasingly marketed to the public [Soyatech, 2008; Soy Foods Association, 2008]. Traditional soy foods are generally recognized as safe, and the cardiovascular benefits of soy consumption have been sufficiently substantiated to warrant approval as a health claim by the Food and Drug Administration . However, the safety and efficacy of many soy and soy isoflavone supplements have not been fully evaluated, and so both the potential risks and potential benefits remain unclear. Soy isoflavones share structural similarities with the mammalian hormone 17-b estradiol (Fig. 1) and in fact are weak estrogens. They are relatively abundant in the circulation of people and animals who consume soy. Given that estrogens are active in the picomolar range of concentration and isoflavones may be present at near micromolar concentrations in blood and tissues of soy consumers, the potential for biologic effect is real. Our studies have focused on the potential for soy isoflavones to serve as r 2009 Wiley-Liss, Inc. endocrine modulators of estrogen action and estrogen metabolism in the breast and reproductive system. In general, we have found little direct effect of isoflavones but clear antagonism of cancer risk markers associated with estrogen exposure. Other investigators have clearly shown that isoflavones can produce estrogen-like promotion of breast cancer cell growth in vitro and in rodent models [Wang et al., 1996; Hsieh et al., 1998]. Regardless of whether their aggregate effect is beneficial or otherwise, the widespread consumption of isoflavones in the human diet and as supplements makes it imperative that we understand their effects in greater detail. Herein we review the evidence for direct and estrogen-modulatContract grant sponsor: NIH; Contract grant number: R01 AT000639. Correspondence to: J. Mark Cline, Wake Forest University Primate Center, Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1040. E-mail: firstname.lastname@example.org Received 21 October 2008; revised 8 February 2009; revision accepted 11 February 2009 DOI 10.1002/ajp.20680 Published online 25 March 2009 in Wiley InterScience (www. interscience.wiley.com). Estrogen/Isoflavone Interactions in Macaques / 723 Estradiol Genistein Fig. 1. Structure of the mammalian sex hormone 17-b estradiol compared with the soy isoflavone genistein. ing effects of soy isoflavones on the reproductive system in human beings and rodents in the light of our recent findings in nonhuman primates. EFFECTS OF SOY ISOFLAVONES ON THE BREAST AND REPRODUCTIVE SYSTEM IN HUMAN SUBJECTS Soy isoflavones appear to decrease breast and endometrial cancer risk in human observational studies. Dietary soy consumption early in life lowers breast cancer risk in women. Cross-cultural studies show three-fold lower age-adjusted rates of breast cancer among Asian women who eat significant amounts of soy compared with women in the United States who eat little soy [Parkin et al., 1992]. Migrant studies [Dunn, 1977; Shimizu et al., 1991; Stanford et al., 1995; Ziegler et al., 1993] have shown that women of Asian origin have higher breast cancer rates when they migrate to the United States and adopt western diets and lifestyle compared with the rates in regions of Asia with high soy consumption. This increase in breast cancer rates is more pronounced in second and third generations residing in the United States, suggesting that a transgenerational or an early-life effect may be present. Migration studies are limited by a number of confounders. The question of dietary soy effects can be focused more closely by examination of case–control and cohort studies within a geographic region. These more controlled but still observational epidemiologic studies [Dai et al., 2002; den Tonkelaar et al., 2001; Greenstein et al., 1996; Hirayama, 1990; Hirose et al., 1995; Horn-Ross et al., 2001, 2002; Ingram et al., 1997; Lee et al., 1991; Shu et al., 2001; Witte et al., 1997; Wu et al., 1996, 1998, 2002b; Yuan et al., 1995; Zheng et al., 1999] tend to show lower rates of breast cancer among those consuming higher amounts of soy, although not all studies have shown statistical significance. Breast cancer effects have recently been reviewed in a meta-analysis by Trock et al. , which reported 30 and 23% lower breast cancer risk estimates among premenopausal and postmenopausal women, respectively, and in our recent commentary [Wood & Messina, 2008], among others. Interventional studies of the breast effects of soy isoflavones in women are limited to short exposures in women scheduled for breast surgery, nipple aspirate studies, and mammographic density measurements. In general, the data from these studies are equivocal. For example, Petrakis et al.  evaluated 24 pre- and postmenopausal women by nipple aspirate and reported increased amounts of aspirate fluid and epithelial hyperplasia in women consuming soy. Unfortunately this study is difficult to interpret in light of effects of time (nipple aspirate fluid volume increased in all women across the study) and low numbers of subjects. Another study, reported in part by McMichael-Phillips et al.  and subsequently in a final report by Hargreaves et al. , evaluated histologic markers in women already scheduled for breast biopsy, who were given a soy muffin delivering 45 mg of isoflavones for 2 weeks prior to the surgery. In the preliminary report [McMichael-Phillips et al., 1998], an increase in cell proliferation was shown, but this effect had disappeared at the time of the final report, although increases in other estrogen-dependent markers such as PS2 were found [Hargreaves et al., 1999]. This study was compromised by the wide range of diagnoses among women scheduled for surgery (from benign fibrocystic disease to invasive carcinoma) and the unavoidable lack of experimental control when using human subjects. Perhaps the most intriguing work to date is that of Maskarinec and Meng , who studied mammographic density. Greater mammographic density is clearly associated with a fourto six-fold increase in breast cancer risk [Boyd et al., 1995]. Maskarinec found no effect of soy isoflavone supplementation on mammographic density [Maskarinec et al., 2004]. Women of Caucasian descent but not women of Asian descent who reported greater lifetime soy consumption tended to have higher mammographic percent densities. This latter observation may indicate a physiologic, genetic, or epigenetic acclimation to soy isoflavone effects in populations consuming them over a long period of time, a notion that harks back to the multigenerational effect seen in the migration studies. Soy isoflavone effects on the lower reproductive tract show a similar pattern. The observational Am. J. Primatol. 724 / Cline and Wood studies of soy consumption show a reduction in endometrial cancer risk [Dai et al., 2007; Goodman et al., 1997]. Short-term soy exposure has been shown in several small studies to lack estrogen-like effects [Murray et al., 2003; Nikander et al., 2005]. The only large intervention study to date was a 5-year study of postmenopausal women randomized to either 150 mg of isoflavones (n 5 179) or placebo (n 5 197) per day consumed as a tablet [Unfer et al., 2004]. Endometrial histology was obtained by biopsy at baseline, 30 months, and 5 years. At the study’s end endometrial hyperplasia was found in 6/154 subjects in the treatment group and none in the placebo group. Although the findings caused some concern, the incidence of hyperplasia was comparable to controls in larger studies such as the Women’s Health Initiative [Rossouw et al., 2002] and the study was flawed by the lack of any consideration of serum estrogens or isoflavone measurements to confirm compliance. The basis for soy isoflavone effects in human subjects may lie in the modification of systemic estrogen concentrations or other estrogen-related mechanisms. Asian women who consume soy and have low risk for breast and endometrial cancer have lower serum estrogens relative to high-risk Caucasian women [Wu et al., 2002a,b]; however, Asian women who have migrated to the United States have estrogen levels comparable to American women [Goodman et al., 1997; Probst-Hensch et al., 2000]. Cassidy et al.  first reported effects of dietary soy (containing 45 mg isoflavones/day) on endogenous pituitary and ovarian hormones in women, demonstrating lowering of the mid-cycle LH and FSH, lower estradiol, and lengthening of the follicular phase of the menstrual cycle. Soy-associated reductions in sex steroids in women have since been found in several studies [Lu et al., 2000, 2001; Wu et al., 2000; Xu et al., 2000]. Some investigators have found no effect of dietary soy consumption on sex steroids [Martini et al., 1999], but the weight of evidence is in the direction of a small suppression of hypothalamo-pituitary gonadal function. Lu et al. , in tightly controlled dietary studies of soy effects on premenopausal women, found that dietary soy containing approximately 150 mg of isoflavone/ day reduced serum progesterone concentrations by 45% and estradiol concentrations by 25%. Isoflavonedepleted supplements also reduced progesterone by 33% and estradiol by 25% [Lu et al., 2001] indicating that other components of soy may also modulate ovarian steroidogenesis. In a longer (7-month) but less tightly controlled study, Wu et al.  found that dietary soy consumption lowered luteal-phase estradiol (17%) but not progesterone, and that this effect was only evident in Asian women. Soy consumption has also been associated with lowering of estrone in postmenopausal women in Shanghai [Wu et al., 2002a]. Other investigators have shown Am. J. Primatol. that diet can affect pubertal sex steroid levels in girls [Dorgan et al., 2003]. EXPERIMENTAL EVIDENCE FOR ESTROGEN/ISOFLAVONE INTERACTIONS There are many mechanistic reasons why soy isoflavones may antagonize estrogenic effects, including a variety of antiproliferative effects, modulation of steroid hormone metabolizing enzymes and binding proteins, induction of apoptosis, and inhibition of angiogenesis [Wood et al., 2002]. In addition, dietary soy or genistein consumption early in life protects the mammary gland of rodents by inducing differentiation of cancer-susceptible terminal end buds within the mammary gland [Lamartiniere, 2000; Lamartiniere et al., 1998; Warri et al., 2008]. The most extensively studied isoflavone is genistein, which can bind to and mediate transcription by estrogen receptors a and b [Kuiper et al., 1998; Miksicek, 1994; Morito et al., 2002]. Several investigators have shown that in the absence of estrogen, isoflavones have weak estrogenic effects, whereas in the presence of estrogen, they may exert an antagonistic effect [Folman & Pope, 1966; Messina et al., 1994], consistent with our own observations in rodents [Cline et al., 1998b] and female macaques [Foth & Cline, 1998; Wood et al., 2006b]. Some investigators have shown antagonism of estrogen-induced proliferation of estrogen-dependent MCF-7 breast cancer cells at physiologic doses [Imhof et al., 2008]. Thus there may be competitive, estrogen receptor-mediated effects. However, it may be that the weak estrogenicity of genistein has little to do with its anticancer effect [Barnes, 1995]. At higher doses (410 mM), genistein has antiproliferative effects in MCF-7 breast cancer cells regardless of their estrogen receptor positivity [Peterson & Barnes, 1991; Tanos et al., 2002; Wang et al., 1996]. This high-dose effect is therefore presumably not dependent on the receptor, and perhaps is mediated by genistein’s potent inhibition of tyrosine kinase activity [Akiyama et al., 1987] or by an epigenetic differentiating effect [Dolinoy et al., 2006]. Genistein is also an inhibitor of DNA topoisomerase [Constantinou & Huberman, 1995], which may be an integral part of its differentiationinducing properties. It is also an antioxidant [Wei et al., 1995] and induces apoptosis in breast cancer cell lines in vitro [Kiguchi et al., 1994]. Furthermore, genistein has antiproliferative and antiangiogenic effects on vascular endothelial cells in vitro [Fotsis et al., 1995] and in tumor models [Buchler et al., 2004; Kim, 2003; Li & Sarkar, 2002; Myoung et al., 2003; Wietrzyk et al., 2005], leading to the hypothesis that its antiangiogenic properties may be responsible for the relatively indolent nature of breast carcinomas in Asian women. Genistein also induces expression of the inhibitory growth Estrogen/Isoflavone Interactions in Macaques / 725 factor transforming growth factor b, which diminishes epithelial cell proliferation in the breast [Sathyamoorthy et al., 1998] and endometrium. TIMING OF EXPOSURE AND ISOFLAVONE EFFECTS Studies evaluating adolescent dietary intakes in human subjects [Shu et al., 2001; Wu et al., 2002b] show that early exposure to dietary soy (before or during pubertal development) produces the clearest protective effect against breast cancer. The observations of Lamartiniere  and Lamartiniere et al.  show that in rodents the critical period of protective soy or soy isoflavone exposure is during early development of the breast. In contrast to an early protective effect, pre-existing neoplasms are promoted. The work of Allred et al.  and Hsieh et al.  has shown that once neoplasms are present, genistein exposure can increase the proliferation of xenografted or chemically induced mammary cancers in rodents. Hilakivi-Clarke et al.  have shown that in utero exposure, in contrast to neonatal exposure, increases the numbers of undifferentiated terminal end buds in the mammary glands of offspring in rats, thus increasing susceptibility to mammary cancer induction. Therefore, it appears that timing of exposure is a critical determinant of isoflavone effects on breast cancer risk [Warri et al., 2008]. The soy isoflavone genistein is a powerful modulator of DNA methylation in utero, leading to altered phenotypes in mice. Dolinoy et al.  in a landmark study demonstrated that in utero exposure to dietary genistein could modify coat color in mice by methylation of a regulatory sequence governing expression of the Agouti coat color gene. This study demonstrated that methylation patterns in a retrotransposal element in the promoter of Agouti were hypermethylated at six CpG sites in offspring. These methylation patterns persisted into adulthood. The effects of this hypermethylation not only altered coat color but also caused lower ectopic Agouti expression, which effectively protected the offspring from adult obesity. Furthermore, exposure of rats to isoflavones during the development of GnRH-producing neurons permanently alters gonadotropin secretion [Bateman & Patisaul, 2008]. On the basis of examples such as this, we believe that soy isoflavones have a high potential as modulators in the ‘‘fetal basis of adult disease’’ or ‘‘Barker hypothesis’’ [Barker, 1992; Barker et al., 2005], which postulates that nutrition and/or environmental factors during prenatal and early development influence cellular plasticity, thereby altering future susceptibility to disease. Our current work is focused on the potential for such effects of dietary soy consumption during the pubertal transition in macaques (Cline, NIH R01 AT000639). OUR STUDIES IN NONHUMAN PRIMATES Why Nonhuman Primates? Old-World monkeys have well-documented similarities to women in terms of reproductive physiology. Endometrial and ovarian physiologies have long been regarded as nearly identical to that of women; only Old-World primates and great apes menstruate [Attia, 1998; Bellino & Wise, 2003; Hodgen et al., 1977], and there are many more subtle similarities in anatomy, development, peripheral steroid hormone metabolism, sex steroid receptor expression, and other features [Cline, 2007; Cline et al., 1996a, 1998b, 2002, 2007, 2008; Kaiserman-Abramof & Padykula, 1989; Mahoney, 1970; Speert, 1948; Wood et al., 2006a]. These features are not shared by rodent models. Although laboratory rodents provide the advantages of ease of use, short life span, and the potential for genetic manipulation, they differ substantially from primates in terms of reproductive biology [Everett, 1989] and mammary gland physiology. The exogenous and endogenous mouse mammary tumor viruses, although experimentally useful [Medina, 2008], have no counterpart in human or nonhuman primates. Prolactin receptor knockout mice have a failure of mammary gland development, along with a variety of other abnormalities [Kelly et al., 2001]. In contrast, prolactin is not an obligate component of mammary growth and development in macaques, although it is required for lactation [Kleinberg et al., 1985]. Rodents do not menstruate, and reproduction differs in that the formation of the corpus luteum is dependent on coitus in rodents but not in primates [Freeman, 1994; Weinbauer et al., 2008]. In rodents reproductive senescence is characterized not by menopause but by ‘‘persistent estrus’’ [Freeman, 1994; Westwood, 2007], which differs markedly from menopause as observed in human beings and Old-World monkeys [Kavanagh, 2005]. We have found in the process of sequencing coding portions of monkey mRNA transcripts for steroidogenic enzymes that there is a high degree of similarity (95–97%) between monkey and human DNA sequences [Scott et al., 2008; Wood et al., 2006b, 2007], compared with a much lower (50–80%) similarity for rodents. The enzymatic ‘‘machinery’’ of peripheral sex steroid metabolism in macaques is similar to that of women [Soderqvist et al., 1994, 1998], and the full complement of aromatase, 17-bhydroxysteroid dehydrogenases, sulfatases, and other metabolic pathways relevant to intratissue sex steroid metabolism are present [Martel et al., 1994; Scott et al., 2008; Wood et al., 2007] and are modified in adult animals by dietary soy isoflavone exposure [Scott et al., 2008; Wood et al., 2007]. Hormones and Cancer Risk We have shown that the primate model of breast and endometrial regulation provides a unique degree Am. J. Primatol. 726 / Cline and Wood of similarity to human breast and endometrium. Our results with respect to breast proliferation as a surrogate marker of cancer risk demonstrated that the progestin medroxyprogesterone acetate augmented estrogen-induced proliferation [Cline et al., 1998a]. We believe that this finding predicted the results of several recent large trials in women indicating that the use of progestins in HRT may increase breast cancer risk [Beral, 2003; Rossouw et al., 1999; Schairer et al., 2000]. Evidence for Estrogen/Isoflavone Interactions We have shown that soy isoflavones are not estrogenic alone at up to 10 times dietary levels (500 mg/person/day equivalent compared with 50 mg/person/day in a high-soy Asian diet) in cynomolgus monkeys [Wood et al., 2006c]. We have also shown that at much lower concentrations (120 mg/person/day equivalent) they reduce estrogen-induced proliferative responses of the breast and endometrium [Wood et al., 2006b]. This effect may be mediated through estrogen receptor interactions and/or modulation of endogenous estrogen metabolism. We have shown in monkeys that there are interactive effects of soy with both endogenous [Scott et al., 2008; Wood et al., 2002] and exogenous [Foth & Cline, 1998; Wood et al., 2006b] estrogens in the postmenopausal period. There is an inverse association between serum estrone and serum isoflavone concentrations [Wood et al., 2006b], and long-term changes in estrogen metabolism induced by prior hormonal and soy treatments [Scott et al., 2008; Wood et al., 2007]. Figure 2 shows the inverse relationship between endogenous estrogens and soy isoflavones in the serum of cynomolgus macaques consuming a soy isoflavone-rich diet providing 129 mg/woman/day equivalent of total isoflavones [Wood et al., 2004]. In our studies of combined exogenous estradiol and dietary isoflavones, we found that dietary isoflavone in the high dietary range could reduce circulating [Wood et al., 2006b] and intrabreast [Wood et al., 2007] estradiol concentrations in adult cynomolgus monkeys, and that lowered circulating estradiol was associated with a decrease in tissue proliferation as measured by Ki67 expression in breast epithelium and endometrial size by ultrasound (Fig. 3). Putative Soy-Derived Selective Estrogens Our findings of dose-dependent estrogen antagonistic effects of soy isoflavones on systemic estrogen concentrations and tissue responses indicate that there are ‘‘anti-estrogens’’ or modulators of mammalian estrogen effects among the isoflavones. Given the well-documented estrogen-like beneficial effect of isoflavones on cardiovascular disease risk [Food and Drug Administration, 1999] and the lack of estrogen agonist effects in primates, we have sought to identify such selective agonists. Glyceollins are a recently discovered class of phytoalexin compounds produced from isoflavones by soybean plants under stressed conditions. Glyceollins do not produce estrogen-like uterine weight gains in mice but do reduce the growth of estrogen-dependent MCF-7 breast cancer xenografts [Burow et al., 2001]. In our studies of glyceollins administered along with estradiol to postmenopausal cynomolgus monkeys, we found a distinct blunting of the estrogen-induced proliferative and transcriptional response in the breast but not in the lower reproductive system [Wood et al., 2006d]. Another potential selective estrogen is equol. This compound is derived by bacterial metabolism from the native soy isoflavone daidzein, and is associated with reduced risk of hormone-dependent cancers. Only about 30% of human subjects produce equol after soy isoflavone consumption [Duncan et al., 2000]. As essentially all macaques are equol producers [Wood et al, 2006b], they mimic this subpopulation of human subjects. Human equol producers have lower-risk estrogen-metabolic profiles, skewed more toward the production of 2-hydroxy estrogen metabolites, which may be 3500 Serum Iso oflavones (n nM) 3000 2500 2000 1500 1000 500 0 0 20 40 60 80 100 120 Serum Estrone (ng/mL) Fig. 2. Inverse relationship between dietary soy isoflavone concentrations in serum and endogenous serum estrone in 60 female ovariectomized control cynomolgus macaques. R 5 0.37, P 5 0.0048. Am. J. Primatol. Estrogen/Isoflavone Interactions in Macaques / 727 Fig. 3. Antagonistic effects of soy isoflavones on breast proliferation and uterine size in either low or high E2 environment. Proliferation was measured by immunohistochemical expression of the proliferation marker Ki67 in breast lobular and ductal epithelium and uterine area was determined by transabdominal ultrasound. The 240 mg isoflavone dose resulted in significantly lower mammary epithelial Ki67 expression and uterine area in the high-estrogen (E2) environment (n 5 29–31 per diet). Horizontal lines, washout values for uterine size measured 1 month after discontinuing treatment. (a) Po0.05 compared with low E2 control; (b) Po0.05 compared with high E2 control. Bars, SE. From Wood et al. [2006b], with permission. cancer preventive [Duncan et al., 2000]. It is currently unclear whether equol is an effector or a marker of a particular estrogen-metabolic phenotype [Xu et al., 2000]. THE POTENTIAL FOR DIETARY ISOFLAVONE EFFECTS IN CAPTIVE PRIMATES Soy isoflavone exposure of rodents alters the onset of puberty; work from the National Toxicology Program indicates an acceleration of puberty as assessed by vaginal opening in rats [Casanova et al., 1999; Delclos et al., 2001, 2007]. The estrogenic effects of other dietary phytoestrogens are well known; for example, ‘‘clover disease,’’ a syndrome of reproductive disruption in sheep, is caused by grazing on pastures rich in coumestrol [Adams, 1995; Bennets, 1946]. These female reproductive tract effects include an increase in thickness and keratinization of vaginal epithelium, enlarged cervix, increased uterine weight, and endometrial hyperplasia, all of which are similar to the changes induced by steroidal estrogens [Adams, 1995]. Adverse effects have been reported in carnivores as well; captive cheetahs experienced a syndrome of hepatic venoocclusive disease attributed to excessive phytoestrogen exposure from soy-based diets [Setchell et al., Am. J. Primatol. 728 / Cline and Wood 1987]. Although there is some evidence that isoflavones alter early development in male marmosets [Sharpe et al., 2002], there is no evidence for longterm adverse effects in male or female primates. Interestingly, most primates are maintained on ‘‘monkey chow,’’ which contains highly variable but generally bioactive levels of isoflavones, as our colleagues have recently reported [Stroud et al., 2006]; levels of exposure were similar to those used for our experimental isoflavone trials [Wood et al., 2004]. These levels are sufficient to alter the behavior of male cynomolgus macaques, resulting in increased aggressive and decreased affiliative behavior [Simon et al., 2004]. It may be that the ‘‘standard’’ colony-reared nonhuman primate has an abnormal pubertal onset as a result of this exposure. The potential for such endocrine disrupting effects of a high-isoflavone soy diet has long been recognized in rodent studies [Boettger-Tong et al., 1998; Thigpen et al., 1999]. 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