Online Issues

ILAR Journal Vol 45(2)

Usefulness of the Monkey Model to Investigate the Role of Soy in Postmenopausal Women's Health

Susan E. Appt

Susan E. Appt, D.V.M., is a Research Fellow in the Department of Comparative Medicine, Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina.

Abstract

Some of the important health issues for postmenopausal women include cardiovascular disease, osteoporosis, breast cancer, and relief of menopausal symptoms. Ovariectomized cynomolgus monkeys (Macaca fascicularis) have many strengths as models for research in this area including a close phylogenetic relationship to humans, similarities in lipid/lipoprotein metabolism and coronary artery anatomy, similar skeletal anatomical and morphological characteristics, mammary glands with similar pathophysiological characteristics, and a 28-day menstrual cycle with similar hormonal fluctuations. Monkeys (macaques) also experience declining ovarian function and irregular menstrual cycles (natural menopause) when they approach 24 to 29 yr of age. However, because of their very short life span after natural menopause, ovariectomized macaques are used to model postmenopausal women. The cynomolgus monkey model has been useful in defining the potential cardiovascular benefits of soy foods and soy supplements; however, it remains unclear whether the observations are generalizable to all women or only to those who, like cynomolgus monkeys, convert the soy isoflavone daidzein to the metabolite equol. Particularly important has been the use of the cynomolgus monkey model to understand the effects of soy on breast health. There is evidence from a cynomolgus monkey trial to suggest that soy/soy phytoestrogens have no estrogen agonist effects for breast. Finally, soy/soy phytoestrogens do not appear to be an adequate alternative to postmenopausal hormone therapy. Nevertheless, important attributes of soy have been identified, and it may have potential as a complementary component to hormone therapy.

Key Words: breast cancer; cardiovascular effects; equol; hormones; menopause; osteoporosis, phytoestrogens; soy

Introduction

Advances in medical care and quality of life have made it possible for North American women to live for ≥ 35 yr beyond the menopause. The number of postmenopausal women living for these extended periods is expanding rapidly with the expectation that there will be more than 40 million postmenopausal women in the United States alone in the next few years. Some of the health issues of concern during this 35-yr postmenopausal period include menopausal symptoms (e.g., hot flushes, vaginal dryness), coronary heart disease and stroke, osteoporosis, breast cancer, cognitive decline, and memory loss. The need to maintain good health and good quality of life during this period has resulted in the search for the "ideal" dietary supplement or hormone therapy regimen. Although traditional hormone therapy is effective for the relief of menopausal symptoms, it has not been judged by either the physician community or menopausal women as being ideal. This sentiment is due primarily to the increased breast cancer risk associated with long-term hormone treatment, ongoing thrombotic risk, and the failure to prevent coronary heart disease in older (~65 yr) women (Rossouw et al. 2002).

The recent widespread disenchantment with hormone therapy has spurned a vigorous attempt to identify "natural" alternative and complementary therapies, such as soy. Soy protein contains three compounds (genistin, daidzein, and glyceitin) called phytoestrogens, because they are plant derived and bind to the estrogen receptor. Daidzein can be converted in the intestinal tract to a metabolite known as equol. Because of their potential estrogen-like properties, there has been a widespread belief that soy-containing phytoestrogens may be useful in improving menopausal symptoms associated with estrogen deficiency and preventing chronic diseases thought to be estrogen dependent (coronary heart disease, osteoporosis, cognitive decline) (see accompanying articles in this issue: Abbott et al. 2004; Archer 2004; Cline 2004; Jerome 2004; Kaplan and Manuck 2004; Bruns and Kemnitz 2004; Shively and Bethea 2004; Story and Kennedy 2004; Williams and Suparto 2004).

Currently, there is a necessity to obtain controlled experimental data to establish the effect or lack of effect of soy protein, and its phytoestrogens, on the clinical conditions associated with estrogen deficiency. Because monkeys have phylogenetic similarities (DNA homology) with women and they share many of the same reproductive biological characteristics, the research group at the Comparative Medicine Clinical Research Center of Wake Forest University has made a comprehensive effort to define their usefulness as models for investigating the risk/benefits of soy supplementation in postmenopausal women (periclinical trials). In this article, the effects of soy/soy phytoestrogens on surgically postmenopausal monkeys (mainly cynomolgus macaques) are summarized, and where possible, an explicit attempt is made to translate the results to what is known currently from clinical trials and observational studies of postmenopausal women.

Cardiovascular Effects

Heart disease is the leading cause of mortality in women in the United States; it accounted for 32% of deaths in the year 2000 (366,000/year). More women die from heart disease than from stroke (103,000/year), lung cancer (65,000/year), and breast cancer (42,000/year) combined (AHA 2002) (Figure 1). Among North American women, the rate of heart disease after menopause is two to three times greater than that of premenopausal women of the same age (NCWHD 2003).

Figure 1 Mortality rates per 100,000 women in the United States according to age and etiology: coronary artery disease, stroke, lung cancer, breast cancer, colon cancer, and endometrial cancer. From the Web (AHA [American Heart Association]. American Heart Association Statistical Update. 2002. http://www.americanheart.org/downloadable/heart/10148328094661013190990123HS_State_02.pdf ; http://www.americanheart.org/presenter.jhtml?identifier=3000941).

Suitability of the Cynomolgus Monkey Model

Ovariectomized cynomolgus monkeys have many strengths as models for research on postmenopausal cardiovascular disease. Among the strengths are their similarities in lipid/lipoprotein metabolism, their comparable coronary artery anatomy to that of women, their relative small size, and an extensive bibliography about their reactions to diets and hormones. Cynomolgus monkeys develop myocardial infarction as a sequela to their coronary artery atherosclerosis at about the same rate as humans (1 per 300 at risk/year) (Bond et al. 1980). Because very large numbers of animals are required to have the statistical power to detect either a positive or negative effect of an intervention, coronary artery atherosclerosis serves as a surrogate for coronary heart disease.

As with all animal models, the ovariectomized cynomolgus monkey has some limitations. The first limitation concerns the appropriateness/validity of extending observations from surgically postmenopausal monkeys to naturally menopausal women. Macaques do develop a natural menopause at around 24 to 29 yr of age (Gilardi et al. 1997), but they have a very short (1- to 2-yr) postmenopausal life span, making study during this period impractical. The principal concern regarding the ovariectomized monkey model relates to their very low plasma estradiol concentrations (<5 pg/mL) compared with those of postmenopausal women (~15-25 pg/mL) (Longcope 1999). They also differ in their plasma androgen concentrations. For example, plasma testosterone concentrations are negligible in the ovariectomized cynomolgus monkey, whereas naturally menopausal women actually have measurable plasma testosterone concentrations (14-19 pg/mL) 24 mo after last menses (Longcope 1999; Speroff 1996).

Soy, Lipids, and Lipoproteins

Plasma lipids/lipoproteins are risk markers for atherosclerosis in humans and monkeys. Elevated plasma tryglycerides (TG¹) and low-density lipoprotein cholesterol (LDLC¹) concentrations, along with reduced high-density lipoprotein cholesterol (HDLC¹) concentrations, are associated with increased risk for coronary artery atherosclerosis (Kannel 1987). After menopause, women's plasma lipid/lipoprotein concentrations tend to shift from a protective profile (high HDLC, low LDLC) to one in which their risk for atherosclerosis is increased (low HDLC, high LDLC) (Speroff 1996). Like postmenopausal women, surgically postmenopausal cynomolgus monkeys fed a "Western" diet have reduced HDLC and increased low-density lipoprotein (LDL¹) + very-low-density lipoprotein cholesterol (VLDLC¹) compared with premenopausal monkeys. These lipoprotein changes are also associated with progressing coronary artery atherosclerosis in the monkeys (Figure 2) (Adams et al. 1985).

Figure 2 Plasma high-density lipoprotein cholesterol (HDLC, mg/dL) concentrations and coronary artery plaque (PQ) area (mm²) in premenopausal versus postmenopausal cynomolgus monkeys, both fed the same moderately atherogenic diet. Data are modified from Adams MR, Kaplan JR, Clarkson TB, Koritnik DR. 1985. Ovariectomy, social status, and atherosclerosis in cynomolgus monkeys. Arteriosclerosis 5:192-200.

Based on current evidence, it appears that dietary soy protein has a beneficial effect on plasma lipids/lipoproteins. In 1999, the US Food and Drug Administration issued a health claim stating, "25 grams of soy protein per day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease" (FDA 1999). This claim was based largely on the results of a meta-analysis by Anderson and colleagues (1995) that reported reductions in LDLC (~13%), and TG (~10%) and slight increases in HDLC (~2%). However, the interpretation of this meta-analysis has come into question recently. The shortcomings of the meta-analysis are described in a review by Nestel (2003), who states, "approximately half of the studies showed minor or no cholesterol lowering effect and three out of every four of the trials included in the meta-analysis had such wide confidence intervals that an alternative conclusion might have been reached with equal validity" (p. 3). In this brief review, the potential reasons for the disparities among the studies of women are discussed, in addition to the disparity in lipid responses to soy between monkeys and women.

Our group has conducted several studies that have compared the plasma lipid/lipoprotein profiles of monkeys fed soy protein versus an animal protein (casein/lactalbumin [CL¹]). Consistently, soy-fed postmenopausal monkeys had increases in plasma HDLC of 20 to 30%, and reductions in plasma LDL + VLDLC of 30 to 40% (Clarkson and Appt 2003). Translation of the monkey findings to human subjects has been confusing and perhaps even controversial. In controlled studies of human subjects, only very small changes in plasma lipid/lipoproteins have been reported (reduced LDLC ~ 6%, no effect on HDLC) (Baum et al. 1998; Crouse et al. 1999; Lichtenstein et al. 2002; Wangen et al. 2001). Several factors that may explain the translational confusion concerning the effects of soy on lipids of postmenopausal women and monkeys are described below.

First, among the human trials, there has been large variation in the composition of the soy products used (e.g., soy protein isolate powders, soymilk, soy flour baked into foods, phytoestrogens pills) and the amount of phytoestrogens present in those products. Monkey studies have shown that beneficial plasma lipid/lipoprotein responses are greater when they are fed soy containing its phytoestrogens (soy (+)¹) than when fed soy from which the phytoestrogens have been removed by alcohol washing (soy (--)¹) (Anthony et al. 1998). It should also be noted that studies in which phytoestrogens were removed from soy and either added back to the diet of monkeys (Greaves et al. 1999) or given as supplements to women (Nestel et al. 1997; Simons et al. 2000) have reported no effect on plasma lipids. This result suggests that soy protein containing its naturally occurring phytoestrogens may provide the maximum benefit for improving plasma lipid concentrations.

Second, we have found that postmenopausal monkeys fed the same "dose" of phytoestrogens in their soy diets have wide variations in their plasma phytoestrogen concentrations. Pertinent to the translational confusion is the finding that monkeys with plasma phytoestrogen concentrations < 400 nmol/L had decreased LDL + VLDLC, increased HDLC, and marked inhibition of coronary atherosclerosis compared with monkeys fed a control diet of soy (--). In contrast, monkeys with plasma phytoestrogen concentrations > 700 nmol/L had no improvement in their plasma lipid profile and no protection against coronary artery atherosclerosis (Clarkson and Anthony 2002). Therefore, there may be an "optimum dose" of phytoestrogens to achieve an "optimum" plasma concentration, both of which may vary from individual to individual.

Third, there may be differences in soy phytoestrogen metabolism between monkeys and women. In nearly all monkeys, the soy phytoestrogen daidzein is converted by bacterial enzymes within the intestine to the metabolite equol, whereas only about 30% of women produce equol ("equol producers") (Setchell et al. 2002.) The biological properties of equol are not well understood, but the implications of separating the equol producers from the "equol nonproducers" with respect to lipid effects has been investigated recently (Setchell et al. 2002). Setchell and colleagues report on the re-evaluation of a randomized, placebo-controlled, lipid-lowering study in which it was initially reported that there was no effect of soy foods on the plasma lipids/lipoproteins of 23 hypercholesterolemic women. However, when plasma lipid responses for equol producers were reported separately, total cholesterol, LDLC, and TG were lowered significantly (8.5%, 10%, and 21%, respectively) (Figure 3). Additional research is needed to determine whether the beneficial effects of soy on the plasma lipids/lipoproteins of monkeys are related to their plasma equol concentrations.

Figure 3 Comparison of the effect of soy on plasma lipid/lipoprotein concentrations of female monkeys (equol producers) and women (reported separately for equol producers and all women). TC, total cholesterol; LDLC, low-density lipoprotein cholesterol; TG, triglycerides. Data are presented as percentage difference from control, and are modified from Clarkson TB, Anthony MS. 2002. A paradoxical association between plasma isoflavone concentration on a soy-containing diet, and both plasma lipoproteins and atherosclerosis. J Nutr 132:583S; and Setchell KD, Brown NM, Lydeking-Olsen E. 2002. The clinical importance of the metabolite equol--A clue to the effectiveness of soy and its isoflavones. J Nutr 132:3577-3584.

Soy and Arterial Function

Studies of monkeys suggest that soy/soy phytoestrogens may inhibit atherosclerosis progression through mechanisms independent of their plasma lipid/lipoprotein concentration effects. One possible mechanism is through direct effects on arterial function (vasodilation and constriction in response to normal physiological stimuli). To date, only vascular reactivity (active dilation or constriction) has been utilized to measure arterial function of monkeys, whereas both vascular reactivity and arterial compliance (arterial stiffness or passive dilation) have been measured in human subjects. These two measures are discussed separately. Additionally, the potential effect of soy phytoestrogens on coronary artery constriction, as a sequela of plaque-associated disturbed blood flow, is reviewed.

Coronary artery reactivity in monkeys has been evaluated by repeated quantitative angiography. Changes in diameter of the left circumflex coronary artery are measured after intracoronary infusion of acetylcholine, which induces endothelium-mediated vasodilation in normal coronary arteries. Results from two studies of the effects of soy on vascular reactivity indicate that coronary arteries of pre- and postmenopausal monkeys respond differently to intracoronary acetylcholine infusion. In the first study, premenopausal moneys were fed either soy (+) or a control diet (soy ( --)), for 6 mo. In this study, arterial dilation of ~6% was observed in the monkeys fed soy (+), whereas the coronary arteries of control-fed animals constricted (~6%) (Honore et al. 1997). In the second study, the arteries of postmenopausal monkeys fed soy (+) constricted ~6%, and those treated with estradiol dilated (~5%) (Williams et al. 2001). Interestingly, when postmenopausal monkeys in that same study were given soy (+) combined with low-dose estradiol (equivalent to a woman's dose of 1 mg/day), their arteries dilated (~12%). The combination of soy and a physiological replacement dose of estradiol resulted in arterial dilation greater than the sum of the dilation observed for either therapy given alone, indicating a significant (p < 0.05) interactive effect of soy and estradiol on vascular reactivity (Figure 4).

Figure 4 Comparison of soy effects on coronary artery vascular reactivity of premenopausal cynomolgus monkeys and postmenopausal cynomolgus monkeys (either treated with estradiol, 1 mg/day of women's equivalent, or not treated). All monkeys were fed either control diet (alcohol washed soy to remove phytoestrogens) or soy containing phytoestrogens (soy (+)). Data are presented as percentages of change in coronary artery diameter, and are modified from Honore EK, Williams JK, Anthony MS, Clarkson TB. 1997. Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques. Fertil Steril 67:148-154; and Williams JK, Anthony MS, Herrington DM. 2001. Interactive effects of soy protein and estradiol on coronary artery reactivity in atherosclerotic, ovariectomized monkeys. Menopause 8:307-313.

As with the monkeys, different effects of soy/soy phytoestrogens on arterial vascular reactivity among pre- and postmenopausal women have been reported. Walker and colleagues (2001) measured flow-mediated dilation of the brachial artery of premenopausal women after intravenous injection of the phytoestrogen genistein. He reported that "Genistein produces acute nitric oxide-dependent vasodilation in the forearm vasculature of men and women with a potency similar to that of 17β-estradiol and potentiates endothelium-dependent vasodilation (p. 262). Conversely, the effects of soy on vascular reactivity of postmenopausal women, without estrogen replacement, appear to be mixed. In two recent trials, no significant increase in brachial artery flow-mediated dilation was observed in postmenopausal women who consumed soy isoflavone concentrate (Hale et al. 2002) or soy protein containing isoflavones (Teede et al. 2001), compared with those receiving placebo. However, Steinberg and colleagues (2003) reported on a study of brachial artery reactivity of postmenopausal women who received soy containing isoflavones, soy without isoflavones, or casein for 6 wk. Brachial artery peak flow velocity (PFV¹) was significantly (37%, p = 0.03) lower after treatment with soy containing isoflavones compared with the casein control. This reduced PFV equates to a vasodilatory response in the postmenopausal women who consumed soy with isoflavones.

Arterial compliance has been reported to be a significant predictor of coronary heart disease (Blacher et al. 1999; Cameron et al. 1996; Guerin et al. 2001; Kingwell et al. 2002; van Popele et al. 2001). Passive changes in the diameter of the artery due to increased pressure in the lumen relate to components of the artery wall that give the arteries their properties of elasticity. Increased arterial compliance after supplementation with soy phytoestrogens has been reported in a study that included both pre- and postmenopausal women (Nestel et al. 1997). Similarly, increasing dietary phytoestrogen intake (recorded by food frequency questionnaires) was associated with decreased aortic stiffness in postmenopausal women as measured by pulse wave velocity (-0.51 m/s, 95% confidence interval -1.00 to -0.03, fourth vs. first quartile, p for trend = 0.07) (van der Schouw et al. 2002). The combined data indicate that soy may exert a beneficial effect on arterial function through its effects on arterial compliance. Future studies of the effects of soy on arterial compliance in postmenopausal monkeys are needed.

The potential effect of soy containing phytoestrogens on myocardial ischemia has been investigated in macaques (Williams and Clarkson 1998). Genistein markedly lowers the serotonin content of platelets (Nakashima et al. 1991). Increased heart rate can result in increased platelet interactions within the coronary artery wall and the subsequent release of serotonin from platelet aggregates, resulting in downstream vasoconstriction and myocardial infarction. Based on that pathophysiological sequence of events, Williams and Clarkson (1998) reasoned that soy isoflavone depletion of platelet serotonin should result in less downstream coronary artery vasoconstriction after platelet aggregation and release. During quantitative angiography of female macaques, collagen was perfused into the left circumflex coronary artery to induce platelet aggregation and serotonin release, and downstream blood flow was quantified (Figure 5). The monkeys treated with soy (+) had only about one third of the reduction in blood flow of that experienced by those fed soy (--). This promising periclinical observation has important implications for older women with complicated coronary artery atherosclerosis and myocardial ischemia associated with stress or physical activity.

Figure 5 Comparison of collagen-induced platelet activation and serotonin release in female macaque monkeys fed either soy containing phytoestrogens (soy (+)) or soy with phytoestrogens removed (soy (--)) for 6 mo. Endpoint data are presented as mean (± standard error) reduction in blood flow and expressed as percentage of control. LCX, left circumflex coronary artery. Adapted from Williams JK, Clarkson TB. 1998. Dietary soy isoflavones inhibit in vivo constrictor responses of coronary arteries to collagen-induced platelet activation. Coron Artery Dis 9:759-764.

Soy and Atherosclerosis

The advantage of using the monkey model, versus human clinical trials, to evaluate coronary artery disease is the ability to obtain detailed information about coronary artery atherosclerotic plaque size and characteristics (inflammatory cells and necrotic cores, regions vulnerable to rupture and thrombosis), using morphometric analyses of tissues taken at necropsy. It is also possible to determine whether certain inflammatory and lipid metabolism pathways within the arterial wall have been upregulated.

Monkeys, as with most animals, must be fed a diet moderately high in cholesterol (similar to a typical Western diet) to develop atherosclerosis. Feeding soy protein in place of animal protein (with added cholesterol) results in a more favorable lipid/lipoprotein profile and reduced atherosclerosis extent in several animal models (mice, rabbits, and monkeys) (Clarkson 2002). Our group has thus focused on determining the role of soy phytoestrogens in the prevention of coronary and internal carotid artery atherosclerosis progression in monkeys. In a randomized prospective trial, surgically postmenopausal cynomolgus monkeys were fed either soy (+) or soy (--) for 3 yr. Monkeys fed soy (+) had significantly reduced internal carotid artery atherosclerosis (p = 0.02) and tended to have reduced amounts of coronary artery atherosclerosis (p = 0.12) compared with the control group fed soy (--) (Figure 6) (Clarkson et al. 2001). We cannot speculate on the effect of soy (--) on atherosclerosis extent because this trial did not include a group fed animal protein (e.g., casein/lactalbumin [CL¹]).

Figure 6 Coronary artery atherosclerosis of surgically postmenopausal monkeys after consuming either soy containing phytoestrogens (soy (+) , 129 mg/day of womens equivalent), conjugated equine estrogens (CEE, 0.625 mg/day of womens equivalent), or control (soy (--), phytoestrogens removed) for 3 yr. Data are presented as mean plaque size (mm² ± standard error) with p < 0.05 considered statistically significant. Data and illustration are adapted from Clarkson TB, Anthony MS, Morgan TM. 2001. Inhibition of postmenopausal atherosclerosis progression: A comparison of the effects of conjugated equine estrogens and soy phytoestrogens. J Clin Endocrinol Metab 86:41-47.

Bone Effects

The National Osteoporosis Foundation recently released a report (NOF 2002) in which it was estimated that more than 10 million people over the age of 50 had osteoporosis, 80% of whom were women. These figures are expected to increase as the population of postmenopausal women in the United States grows from approximately 30 million in 2002, to more than 40 million in 2020. In the report, it is concluded that "A comprehensive national effort aimed at the prevention, diagnosis and treatment of osteoporosis and related fractures is necessary to address this debilitating and costly disease."

Suitability of the Cynomolgus Monkey Model

Macaques have been studied extensively to evaluate their suitability as an animal model of osteoporosis. An important similarity to human skeletal biology is the presence of haversian osteonal remodeling in cortical bone, a characteristic not present in rodents (Burr 1992; Jerome and Peterson 2001). Female cynomolgus monkeys reach peak bone mass by about 10 yr (Champ et al. 1996; Jayo et al. 1994) and experience a decrease in bone turnover with age (Lees and Ramsay 1999). Colony-housed female cynomolgus monkeys (>10.5 yr of age) tended to experience 3 to 4% vertebral bone loss per year, and osteopenia was observed in aging cynomolgus monkeys (Jayo et al. 1994). Furthermore, cynomolgus monkeys have bone biomarker responses to fluctuations in estradiol that are similar to those of humans throughout menstruation, pregnancy, and lactation (Hotchkiss and Brommage 2000; Lees et al. 1998b).

Because cynomolgus monkeys do not experience natural menopause until their third decade of life, surgically postmenopausal cynomolgus monkeys have been used to study the effect of long-term ovariectomy on bone. Most of these studies indicate that animals ovariectomized at 10 yr or older have lower (~9%) spine bone mineral density than premenopausal animals (Jerome and Peterson 2001), and that most of the bone loss occurs within the first year after ovariectomy (Jerome et al. 1997b). Monkeys ovariectomized before peak bone mass (<10 yr) have arrested bone development but not bone loss. Failure to consider age at ovariectomy has resulted in criticism of drug studies performed with the cynomolgus model (McClung 2002).

Soy and Osteoporosis

Interest in the use of soy/soy phytoestrogens to prevent and treat osteoporosis stems from reports of its estrogen-like effects in other tissues. Both alpha and beta estrogen receptors are present in bone (Arts et al. 1997; Onoe et al. 1997), and estrogen replacement in women (Lindsay et al. 1976) and monkeys, ovariectomized after peak bone mass has been reached, is effective in slowing postmenopausal bone loss (Jayo et al. 1990; Jayo et al. 1998; Jerome et al. 1994, 1997a). The results of trials designed to study the effect of soy/soy phytoestrogens on bone loss in postmenopausal women are mixed. Little or no effect of soy on bone biomarkers or bone mineral density is reported in several studies (Dalais et al. 1998, 2003; Gallagher 1999), whereas others report a beneficial effect (Alekel et al. 2000; Potter et al. 1998; Setchell et al. 2002).

Most animal studies undertaken to evaluate soy's effects on bone have been conducted in rats and have focused on effects of phytoestrogens. Although results from the rat studies are mixed, and there is some variation in study design and route of administration, they generally indicate a bone-sparing effect of soy (Arjmandi and Smith 2002, Setchell and Lydeking-Olsen 2003). Soy/soy phytoestrogen effects on the bones of surgically postmenopausal monkeys have been studied by our group. In one study, postmenopausal monkeys were treated with either estradiol (1 mg/day of women's equivalent), soy protein (148 mg/day of women's equivalent), or a control diet of CL (Lees and Ginn 1998). In that study, estradiol--but not soy--prevented ovariectomy-induced increases in bone turnover. In another recent trial (Register et al. 2003), postmenopausal monkeys were treated with either conjugated equine estrogens (CEEs,1 0.625 mg/day of women's equivalent), soy protein isolate (129 mg/day of women's equivalent), or CL. Bone protective effects (higher bone mass and decreased bone turnover) were observed in the CEE-treated animals. The soy-treated animals, however, had a decrease in bone mass similar to those treated with the control diet. Taken together, these studies indicate that soy/soy phytoestrogens do not prevent bone loss in surgically postmenopausal monkeys, and they may not be effective in preventing osteoporosis in women.

Breast Effects

Breast cancer is one of the most common forms of cancer that affects women in the United States. The National Cancer Institute estimates that one in eight women will develop breast cancer in their lifetime. Recent findings of increased breast cancer risk with certain hormone formulations (Rossouw et al. 2002) have persuaded many women to seek alternatives to hormone therapies for menopausal symptom relief. Many women are choosing soy as an alternative therapy in the belief that it will effectively treat menopausal symptoms and possibly provide some breast cancer protection. However, the extent of reduction in menopausal symptoms may not be adequate for many women.

Suitability of the Cynomolgus Monkey

The mammary glands of cynomolgus monkeys and women share many pathophysiological characteristics (Cline et al. 1996; Labrie et al. 1995; Mahoney 1970; Speert 1948; Tsubura et al. 1991). They are anatomically similar in terms of nerve innervations, mechanisms of glandular secretion, and lobuloalveolar and ductal cytokeratin, as well as sex steroid receptor patterns (Macpherson and Montagna 1974). Like women, cynomolgus monkeys have a 28-day menstrual cycle with similar fluctuations in estrogen and progesterone throughout the cycle. They also experience declining ovarian function and irregular menstrual cycles, similar to women, when they approach 30 yr of age. Breast cancer has been reported in macaques (Uno 1997), but given the paucity of aged monkey populations, it is difficult to determine the incidence of breast cancer in cynomolgus monkeys. For these reasons, our group has used mammary gland expression of the proliferation marker Ki67 and epithelial area as surrogate markers for breast cancer promotion.

Soy and Breast Cancer

Interest in soy/soy phytoestrogens as breast cancer protective agents originated from epidemiological studies of Japanese women. Breast cancer rates are lower among Japanese women who live in Japan and consume a diet with a large percentage of soy than among women who live in the United States and consume little soy. When Japanese women migrate to the United States, however, their incidence of breast cancer becomes equivalent to Western women (Shimizu et al. 1991;Wu et al. 1996). This observation led to speculation that dietary factors, particularly soy, may be responsible for the breast protection seen in Japanese women. Furthermore, supportive evidence for the role of soy in breast cancer prevention has been provided by several observational studies of women who consume soy (Anderson et al. 1999; Dai Qet et al. 2001; Wu et al. 2002; Yamamoto et al. 2003).

In a trial designed to study the effects of mammalian and plant estrogens on mammary glands of macaques, investigators (Foth and Cline 1998) surgically treated postmenopausal monkeys with placebo, E2 (1 mg/day of women's equivalent), soy protein isolate (soy (+), containing 148 mg/day of women's equivalent phytoestrogens), or both soy (+) and E2, for 6 mo. As expected, E2-treated animals had a significant increase in Ki67 labeling compared with placebo-treated animals (p <0.05). A statistically significant effect on proliferation was not seen in monkeys treated with soy (+). Perhaps the most intriguing finding in this study was that the combination of soy (+) and E2 resulted in the attenuation of the E2-induced proliferation. A larger, randomized crossover periclinical trial is currently under way at our institution to investigate further this apparent antagonistic effect of soy (when given with exogenous estradiol) on mammary proliferation of ovariectomized cynomolgus macaques.

Hot Flushes

More than 75% of women in the menopausal transition experience hot flushes, which are episodes of flushing, increased skin blood flow and temperature, increased sensation of heat with sweating on the face, neck, and chest, and increased heart rate. These symptoms are the primary reason that women at menopause seek medical attention, and many of them (~25%) remain symptomatic for more than 5 yr (Guthrie et al. 2003). These vasomotor symptoms are associated with difficulty sleeping (Kravitz et al. 2003) and have adverse effects on quality of life. Women experiencing hot flushes may also be at more risk of unsteadiness and rotary vertigo than women without symptoms (Ekblad et al. 2000). They also may have an increased level of cardiovascular reactivity to stress situations, potentially increasing their risk for cardiovascular disease (Leal et al. 2002).

Suitability of the Cynomolgus Monkey

The cause of hot flushes in women is not completely understood. It is clear that the declining estrogen production that occurs at menopause or after bilateral oophorectomy precipitates the vasomotor symptoms. It has been reported that women who experience these symptoms are lacking a thermoneutral zone (the temperature range within which sweating, peripheral vasodilation, and shivering do not occur) and that small temperature elevations preceding the hot flushes are the triggering mechanism (Freedman 2001).

Rhesus monkeys (Macaca mulata) have a thermoregulatory system that is similar to that of humans, with a defined thermoneutral zone. Sweating is their primary means of decreasing core body temperature, and likewise, shivering increases body temperature (Bellino and Wise 2003). At least two studies have evaluated the monkey as a model of postmenopausal vasomotor changes. Dierschke (1985) reported temperature fluctuations at the ear pinna of three ovariectomized rhesus monkeys. Temperature fluctuations tended to be reduced with estrogen therapy. In another study (Jelinek et al. 1984), skin temperatures of two-stump tail macaques (Macaca arctoides) were measured before and after ovariectomy. A hairless area of the head over the frontal region was used, and temperatures were taken at 60-min intervals over 6.5-hr time periods. There was very little variation in the skin temperatures of premenopausal monkeys, however, skin temperature fluctuated significantly in the postmenopausal animals. Furthermore, treatment with two different hormone therapies (ethinyl estradiol or tibolone) removed the temperature fluctuations. The authors of this study also refer to less reliable findings from a separate, unpublished study using rhesus monkeys. They suggest that the relative hypersensitivity of those rhesus monkeys to noise and movement, compared with the docile, hand-reared stump tail monkeys, made them unreliable as a model of postmenopausal vasomotor change.

Our group has measured "hot flushes" in postmenopausal cynomolgus monkeys treated with either placebo or estrogen. Monkeys not receiving estrogen treatment experienced more frequent and prolonged episodes of temperature increase (≈1.5°F) than those receiving estrogen (J.R. Kaplan, Wake Forest University, Winston-Salem, NC, personal communication, 2003).

Soy and Hot Flushes

Although there have been several trials designed to investigate the effect of soy/soy phytoestrogens on hot flush control in postmenopausal women, no data have been published regarding the effects of soy on vasomotor changes using nonhuman primate models. Despite the fact that some recent human trials report a beneficial effect of soy, the results are mixed, and most of these trials report only a modest effect of soy on hot flushes compared with placebo (Albertazzi et al. 1998; Burke et al. 2003; Faure et al. 2002; Han et al. 2002; Messina and Hughes 2003; Nagata et al. 2001; Penotti et al. 2003; Scambia et al. 2000; Upmalis et al. 2000). These mixed results may be due to individual differences in soy metabolism among women (e.g., equol producers vs. nonproducers), and there may exist an important research opportunity to investigate the effects of soy phytoestrogen metabolism on frequency and severity of hot flushes using the monkey model.

Soy as Complementary Versus Alternative Hormone Therapy for Postmenopausal Women

Since the early 1990s, the predominant research effort has been to explore whether, and to what extent, soy could be used as an alternative to hormone therapy (HT¹) for postmenopausal women. As discussed above, soy resulted in moderate inhibition of coronary artery atherosclerosis in studies of monkeys. However, compared with HT, soy has marginal, if any, effects on postmenopausal bone loss in monkeys and women, and is inadequate controlling hot flushes. Although some women may still choose soy as an alternative therapy due to fear of HT side effects, those women will require additional therapies to prevent bone loss and treat menopausal symptoms adequately.

There is increasing evidence that soy may have considerable advantages as a complementary therapy for HT. Comprising an important aspect of that evidence are the significant interactions that occur between plasma estradiol and soy phytoestrogens, which may decrease the risk factors for cardiovascular disease. Among cynomolgus macaque females with pre-existing coronary artery atherosclerosis, treatment with soy resulted in a slight increase in aortic cholesteryl content (nonsignificant) compared with the control, whereas estradiol treatment resulted in a significant (p = 0.001) reduction in aortic cholesteryl ester content. More importantly, as can be seen in Figure 7, monkeys treated with a combination of soy and estradiol had a larger reduction in aortic cholesteryl ester concentration than would be expected from the sum of the two treatments when given alone (soy protein × estradiol interaction, p = 0.02) (Wagner et al. 1997). Additionally, a significant (p <0.05) interactive effect of soy and estradiol on vascular reactivity of postmenopausal monkeys has also been reported (Williams et al. 2001).

Figure 7 Aortic cholesteryl ester content (mg/g) of surgically postmenopausal cynomolgus monkeys treated with (1) casein control, (2) soy (148 mg of women's equivalent phytoestrogens /day), (3) casein + estradiol (E2, 1 mg/day of women's equivalent), and (4) soy + E2. Data are expressed as means adjusted for baseline total cholesterol:high density cholesterol ratio. Note significant E2 main effect (p = 0.001) and significant soy × E2 interaction (p = 0.02). Data are modified from Wagner JD et al. Metabolism, 1997. Illustration from Clarkson TB, Appt SE. 2003. Cardiovascular effects of dietary soy. In: Watson RR, Preedy V, eds. Nutrition and Heart Disease: Prevention and Treatment. Boca Raton: CRC Press (In Press).

Important interactions have also been described between changing plasma estradiol concentrations of premenopausal women and the extent to which relatively high doses of soy phytoestrogens (129 mg/day) reduced plasma LDLC concentrations. In those studies, Merz-Demlow and coworkers (2000) compared the reductions in plasma LDLC concentration among women who consumed a moderate dose of soy phytoestrogens (65 mg/day) and of a higher soy phytoestrogen dose (129 mg/day) across the menstrual cycle (Merz-Demlow et al. 2000). In the same subset of women, Duncan and colleagues (1999) reported on the plasma estradiol concentration of the women in each of the cycle stages evaluated by Merz-Demlow and coworkers (Duncan et al. 1999). The only significant reductions in LDLC occurred at the 129 mg/day dose of soy isoflavone, and the significant decreases were observed only at midfollicular and perifollicular stages of the menstrual cycle. Because nonovarian-derived plasma estradiol concentrations of postmenopausal women vary from 2 to 50 pg/mL, it may also be important to consider estradiol concentration as a potential modifier of the effects of soy on LDLC concentration in postmenopausal women. Whether such estrogen-phytoestrogen interactions occur among pre- and postmenopausal cynomolgus monkeys is important and is currently under study by our group.

Recently our group used the cynomolgus model to study the possible interaction between soy and the synthetic steroid tibolone (used in several countries to treat menopausal symptoms and osteoporosis) (Appt et al. 2003). Tibolone is metabolized into two compounds with estrogenic activity (3α-hydroxy and 3β-hydroxy metabolites) and one compound with progestogenic and androgenic activity (4-ketoisomer). Due to reductions in plasma HDLC that have been reported in women taking tibolone, concern has arisen about its cardiovascular safety. Because soy has been shown to increase plasma HDLC, we sought to determine whether coadministration of soy with tibolone would attenuate the tibolone-induced reductions in HDLC. Surgically postmenopausal cynomolgus macaques were assigned randomly to one of four treatment diets. The diets contained either CL or soy (+), either with or without tibolone (women's equivalent of 1.25 mg/day). As expected, HDLC concentration was significantly (p <0.01) reduced with tibolone treatment and was increased with soy. However, when soy was combined with tibolone, there was no reduction in HDLC concentration as compared with the control (Appt et al. 2003) (Figure 8). Further study is required to identify the mechanism responsible for this potential soy/tibolone interaction.

Figure 8 Plasma high-density lipoprotein cholesterol (HDLC) concentrations of surgically postmenopausal cynomolgus monkeys treated with (1) tibolone (1.25 mg/day of women's equivalent), (2) soy containing phytoestrogens (soy (+), 137 mg/day of women's equivalent), or (3) soy (+) + tibolone. Only monkeys given tibolone alone had a significant (p = 0.0001) reduction in HDLC compared with control (casein/lactalbumin). Data are expressed as percentage of control (casein diet) and are adjusted for baseline plasma HDLC. Data are adapted from Appt SE, Clarkson TB, Anthony MS, St. Clair RW. 2003. Complementary soy therapy prevents dyslipoproteinemia associated with tibolone treatment of postmenopausal cynomolgus monkeys. In: Proceedings of the 5th International Symposium on Soy, held in Orlando, Florida, September 2003. J Nutr (In Press).

Conclusions

Surgically postmenopausal cynomolgus monkeys have been used extensively to investigate the potential health benefits of soy for postmenopausal women. Studies of the cynomolgus model have provided evidence for beneficial effects of soy on cardiovascular risk factors (plasma lipids/lipoproteins, arterial function) with subsequent moderate reductions in atherosclerosis progression. Observations of soy/soy phytoestrogen effects on breast tissue of cynomolgus monkeys suggest a beneficial antiestrogen effect and indicate that soy has potential as a complementary therapy to traditional hormone therapy. Further investigation in this area is needed.

The cynomolgus monkey model has also been useful in identifying areas in which soy has little or no effect. Key among those areas have been the periclinical trials, which have shown a lack of effect on postmenopausal bone loss.

A challenging research goal for the future is to identify whether, and to what extent, the beneficial effects of soy/soy phytoestrogens observed in the cynomolgus monkey relate to the conversion of daidzein to equol. This monkey model has high plasma concentrations of equol, and it is unknown whether the effects that have been noted relate to equol or to some of the primary soy phytoestrogens (daidzein, genistein, glyceitin). It is important to know whether the results obtained from the cynomolgus monkeys are translatable to all women or only to those women who are equol producers (~25-30% of the US population).

¹Abbreviations used in this article: CEE, conjugated equine estrogen; CL, casein/lactalbumin; HDLC, high-density lipoprotein cholesterol; HT, hormone therapy; LDL, low-density lipoprotein; LDLC, low-density lipoprotein cholesterol; PFV, peak flow velocity; soy (+), soy containing phytoestrogens; soy (--), soy from which the phytoestrogens have been removed; TG, triglycerides; VLDLC, very-low-density lipoprotein cholesterol.

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