Online Issues

ILAR Journal Vol 45(4)

Behavioral Effects of Endocrine-disrupting Substances: Phytoestrogens

Edwin D. Lephart, Kenneth D. R. Setchell, Robert J. Handa, and Trent D. Lund

Edwin D. Lephart, Ph.D., is a Professor in the Department of Physiology and Developmental Biology and Director of the Neuroscience Center at Brigham Young University, Provo, Utah. Kenneth D. R. Setchell, Ph.D., is a Professor in the Department of Pediatrics and Director of the Clinical Mass Spectrometry Laboratory at Children's Hospital Medical Center, Cincinnati, Ohio. Robert J. Handa, Ph.D., is a Professor, and Trent D. Lund, Ph.D., is an Assistant Professor in the Department of Biomedical Sciences at Colorado State University, Fort Collins, Colorado.

Abstract

A major source of endocrine-disrupting substances, usually not considered in laboratory animal experiments, is the diet used in research investigations. Soy represents the main protein source in almost all natural-ingredient commercially available formulated diets. Soy-derived isoflavones are the most abundant and in many ways the most studied phytoestrogens, and phytoestrogens (isoflavones) are known endocrine disruptors. Research is reviewed that ide.jpgies the physiological and behavioral endocrine-disrupting effects of dietary phytoestrogens (isoflavones) in animal diets, including most of the isoflavones, which are in a glycoside form and biologically inactive, and those in the gastrointestinal tract, which are biologically active. The isoflavones genistein and daidzein have similar molecular weights and structural characteristics to that of 17-beta estradiol, which may enable them to exert estrogenic and antiestrogenic properties are described and characterized. Daidzein can be further metabolized to the potent and abundant molecule equol, which in rodents is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones. Equol has the unique and important ability to specifically bind 5 alpha-dihydro-testosterone, and to act in turn to inhibit the action of this potent androgen. The specific influence of dietary soy phytoestrogens on consumptive, learning and memory, and anxiety-related behaviors is ide.jpgied. Regulatory behaviors such as food and water intake, adipose deposition and leptin, and insulin levels affected by dietary isoflavones are also discussed.

Key Words: anxiety; body adipose weight; equol; food and water intake; isoflavones; learning and memory; leptin; rodents

Impact of Diet on Animal Research Results

Since the early 1990s, environmental estrogens, or xenoestrogens, have received extensive attention due to their widespread use in a variety of products to which humans and animals are exposed on a regular and sometimes continual basis (Brown and Setchell 2001; Naz 1999; Singleton and Khan 2003; Thigpen et al. 1999; Turner and Sharpe 1997). However, one source of endocrine-disrupting substances that is usually not considered in laboratory animal experiments is the diet used in research investigations. The article by Dr. Thigpen in this issue covers this topic in detail (Thigpen 2004). In addition, because soy or soy meal is the first or second ingredient by quantity in most laboratory animal diet formulations, the focus on soy-derived phytoestrogen molecules that appears in the text below is also warranted.

Of the hundreds of molecules that are classified as phytoestrogens, soy-derived isoflavones are the most abundant and in many ways the most studied (Barnes 1998; Setchell 1998; Whitten and Patisaul 2001). These characteristics apply not only to animal studies but also to results from clinical investigations, which suggest that consumption of soy-derived isoflavones protect against age-related diseases (e.g., cardiovascular disease and osteoporosis), certain cancers (e.g., breast and prostate), and postmenpausal symptoms (e.g., hot flushes) (Appt 2004; Barnes 1998; Mazur and Adlercreutz 2000; Patisaul and Whitten 1998; Setchell 1998).

In animal diets, most of the isoflavones are in a glycoside form that is biologically inactive (e.g., genistin or daidzin) (Brown and Setchell 2001; Thigpen et al. 1999). Once in the gastrointestinal tract, the glycoside portion of these molecules are cleaved by intestinal bacteria to the aglycone forms (e.g., genistein and daidzein), which are then biologically active. Daidzein can be further metabolized to a potent and abundant molecule, equol (Setchell et al. 2002) (Figure 1). The molecular weights and structural characteristics of the isoflavones are similar to those of 17-beta estradiol, which may enable them to exert estrogenic or antiestrogenic properties (Figure 1). Especially in rodents, equol is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones (in the aglycone forms; Table 1). In most cases, equol represents 70 to 90% of all of the circulating isoflavones. For this reason, the endocrine-altering properties of equol are potentially very important.

Figure 1 Conversion of the glycones (genistin and daidzin, beta-glycoside forms) into the aglycones (genistein and daidzein) by intestinal bacteria. Daidzein can be converted to equol subsequently. The similar planar molecular structure of the potent sex steroid 17-beta estradiol is compared with that of the aglycones and equol.

In contrast to rodents, humans produce relatively low levels of equol. In addition, only about 30% of men and women who consume soy foods produce equol (Setchell et al. 2002). However, the issue of equol producers in reference to human health benefits is becoming a novel and emerging story (Setchell et al. 2002).

The primary mechanism of action that these isoflavone molecules exert in their endocrine-disrupting effect is via the mammalian estrogen receptor (ER¹) system. It is well established that genistein and other isoflavones have a higher affinity for ER beta than for ER alpha due to their ability to mimic the structural confirmation of estradiol (Kuiper et al. 1997, 1998). Several studies have clearly documented that isoflavones can influence neurobehavioral parameters that are most likely explained by mediation via ER beta (Lephart et al. 2002; Patisaul et al. 2001, 2002).

Among the endocrine-altering influences of isoflavones, one important recently reported influence is the ability of equol to specifically bind 5 alpha-dihydrotestosterone (DHT¹) (but not testosterone) in the circulation and presumably within cells (Lund et al. 2004). In addition, equol does not bind the androgen receptor, yet it has the ability to bind ER beta but not ER alpha. From in vitro binding studies, it is known that the affinity of equol for ER beta is approximately 1/200 that of estradiol (Lund et al. 2004). However, because rodents produce equol at high levels (see Figure 2), and when a soy-rich diet is fed, this molecule circulates in the 1,000- to 2,500-ng/mL range--compared with the 10- to 100-pg/mL range of circulating estradiol (in males and females)--it is clear that this estrogenic mimic can have profound influences on neuroendocrine and behavioral parameters by mass action.

Figure 2 Influence of dietary soy phytoestrogen (Phyto) on a four-arm (working and reference memory) task (baited/unbaited) in the radial arm maze. Male and female Long-Evans rats received lifelong exposure to a high phytoestrogen-containing diet (Phyto-600) from conception to adulthood. In adulthood, one half the total number of male or female (random cycling) rats were either (1) kept on the original Phyto-600 diet, or (2) changed to a Phyto-free diet. The number of correct arm choices made in the first four arm entries was recorded (mean ± SEM; average of three trials). A correct choice was defined as an entry into a baited arm not yet visited in the trial. (a) Males (regardless of diet) made significantly more correct choices than females (p < 0.05) on trials 7-9. (b) Phyto-600 females and Phyto-free males made significantly more correct choices than Phyto-free males and Phyto-600 males (p < 0.05) on trials 10-12 and 13-15.

Thus, equol represents an extremely novel molecule that has the ability to bind ER-beta and specifically bind the potent androgen 5 alpha DHT (Lund et al. 2004). This combined altering of estrogen and androgen hormone actions by one molecule in such a specific way has important implications for endocrine disruption not only in animal models but also potentially in human neuroendocrine health issues such as female- and male-pattern baldness, facial and body hair growth, skin integrity, and prostate physiology (Lund et al. 2004) (equol technology, patent pending: E.D.L., R.J.H., D.R.S., and T.D.L.).

Rodent-Animal Studies

The behavioral research areas described below represent data from our laboratory and others on the endocrine-disrupting effects of phytoestrogens (isoflavones) in the diet of laboratory animals. Documented results are pertinent to body weight and adipose deposition effects of estrogens as well as food/water intake and changes in body weight.

Background: Body Weight and Adipose Deposition Effects of Estrogens

It has been known for more than 30 yr that estrogen profoundly affects body weight (Wade 1972) and that ovariectomy shifts body weight patterns toward male-like profiles (Leshner 1978). In general, estrogen plays a dual role in regard to body weight and adipose tissue deposition. On the one hand, estrogens decrease food intake, increase locomotor activity, and hence decrease body weight (Lindsay et al. 1997; Yen et al. 1999). On the other hand, adipose tissue deposition increases with puberty and early pregnancy in women, suggesting that estrogens influence body fat accumulation (O'Sullivan et al. 2001). Additionally, in aging, estrogens promote adipose deposition and insulin resistance (Cohen 2001). Conversely, results from aromatase, follicle-stimulating hormone, and ER-knockout studies indicate that estrogens regulate adiposity and that the complete lack of estrogens or blocking estrogen hormone action increases adipose tissue deposition (Cooke et al. 2000; Danilovich et al. 2000; Jones et al. 2000; Ohlsson et al. 2000; Rissman et al. 1999) whereas estrogen replacement in these models decreases adiposity. Interestingly, the phytoestrogen genistein has been shown to affect lipogenesis and lipolysis in isolated rat adipocytes (Szkudelska et al. 2000). Genistein augmented basal lipolysis, but this effect was reduced by insulin (Szkudelska et al. 2000). Preliminary data suggest that animals fed soy phytoestrogens have significantly decreased body and adipose tissue weights compared with animals fed a phytoestrogen-free diet. This suggestion implies that the estrogenic hormone action of phytoestrogens is beneficial to body fat regulation; however, we do not know the influence of phytoestrogens on body weight and fat regulation with aging.

Food/Water Intake and Changes in Body Weight

The primary objectives of regulatory behaviors are the following: (1) to supply the body with energy it derives from nutrients (e.g., carbohydrates, proteins, and fats, including important vitamins and minerals) via consumption; and (2) maintaining a homeostatic balance of intra- and especially extracellular fluids by water intake. Although good nutrition and available water are continually monitored in laboratory settings, the impact of phytochemicals (isoflavones) in diet formulations is rarely considered in the experimental design of most research investigations. Again, this impact is due to the endocrine-active properties of these molecules, which can alter basic regulatory behaviors such as food and water intake. We have examined these regulatory parameters in several studies using different rat strains (both outbred and inbred models) and animals under various hormonal conditions (Lephart et al. 2001a, 2002; Lund and Lephart 2001a; Weber at al. 2001). In general, a common thread representing many data sets suggests that different diet formulations of varying isoflavone content have a significant impact on food and water intake.

We have utilized four different diets of food and water that have contained varying concentrations of isoflavones. Male Long-Evans rats (Crl(LE)BR) were randomly assigned to one of the following four diet treatment groups at 50 days of age: (1) the AIN-76 diet (Test Diets, Richmond, IN), which contained approximately 0 ≤ 5 ppm of isoflavones; (2) a phytoestrogen (Phyto¹)-"free" diet (Ziegler Bros., Gardner, PA), which contained approximately 10 to 15 ppm of isoflavones; (3) a Phyto-200 diet (NIH-07, Ziegler Bros.), which contained approximately 200 ppm of isoflavones; and (4) a Phyto-600 diet (Harlen Teklad 8604, City, State), which contained approximately 600 ppm of isoflavones. The rats displayed significant differences in food and water intake at mid-age depending on the diet isoflavone formulation (Table 2). The Phyto-600-fed males displayed significantly greater water intake compared with all other groups, and food intake was significantly less in the AIN-76 and Phyto-free-fed males compared with Phyto-200 and Phyto-600 values (Table 2). These observations led us to examine the potential impact on body weight and white adipose tissue deposition based on these diet treatments.

When body and white adipose tissue weights were recorded at 350 days of age, it was surprising to discover that animals having the highest intakes of food and water displayed the lowest body and white adipose tissue weights (Table 2). In fact, the animals fed the Phyto-free diet had the heaviest body weights and white adipose tissue deposition whereas animals fed the highest isoflavone diet (Phyto-600) displayed the lowest body and adipose tissue weights (Table 2).

Although the literature is replete regarding dietary ingredients that influence regulatory behaviors, the available data to explain the findings above are sparse. Nevertheless, some insight can be gained by several studies that have examined specifically dietary isoflavones. We had previously observed that food and water intake and body weights in young adult Sprague-Dawley rats (SAS:VAF(SD)) were not significantly altered when animals were fed the Phyto-200 compared with the Phyto-free diet (Weber et al. 1999). This observation suggests that in addition to age as a factor, a threshold concentration of dietary isoflavones may be needed to influence these regulatory parameters--especially because data derived from our studies utilizing the Phyto-free versus the Phyto-600 diets consistently demonstrated alterations in food/water intake and clear reductions in body weights of Phyto-600-fed animals (Lephart et al. 2002). Presumably this result is due to the ability of isoflavones to inhibit lipogenesis and stimulate lipolysis (Naaz et al. 2003; Szkudelska et al. 2000). This result has been reported not only in animals but also in clinical studies, in which consumption of dietary soy-derived phytoestrogens significantly decreased body weight in humans (Bhathena and Velasquez 2002). We have also observed significantly increased levels of leptin (produced in adipose tissue) (Ahima et al. 2000) and insulin in AIN-76 and Phyto-free versus Phyto-200 and Phyto-600-fed animals. The increased levels in turn influence hypothalamic neuropeptide Y levels, which regulate feeding behavior (Ahima et al. 2000) (Table 2).

When considering alterations in water intake with isoflavone consumption, Slikker and colleagues (2001) reported that rats treated with genistein consumed more of a sodium-flavored solution compared with controls. These findings are similar to other studies in which dietary ethinyl estradiol exposure during development caused increased voluntary sodium intake (Ferguson et al. 2003). Moreover, dietary genistein exposure increased vasopressin levels in the rat hypothalamic region (Scallet et al. 2003), suggesting a shift in fluid balance and, potentially, alterations in blood pressure (Lephart et al. 2004; West 2003). Specifically, slight but significant decreases in blood pressure have been noted with soy consumption in animal and human studies (Lephart et al. 2004; West 2003).

Taking into consideration the influence of dietary isoflavones on regulatory parameters, we have observed slight but significant increases in locomotor behaviors in Phyto-600- versus Phyto-free-fed animals (dependent on the experimental task examined; Lund et al. 2001; Weber et al. 2001) along with alterations in thyroid hormone (E.D.L., unpublished observations), where consumption of isoflavone-rich diets have slight but significant effects on metabolism. In this regard, we have observed a significant increase in thyroid levels with soy consumption.

Notably, the influence of endocrine-disrupting substances such as dietary isoflavones require further study when considering their potential influence on basic regulatory behaviors such as food/water intake. This intake in turn modulates body weight and adipose tissue deposition, which cascades into other physiological and neuroendocrine hormonal mechanisms of homeostatic balance.

Learning and Memory

It is well established that estrogen acts as a neuroprotective and neurotrophic factor and plays an important role in influencing memory and cognition (Fugger et al. 2000; Juraska 1991; Luine et al. 1998; Shughrue and Merchenthaler 2000; Warren and Juraska 1997, 2000; Wise and Dubal 2000). Among the estrogenic endocrine disrupters examined to date, phytoestrogens have been investigated the most extensively; however, little research has been conducted to examine visual spatial memory in relation to phytoestrogens. In this section, we briefly review general characteristics of spatial skills, sex differences during trial performance, and the influence of isoflavones on visual spatial memory and learning.

Spatial Skills

In tasks requiring the use of spatial skills, many investigators have reported a consistent, sexually dimorphic difference in which males reliably outperform females (Frye and Sturgis 1995; Halpern 2000; Harris 1981; Juraska 1991; Luine et al. 1998; Rissman et al. 1999; Warren and Juraska 1997). This sex difference is most likely due to the presence in the brain of testosterone or, more likely, its metabolite estradiol (Frye and Sturgis 1995; Halpern 2000; Harris 1981; Juraska 1991; Luine et al. 1998; Rissman et al. 1999; Warren and Juraska 1997). Because phytoestrogens have the ability to bind estrogen receptors and alter many of the biological responses that are evoked by physiological estrogens (Kuiper et al. 1998; Lephart et al. 2001a,b; Lund and Lephart 2001a,b; Lund et al. 2001; Pan et al. 2000), many researchers have endeavored to determine the effects of phytoestrogens on visual spatial memory in adult rats. This mechanism is particularly important because the affinity of isoflavones for ER-beta is greater than it is for ER-alpha. Rissman and coworkers (2002) recently demonstrated that ER-beta is required for optimal spatial learning in ER-beta knockout mice. In examining the influence of phytoestrogens, the pioneering studies of Pan and colleagues (2000) ide.jpgied that ovariectomized female rats treated with phytoestrogens showed a dose-dependent improvement of visual spatial memory.

Sex Differences in Performance of Trials

We have expanded the research related to sex differences by comparing male and female rats on varying feeding schedules in behavioral paradigms (Lephart et al. 2002; Lund and Lephart 2001b; Lund et al. 2001). In these investigations, we ide.jpgied sex differences dependent on diet and behavioral paradigm. Measurements of accuracy in acquisition (determined as trials required for shaping to a predetermined criterion) of an eight-arm radial maze were shown to be diet dependent in that males fed a diet free of phytoestrogens (Phyto-free) and females fed a diet rich in phytoestrogens (Phyto-600) acquired the maze in fewer days than females or males fed the alternate diet (Lephart et al. 2002; Lund and Lephart 2001b; Lund et al. 2001;). There were, however, no significant dietary effects observed in an eight-arm task performance (in which food deprived animals retrieve a food reward from each of eight arms in an eight-arm radial arm maze) in either males or females (Lephart et al. 2002; Lund and Lephart 2001b; Lund et al. 2001).

Influence of Isoflavones

Measures of accuracy on a baited/unbaited four-arm task (a task in which food-deprived rats retrieve a food reward from only four predetermined arms in an eight-arm radial arm maze) demonstrated that a diet change in young adult animals (a change from phytoestrogen-rich [Phyto-600] to Phyto-free) had a positive influence on the accuracy in males but a negative influence on the accuracy in females, as shown in Figure 2 (Lephart et al. 2002; Lund and Lephart 2001b; Lund et al. 2001). The discrepancies in maze performance were expressed in two ways: (1) Males fed a lifelong diet rich in phytoestrogens (Phyto-600) committed more reference² errors than males switched to the Phyto-free diet; and (2) lifelong Phyto-600-fed males and females had increased mobility within the maze, which appeared to be advantageous to females but disruptive to males (Lephart et al. 2002; Lund and Lephart 2001b; Lund et al. 2001).

It is intriguing to speculate that the increase in reference² errors, but not working or working/reference² errors, in these studies suggests that disruption of maze performance occurred within the frontal cortex (but not the hippocampus) of lifelong Phyto-600-fed males (Lund et al. 2001). It is noteworthy that these changes in behavior and presumably in brain structure and function took place over a relatively short time interval (approximately 50 days for the diet changes) (Lephart et al. 2002, 2003; Lund et al. 2001b). This time factor demonstrates the plasticity of the brain during adulthood, especially in the hippocampus, where Gould and colleagues and others have shown neurogenesis in animals and humans (Eriksson et al. 1998; Gould et al. 2000). Furthermore, the demonstrated high affinity that isoflavones have for ER-beta, and the very high content of isoflavones in brain regions that express this ER subtype (e.g., 50- and 20-fold in frontal cortex and cerebellum, respectively), suggest a functional link in the regulation of behavior such as learning and memory (Lephart et al. 2002; Lund et al. 2001).

Taken together, these findings suggest that soy dietary phytoestrogens present in the animal diets, or the lack thereof, for a relatively short interval (approximately 50 days) even in young adult animals, significantly influence sexually dimorphic cognitive behavior. To date we are unaware that any other studies have demonstrated such profound effects on learning and memory by diet over a short exposure interval during adulthood. Dietary phytoestrogens sex-reversed visual spatial memory, as expressed in the radial-arm maze by enhancing spatial memory in females but inhibiting this ability in males.

Anxiety-related Behaviors and Phytoestrogens (Isoflavones)

In rodents, anxiety-related behaviors are tested by an elevated plus maze, which qua.jpgies the inherent conflict between exploration into the open arms of the maze, or moving into and remaining in the closed arms of the maze, which provides an environment of increased security. Movement into or time spent in the open arm is an index of decreased anxiety, as expressed in the elevated plus maze (Lund et al. 2001a). It is well recognized that the ovarian hormones estradiol and progesterone alter behavioral indices of anxiety in male and female rats (Imhof et al. 1993; Johnston and File 1991; Mora et al. 1996). Both estrogen and progesterone decrease anxiety by divergent mechanisms. Furthermore, neonatal tamoxifen administration and ovariectomy reduced the amount of time females spent on the open arms of the elevated plus maze (Zimmerberg and Farley 1993), whereas the absence of male gonadal hormones during the perinatal period decreased elevated plus maze-assessed anxiety and resulted in maze behavior resembling that of females. In addition to hormones, a number of plant extracts have been shown to have anxiolytic properties (Ang and Cheang 1999; Jaiswal et al. 1994; Peng et al. 2000; Thongsaard et al. 1996).

With regard to anxiety, the amygdala is involved in behavioral, autonomic, and neuroendocrine responses to stressful stimuli (Kim et al. 2001). The central nucleus of the amygdala is a component of the limbic fear-anxiety circuit, and has also been implicated in regulation of the hypothalamic-pituitary-adrenal (HPA¹) stress axis. Additionally, corticotropin-releasing factor (CRF¹), anterior pituitary adrenocorticotrophic hormone, and corticosterone are released in response to amygdala stimulation (Feldman and Weidenfeld 1998; Gabr et al. 1995) whereas lesions of the central or medial amygdaloid nuclei, sources of major efferent pathways from the amygdala, block or greatly attenuate HPA responses to stress (Beaulieu et al. 1986). CRF and arginine vasopressin-expressing neurons in the medial parvocellular paraventricular nucleus receive multisynaptic innervation from the amygdala, providing a neuroanatomical basis for amygdala regulation of the HPA axis (Prewitt and Herman 1998). Finally, it is reasonable to consider that other chemical messengers may influence anxiety-related behaviors via isoflavone hormonal actions. For example, oxytocin and vasopressin have been implicated in regulating anxiety (Bale et al. 2001; Mantella et al. 2003; Windle et al. 1997).

In specifically examining dietary soy-derived isoflavones and their influence on anxiety (as expressed in the elevated plus maze), our laboratory previously reported that exposure to dietary isoflavones throughout life, from conception until young adulthood, decreases anxiety in intact male and female rats (Lund and Lephart 2001a). When the anxiety-related behaviors were qua.jpgied, Phyto-600-fed animals spent significantly more time on the open arms and exhibited significantly more entries into the open arms.

In reference to our previous investigations in a recent study (Lund and Lephart 2001a), we examined the influence of different isoflavone diets on anxiety-related behaviors (expressed in the elevated plus maze) in mid-age (~10- to 11-mo-old) male or female rats (Figures 3-8). The Long-Evans rats were exposed to the AIN-76, Phyto-free, Phyto-200, or Phyto-600 diets from postnatal day 50 until approximately 300 days of age. In males, there was a dose-dependent reduction of anxiety-related behaviors in which animals fed the highest concentration of isoflavones displayed the lowest anxiety parameters, whereas animals fed the AIN-76 diet with essentially no isoflavones displayed the highest levels of anxiety (Figure 3). Analysis of the percentage of time spent in the open arms revealed a pattern that was similar to the number of entries into the open arms (Figure 4). Determination of serum phytoestrogen concentrations by gas chromatography/mass spectrometry (GC/MS¹) from these animals revealed a striking pattern in which AIN-76 displayed the lowest (or nearly nondetectable) levels, and the Phyto-600 males displayed the highest levels of isoflavones (Figure 5). In fact, the correspondence between the circulating isoflavone levels and display of anxiety-related behaviors in the elevated plus maze was 0.90, suggesting that high plasma isoflavone levels decrease the expression of anxiety.

Figure 3 Anxiety-related behaviors in the elevated plus maze of mid-aged (300-day-old) male Long-Evans rats fed four different diets from 50 to 300 days of age. *, Significantly increased the number of entries compared with AIN-76 or phytoestrogen-free-fed male values. ●, Significantly increased the number of entries compared with all other diet treatment groups. Animals per diet treatment, n = 5.

Figure 4 Anxiety-related behavior in the elevated plus maze of mid-aged (300-day-old) male Long-Evans rats fed four different diets from 50 to 300 days of age. *, Significantly increased time in the open arms compared with AIN-76 or phytoestrogen-free-fed males. ●, Significantly increased the time in the open arms compared with all other diet treatment groups. Animals per treatment group, n = 5.

Figure 5 Serum (total) phytoestrogen (Phyto) levels in mid-aged (300-day-old) male Long-Evans rats fed four different diets from 50 to 300 days of age. Animals per diet treatment, n = 5. Serum isoflavone concentrations were qua.jpgied by gas chromatography/mass spectometry. Individual isoflavone levels for each diet group are shown. AIN-76: <1 ng/mL; Phyto-free: genistein = 3.3 + 0.3; daidzein = 2.3 + 0.3; equol = 25 + 7; Phyto-200: genistein = 125 + 20; daidzein = 121 + 22; equol = 280 + 39; Phyto-600: genistein = 134 + 10; daidzein = 144 + 15; equol = 592 + 79.

Figure 6 Anxiety-related behaviors in the elevated plus maze of mid-aged (330-day-old) female Long-Evans rats fed four different diets from 50 to 300 days of age. □, Significantly increased number of entries compared with AIN-76 values; *, Significantly increased number of entries compared with all other diet treatment groups. Animals per diet treatment, n = 7.

Figure 7 Anxiety-related behavior in the elevated plus maze of mid-aged (330-day-old) female Long-Evans rats fed four different diets from 50 to 330 days of age. □, Significantly increased time spent in open arms compared to AIN- 76 values; ▼, Significantly increased time spent in open arms compared with AIN-76 and phytoestrogen-free values. Animals per group, n = 7.

Figure 8 Serum (total) phytoestrogen (Phyto) levels in mid-aged (330-day-old) female Long-Evans rats fed four different diets from 50 to 300 days of age. Animals per diet treatment, n = 7. Serum isoflavone concentrations were qua.jpgied by gas chromatography/mass spectometry). Individual isoflavone levels for each diet group are as follows: AIN-76: <1 ng/mL; Phyto-free: genistein = 2.3 + 0.1; daidzein = 3.5 + 0.2; equol = 13 + 1; Phyto-200: genistein = 48 + 5; daidzein = 42 + 3; equol = 243 + 28; Phyto-600: genistein = 53 + 6; daidzein = 58 + 4; equol = 733 + 35.

Examination of the female rats revealed a pattern of anxiety-related behaviors that was similar to that of the male rats (Figure 6). However, the influence of dietary isoflavones was not as robust as that seen in males, although the highest percentage of the number of entries into or time spent in the open arms was seen in Phyto-600-fed females, with a stepwise pattern of decline until the lowest percentage of entries were seen in the AIN-76-fed females (Figures 6 and 7). It should be noted that before testing, the rats were monitored by vaginal smears for 12 consecutive days, and none of these animals were cycling. It is well established that Long-Evans rats approximately 270 days of age display irregular cycles and stop cycling shortly thereafter (Mills et al. 2002). Thus, it is not possible that the estrous cycle has an influence on the results. In fact, we measured the total distance the males and females travel (by diet treatment groups) during the elevated plus maze test, and there are no significant differences in any of the values among the treatment groups (by sex; data not shown). Again, determination of serum phytoestrogen levels by GC/MS revealed a high correlation between circulating isoflavone concentrations within each diet treatment group and the expression of anxiety (r = 0.81) (Figure 8).

These preliminary data sets are novel, and suggest that diet alone can influence anxiety-related behaviors. In other words, the isoflavone content of a diet can have significant effects on anxiety presumably by these estrogen-like molecules binding estrogen receptors within the brain, and subsequently changing the expression and/or distribution of these receptor subtypes. However, it is also intriguing to consider that because the most abundant circulating isoflavone in rodents is equol, which recently has been shown to specifically bind the most potent 5 alpha-reduced androgen (i.e., 5 alpha-DHT) and 5 alpha-reduced progesterone metabolites (e.g., tetrahydroprogesterone [Lund et al. 2004]), this finding may represent an alteration of hormone balance that would stimulate exploration of a novel area over expression of anxiety-related behavior. Assuredly, further research is required to unravel the complex hormonal actions of isoflavones (especially in the brain) and the expression of behavior such as anxiety.

Summary and Conclusions

This brief article focuses mainly on our investigations of the influence of dietary soy-derived isoflavones on certain aspects of regulated behaviors (e.g., food and water intake) and on resulting changes in body weight and adipose tissue deposition as well as on learning and memory and on the expression of anxiety-related behaviors. Because soy products are used as the main protein source in all natural-ingredient commercially available animal formulated diets, we characterized the influence of a commercially available diet rich in phytoestrogens (Phyto-600, containing 600 µg of isoflavones or 600 ppm) and compared it with diets containing 200 ppm (Phyto-200), 10-15 ppm (Phyto-free), or almost no isoflavones (0 < 5 ppm, AIN-76). Most of our studies have utilized the Phyto-600 and Phyto-free diets. To validate the soy dietary effects on behavioral parameters, phytoestrogen levels in each diet were measured by high-performance liquid chromatography analysis (Lephart et al. 2002). After consumption of these diets, circulating phytoestrogen plasma levels were qua.jpgied by GC/MS methods and in some cases, brain phytoestrogen concentrations were determined by time-resolved radioimmunoassay analysis in certain brain regions (Lephart et al. 2004; Lund et al. 2001).

From several studies, Phyto-rich (Phyto-600)-fed animals displayed circulating phytoestrogen levels approximately 30- to 60-fold higher compared with Phyto-free-fed animals. In certain brain regions, especially where ER-beta is abundant, isoflavone concentrations were dramatically greater (8- to 50-fold higher) in Phyto-600- versus Phyto-free-fed animals (Lephart et al. 2004; Lund et al. 2001), as discussed above.

We have examined brain aromatase under different developmental intervals (perinatal, maternal and during adulthood) and have observed no significant alterations (Lephart et al. 2002). This observation suggests that production of estrogen via the conversion of androgen in brain is not altered by phytoestrogens, and that the most likely action of isoflavones in brain is at the ER system (Lephart et al. 2002). However, the recent finding that equol, the most abundant circulating isoflavone in rodents that possesses the ability to specifically bind 5 alpha-DHT, but not testosterone, places an additional modification or connection in the hormonal action of these potent dietary-derived molecules, with important implications for brain function, reproductive/endocrine physiology, and behavior (Lund et al. 2004).

In experiments examining the impact of isoflavones on learning and memory, animals were tested in the radial arm maze to determine visual spatial memory parameters such as reference and working memories. In one study, female rats fed the Phyto-600 diet acquired the maze significantly faster than females fed the Phyto-free diet, whereas in males, the opposite diet effect was observed (i.e., Phyto-free-fed males acquired the maze significantly faster than males fed the Phyto-600 diet). In a similar experimental design in which the diet was switched during adulthood, significant alterations were observed in visual spatial memory performance by sex and diet. Before the diet changed, males fed the Phyto-600 diet acquired the maze significantly faster than females, and males outperformed females on the same diet (in working memory tasks). After the diet switch, in a reference memory task, Phyto-600-fed males performed significantly better than Phyto-free-fed females; and surprisingly, males displayed an opposite pattern (i.e., males switched to the Phyto-free diet significantly outperformed males fed the Phyto-600 diet lifelong).

The findings described above suggest that dietary isoflavones may sex-reverse the typical sexually dimorphic expression of visual spatial memory performance. Specifically, tasks requiring reference memory appear to be affected more than working memory, possibly due to the abundance of ER-beta in the frontal cortical area, where approximately 50-fold higher concentrations of isoflavones have been observed in Phyto-600 versus Phyto-free-fed animals (Lephart et al. 2004; Lund et al. 2001). However, further research is necessary to unravel the complex mechanisms of how isoflavones influence learning and memory, especially in males. For example, more recent data (unpublished, from our laboratory) suggest that isoflavones are neuroprotective within the hippocampus, whereas consuming a Phyto-free diet may act as a memory enhancer by inhibiting GABA mechanisms in the hippocampus and possibly causing cellular damage in the long term.

Finally, in studies characterizing the effects of dietary phytoestrogens on the expression of anxiety (as determined in the elevated plus maze), isoflavone consumption of the Phyto-600 versus the Phyto-free diet in both mid-aged male and female Long-Evans rats produced presumably anxiolytic effects. The Phyto-600-fed animals appeared to be less anxious than the Phyto-free-fed animals in that they spent significantly more time on and made significantly more entries into the open arms of the maze. This behavior suggests that consumption of dietary isoflavones can alter anxiety levels in animal models as expressed in the elevated plus maze.

As reviewed by Setchell and coworkers (2002), isoflavones are important estrogen mimics, and rodents produce equol at high levels when they consume a diet rich in soy ingredients. Humans, in contrast, generate relatively low levels of equol, and only approximately 30% of individuals consuming a soy diet produce equol. Although a divergent characteristic between humans and rodent in isoflavone metabolism is clear, the importance of equol in rodent and human health issues is only now emerging (Setchell et al. 2002).

The findings discussed herein indicate that dietary soy-derived isoflavones can influence the expression of behavioral parameters that previously were unknown. Increased awareness and understanding of behavioral plasticity are evolving with advancements in technology and exchange of scie.jpgic information. Because all commercially available rodent diets use soy as the main source of protein in their formulations, it is critical to learn the exact effect of dietary isoflavones on brain and the expression of behavior.

Acknowledgments

This manuscript was developed with support, in part, from US Department of Agriculture grant 2002-00798 (E.D.L.). The authors thank L. Bu, D. Bu, and L. Gonzalez for their valuable assistance in the performance of behavioral and biochemical assays and statistical analysis of certain aspects of the studies presented herein.

¹Abbreviations used in this article: ER, estrogen receptor; CRF, corticotropin-releasing factor; DHT, dihydrotestosterone; GC/MS, gas chromatography/mass spectrometry; HPA, hypothalamic-pituitary-adrenal; Phyto, phytoestrogen.

²The basic definitions for working and reference memory in the radial arm maze include the following: Reference memory is information that should be retained until the next trial (memory that persists from trial to trial). Working memory is information that disappears in a short time (trial-dependent memory).

References

Ahima RS, Saper CB, Flier JS, Elmquist JK. 2000. Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 3:263-307.

Ang HH, Cheang HS. 1999. Studies on the anziolytic activity of Eurycoma lo.jpgolia jack roots in mice. Jpn J Pharmacol 79:497-500.

Appt SE. 2004. Usefulness of the monkey model to investigate the role of soy in postmenopausal women's health. ILAR J 45:200-211.

Bale T, Davis AM, Auger AP, Dorsa DM, McCarthy, CNS. 2001. Region-specific oxytocin receptor expression: Importance in regulation of anxiety and sex behavior. J Neurosci 21:2546-2552.

Barnes S. 1998. Evolution of the health benefits of soy isoflavones. Proc Soc Exp Biol Med 217:386-392.

Beaulieu S, Di Paolo T, Barden N. 1986. Control of ACTH secretion by the central nucleus of the amygdala: Implication of the serotoninergic systems and its relevance to the glucocorticoid delayed negative feedback mechanism. Neuroendocrinology 44:247-254.

Bhathena SJ, Velasquez MT. 2002. Beneficial role of dietary phytoestrogens in obesity and dieabetes. Am J Clin Nutr 76:1191-201.

Brown NM, Setchell KD. 2001. Animals models impacted by phytoestrogens in commercial chow: Implications for pathways influenced by hormones. Lab Invest 81:735-747.

Cohen PG. 2001. Aromatase, adiposity, aging and disease: The hypogonadal-metabolic-atherogenic-disease and aging connection. Med Hypotheses 56:702-708.

Cooke PS, Heine PA, Taylor JA, Lubahn DB. 2000. The role of estrogen and estrogen receptor alpha in male adipose tissue. Mol Cell Endocrinol 178:117-154.

Danilovich N, Babu PS, Xing W, Gerdes M, Krishnamurthy H, Sairam MR. 2000. Estrogen deficiency, obesity, and skeletal abnormalities in FSH receptor knockout (FORKO) female mice. Endocrinology 141:4295-4308.

Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH. 1998. Neurogenesis in the adult human hippocampus. Nat Med 4:1313-1317.

Feldman S, Weidenfeld J. 1998. The excitatory effects of the amygdala on the hypothalamo-pituitary-adrenocortical responses are mediated by hypothalamic norepinephrine, serotonin, and CRF-41. Brain Res Bull 45:389-393.

Ferguson SA, Delclos KB, Newbold RR, Flynn KM. 2003. Dietary ethinyl estradiol exposure during development causes increased voluntary sodium intake and mild maternal and offspring toxicity in rats. Neurotoxicol Teratol 25:491-501.

Frye CA. Sturgis JD. 1995. Neurosteroids affect spatial/reference, working, and long-term memory of female rats. Neurobio Learn Mem 64:83-96.

Fugger HN, Foster TC, Gustafsson J, Rissman EF. 2000. Novel effects of estradiol and estrogen receptor alpha and beta on cognitive function. Brain Res 883:258-264.

Gabr RW, Birkle DL, Azzaro AJ. 1995. Stimulation of the amygdala by glutamate facilitates corticotropin-releasing factor release from the median eminence and activation of the hypothalamic-pituitary-adrenal axis in stressed rats. Neuroendocrinology 62:333-339.

Gould E, Tanapat P, Rydel T, Hastings N. 2000. Regulation of hippocampal neurogenesis in adulthood. Biol Psychiatry 48:715-720.

Halpern DF. 2000. Sex Differences in Cognitive Abilities. 3rd ed. San Bernadino CA: LEA Press.

Harris LJ. 1981. Sex differences in spatial ability: Possible environmental, genetic, and neurological factors. In: Kinsbourne M, ed. Asymmetrical Function of the Brain. New York: Cambridge University Press.

Imhof JT, Coelho ZMI, Schmitt ML, Morato GS, Carobrez AP. 1993. Influence of gender and age on performance of rats in the elevated plus maze apparatus. Behav Brain Res 56:177-180.

Jaiswal AK, Bhattacharya SK, Acharya SB. 1994. Anxiolytic activity of Azadirachta indica leaf extracts in rats. Ind J Exp Biol 32:489-491.

Johnston AL, File SE. 1991. Sex differences in animal tests of anxiety. Physiol Behav 49:254-250.

Jones ME, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto OK, Leury BJ, Robertson KM, Yao S, Simpson ER. 2000. Aromatase-deficient (ArKO) mice have a phenotype of increased obesity. Proc Natl Acad Sci U S A 97:12735-12740.

Juraska JM. 1991. Sex differences in "cognitive" regions of the rat brain. Psychoneuroendocrinology 16:105-119.

Kim JJ, Lee HJ, Han J-S, Packard MG. 2001. Amygdala is critical for stress-induced modulation of hippocampal long-term potentiation and learning. J Neurosci 21:5222-5228.

Kuiper GG, Carlsson, B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870.

Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA. 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263.

Lephart ED, Adlercreutz H, Lund TD. 2001a. Dietary soy phytoestrogen effects on brain structure and aromatase in Long-Evans rats. NeuroRep 12:3451-3455.

Lephart ED, Lund TD, Horvath TL. 2001b. Brain androgen and progesterone metabolizing enzymes: Biosynthesis distribution and function. Brain Res Rev 37:25-37.

Lephart ED, Porter JP, Hedges DW, Lund TD, Setchell KDR. 2004. Phytoestrogens: Implications in neurovascular research. Neurovas Res (In Press).

Lephart ED, Rhees RW, Setchell KDR, Bu L, Lund TD. 2003. Estrogens and phytoestrogens: Brain plasticity of sexually dimorphic brain volumes. J Steroid Biochem Mol Biol 85:299-309.

Lephart ED, West TW, Weber KS, Rhees RW, Setchell KDR, Adlercreutz H, Lund TD. 2002. Neurobehavioral effects of dietary soy phytoestrogens. Neurotoxicol Teratol 24:1-12.

Leshner AI. 1978. Regulatory behaviors. In: An Introduction to Behavioral Endocrinology. New York: Oxford University Press. p 39-48.

Lindsay R, Dempster DW, Jordan VC. 1997. Estrogens and Antiestrogens. New York: Lippincott-Raven. p 122-123.

Luine VN, Richards ST, Wu VY, Beck KD. 1998. Estradiol enhances learning and memory in a spatial memory task and effects levels of monoaminergic neurotransmitters. Horm Behav 34:149-162.

Lund TD, Lephart ED. 2001a. Dietary soy phytoestrogens produce anxiolytic effects in the elevated plus-maze. Brain Res 913:180-184.

Lund TD, Lephart ED. 2001b. Manipulation of prenatal hormones and dietary phytoestrogens during adulthood alter the sexually dimorphic expression of visual spatial memory. BMC Neurosci 2:21.

Lund TD, Munson DJ, Haldy ME, Setchell KDR, Lephart ED and Handa RJ. 2004. Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod 70:1188-1195.

Lund TD, West TW, Tian LY, Bu LH, Simmons DL, Setchell KDR, Adlercreutz H, Lephart ED. 2001. Visual spatial memory is enhanced in females (but inhibited in males) by dietary soy phytoestrogens. BMC Neurosci 2:20.

Mantella RC, Vollmer RR, Xia L, Amico JA. 2003. Female oxytocin-deficient mice display enhanced anxiety-related behavior. Endocrinology 144:2291-2296.

Mazur W, Adlercreutz H. 2000. Overview of naturally occurring endocrine-active substances in the human diet in relation to human health. Nutrition 16:654-658.

Mills RH, Romeo HE, Lu JK, Micevych PE. 2002. Site-specific decrease of progesterone receptor mRNA expression in the hypothalamus of middle-aged persistently estrous rats. Brain Res 955:200-206.

Mora S, Dussaubat N, Diaz-Veliz G. 1996. Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats. Psychoneuroendocrinology 21:609-620.

Naaz A, Yellayi S, Zakroczymski MA, Bunick D, Doerge DR, Lubahn DB, Helferich WG, Cooke PS. 2003. The soy isoflavone genistein decreases adipose deposition in mice. Endocrinology 144:3315-3320.

Naz RK. 1999. Endocrine Distruptor-Effects on Male and Female Reproductive Systems. Boca Raton: CRC Press.

Ohlsson C, Hellberg N, Parini P, Vidal O, Bohlooly M, Rudling M, Lindberg MK, Warner M, Angelin B, Gustaffson JA. 2000. Obesity and distributed lipoprotein profile in estrogen receptor-alpha-deficient male mice. Biochem Biophys Res Commun 278:640-645.

O'Sullivan AJ, Martin A, Brown MA. 2001. Efficient fat storage in premenopausal women and in early pregnancy: A role for estrogen. J Clin Endocrinol Metab 86:4950-4960.

Pan Y, Anthony M, Watson S, Clarkson TB. 2000. Soy phytoestrogens improve radial arm maze performance in ovariectomized retired breeder rats and do not attenuate benefits of 17beta-estradiol treatement. Menopause 7:230-235.

Patisaul HB, Dindo M, Whitten PL, Young LJ. 2001. Soy isoflavone supplements antagonize reproductive behavior and estrogen receptor alpha- and beta-receptor dependent gene expression in the brain. Endocrinology 142:2946-52.

Patisaul HB, Melby M, Whitten PL, Young LJ.2002. Genistin affects ER beta--But not ER alpha-dependent gene expression in the hypothalamus. Endocrinology 143:2189-2197.

Patisaul HB, Whitten PL. 1998. Dietary phytoestrogens. In: Naz RK, ed. Endocrine Disruptors. New York: CRC Press. p 100-123.

Peng WH, Hsieh MT, Lee YS, Lin YC, Liao J. 2000. Anxiolytic effect of seed of Ziziphus jujuba in mouse models of anxiety. J Ethnopharmacol 72:435-441.

Prewitt CM, Herman JP. 1998. Anatomical interactions between the central amygdaloid nucleus and the hypothalamic paraventricular nucleus of the rat: A dual tract-tracing analysis. J Chem Neuroanat 15:173-185.

Rissman EF, Wersinger SR, Fugger HN, Foster TC. 1999. Sex with knockout models: Behavioral studies of estrogen receptor alpha. Brain Res 835:80-90.

Rissman EF, Heck AL, Leonard JE, Shupnik MA, Gustafsson J-A. 2002. Disruption of estrogen receptor beta gene impairs spatial learning in female mice. Proc Natl Acad Sci U S A 99:3996-4001.

Scallet AC, Wofford M, Meredith JC, Allaben WT, Ferguson SA. 2003. Dietary exposure to genistein increases vasoporessin but does not alter beta-endorphin in the rat hypothalamus. Toxicol Sci 72:296-300.

Setchell KDR. 1998. Phytoestrogens: Biochemistry, physiology and implications for human health of soy isoflavones. Am J Clin Nutr 129:333S-1346S.

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.

Shughrue PJ, Merchenthaler I. 2000. Estrogen is more than just a "sex hormone": Novel sites for estrogen action in the hippocampus and cerebral cortex. Front Neuroendocrinol 21:95-101.

Singleton DW, Khan SA. 2003. Xenoestrogen exposure and mechanism of endocrine disruption. Front Biosci 8:110-118.

Slikker W Jr, Scallet AC, Doerge DR, Ferguson SA. 2001. Gender-based differences in rats after chronic dietary exposure to genistiein. Int J Toxicol 20:175-179.

Szkudelska K, Nogowski L, Szkudelski T. 2000. Genestein affects lipogenesis and lipolysis in isolated rat adipocytes. J Steroid Biochem Mol Biol 75:265-271.

Thigpen JE. 2004. Selecting appropriate diets for endocrine disruptor research and testing studies. ILAR J 45:401-416.

Thigpen JE, Setchell KDR, Ahlmark KB, Locklear J, Spahr T, Caviness GF, Goelz MF, Haseman JK, Newbold RR, Forsythe DB. 1999. Phytoestrogen content of purified, open- and closed-formula laboratory animal diets. J Anim Sci 49:530-536.

Thongsaard W, Deachapunya C, Pongsakorn S, Boyd EA, Bennett GW, Marsden CA. 1996. Barakol: A potential anxiolytic extracted from Cassia siamea. Pharmacol Biochem Behav 53:753-758.

Turner KJ, Sharpe RM. 1997. Environmental oestrogens--Present understanding. Rev Reprod 2:69-73.

Wade GN. 1972. Gonadal hormones and behavioral regulation of body weight. Phys Behav 8:523-534.

Warren SG, Juraska, JM. 1997. Spatial and nonspatial learning across the rat estrous cycle. Behav Neurosci 111:259-266.

Warren SG, Juraska, JM. 2000. Sex differences and estropausal phase effects on water maze performance in aged rats. Neurobiol Learn Mem 74:229-240.

Weber KS, Jacobson NA, Setchell KDR, Lephart ED. 1999. Brain aromatase and 5α-reductase, regulatory behaviors and testosterone levels in adult rats on phytoestrogen diets. Proc Soc Exp Biol Med 22:131-135.

Weber KS, Setchell KDR, Stocco DM, Lephart ED. 2001. Dietary soy-phytoestrogens decrease testosterone levels and prostate weight, without altering LH, prostate 5α-reductase or testicular StAR levels in adult male Sprague-Dawley rats. J Endocrinol 170:591-599.

West SG. 2003. Blood pressure and vascular effects of soy: How strong is the evidence? Curr Top Nutraceut Res 1:17-30.

Whitten PL, Patisaul HB. 2001. Cross-species and interassay comparisons of phytoestrogen action. Environ Health Perspect 109:5-20.

Windle RJ, Shanks N, Lightman SL, Ingram CD. 1997. Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology 138:2829-34.

Wise PM, Dubal, DB. 2000. Estradiol protects against ischemic brain injury in middle-aged rats. Biol Reprod 63:982-985.

Yen SSC, Jaffe RL, Barbieri RL. 1999. Reproductive Endocrinology. Philadelphia: WB Saunders.

Zimmerberg B, Farley MJ. 1993. Sex differences in anxiety behavior in rats: Role of gonadal hormones. Physiol Behav 54:–1119-1124.