| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Female Reproductive Tract |
Departments of Veterinary Biosciences,3
Food Science and Human Nutrition,4
and Chemistry5
and Division of Nutritional Sciences,6 University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
National Center for Toxicological Research,7 Food and Drug Administration, Jefferson, Arkansas 72079
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.
female reproductive tract, steroid hormone receptors, uterus, vagina
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß [6, 7], with higher affinity for ERß [8]. Studies on soy isoflavones have focused primarily on genistein because it is a more potent estrogen than daidzein [9, 10], is typically present in higher quantities than daidzein, and has other properties such as inhibition of protein tyrosine kinases [11] and topoisomerase II [12, 13], as well as induction of TGFß [9].
Despite the limited estrogenicity of daidzein, some recent evidence suggests that its metabolite equol may play a critical role in phytoestrogen effects [2]. Daidzein is converted by microbial biotransformation in the intestine to the isoflavan equol, a biologically active metabolite [14]. Equol has a significantly longer half life in the circulation than genistein or daidzein [15] and binds to both ER and ERß, although similar to genistein and daidzein its binding affinity for ERß is greater than that for ER [16]. Approximately one third of humans produce equol; this may reflect differences in the intestinal microbial flora of this group compared with humans who do not produce equol [17, 18]. Setchell et al. [2] have postulated that the beneficial responses from soy consumption may correlate with the equol-producing status of individuals, suggesting a major role for equol as a biomarker for the effectiveness of soy [2]. Further, rodents on commercial diets formulated with soy meal are continuously exposed to isoflavones [19] and have sustained high serum equol concentrations [20]. Such high equol levels may potentially have important implications in experiments measuring endpoints influenced by isoflavones [21].
Despite the potential importance of equol, there have been limited studies of equol effects in vivo because of the high cost of this compound and its limited availability. In addition, studies of equol effects in vivo [22, 23] have utilized equol injection, rather than the more physiological administration of equol in the diet, again because of the high cost of equol precluding dietary studies. Furthermore, due to difficulties in quantitating equol in biological fluids [2], there have been no studies correlating various serum equol concentrations with estrogenic or other biological effects.
Development of a technique for producing high quantities of a racemic mixture of equol [16] has provided a unique opportunity to more fully characterize the effects of equol in vivo on a variety of estrogen-responsive endpoints. In this study, we examined effects of injected equol and dietary equol exposure that produce levels of equol exposure similar to those reported in animals consuming standard soy-based diets. Our results show that this racemic equol can induce estrogenic effects on various parameters by acting through ER when present at concentrations routinely obtained in rodents fed soy-based diets, although its potency is substantially less than genistein.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Juvenile age-matched female C57BL/6 mice (21-day-old weanlings; 12–14 g) were purchased from Harlan (Indianapolis, IN). Individually caged mice were ovariectomized on Day 26 to minimize endogenous estrogen production and placed on a phytoestrogen-free diet (AIN-93G purified rodent diet; Dyets Inc., Bethlehem, PA); treatment was started on Day 30. Mice were housed under controlled lighting (12L:12D) and temperature (21°–22°C) and maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois.
Treatments
The effects of a racemic mixture of equol [16] on a number of estrogen-sensitive endpoints were evaluated in this study. Mice were given daily subcutaneous injections of vehicle (control) or equol at 4, 8, 12, and 20 mg (kg body weight [BW])–1 day–1 in a DMSO–corn oil suspension (50 µl DMSO in 1 ml corn oil). Equol dissolved in DMSO was used as a suspension in oil for the injections to create a more sustained release and to avoid rapid increases in serum followed by a log decline as observed in previous experiments with injected phytoestrogens [24]. As a positive control for the effects of estrogen on uterine weight, one group of animals was given daily injections of 1.5 µg E2/kg BW. To determine the effects of dietary equol, mice were fed ad libitum with AIN-93G purified rodent diet (control) or this diet supplemented with equol at 500 or 1000 ppm for 12 days (n = 5 per group). Individual feed intake was measured during the treatment period to control for possible changes in consumption levels of equol-supplemented feed compared with control.
Tissue Collection
At the end of treatment, mice were weighed and killed by CO2 overdose. Blood was collected by cardiac puncture, and the serum was separated following coagulation at 4°C and frozen at –20°C for subsequent equol assay. Mice were killed 2 h after the last injection [25]. Mice given dietary equol were killed at lights on (0800 h) on Day 12 to get a representative serum measurement of equol [26]. The right parametrial and inguinal fat pads were removed and weighed, and uterus, vagina, and thymus were removed, weighed, and fixed in neutral buffered formalin (NBF) for histological processing.
Histology and Immunohistochemistry
Tissues were embedded in paraffin and sectioned at 4 µm. For routine histological examination, sections were stained with hematoxylin and eosin. To quantify the effect of equol on uterine epithelial proliferation, uterine cross-sections were immunostained with anti-mouse Ki-67 monoclonal antibody (BD Pharmingen, San Diego, CA). Paraffin sections were hydrated and subjected to antigen retrieval by immersing in 0.01 M citrate buffer and microwaving for 20 min. Endogenous peroxidase was inactivated using 0.3% H2O2 in methanol. Nonspecific binding of antibodies was blocked using 5% normal goat serum, and then samples were incubated with anti-mouse Ki-67 antibody (1:1200) in a moist chamber overnight. After incubation with a biotinylated secondary antibody, the signal was amplified by using the streptavidin-biotin system with the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA). The samples were then incubated with substrate Tris-HCl/H2O2/DAB for 8 min. The sections were mounted after slight counterstaining with hematoxylin. Vaginal epithelial thickness was measured after digital imaging using the software package Image J, 1.30v (National Institutes of Health, Bethesda, MD).
Serum Equol Measurements
Total equol and the concentration of the aglycone form of equol were measured in the serum using a validated isotope dilution liquid chromatography electrospray mass spectrometry (LC-ES/MS) method [27].
Statistical Analyses
Data were analyzed by ANOVA and Dunnet post hoc two-sided comparisons using Systat version 10 (SPSS Inc., Chicago, IL; 2000). Data are represented as mean ± SEM. Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There was a dose-dependent increase in total serum equol levels following equol injection or dietary equol exposure (Fig. 1). Serum equol was measured 2 h after the last injection, and the concentrations of equol were 1.4 ± 0.1, 2.6 ± 0.3, 5.1 ± 0.2, and 7.5 ± 1.1 µM for the 4, 8, 12, and 20 mg (kg BW)–1 day–1 groups, respectively (n = 5/group; Fig. 1). In the feeding study with dietary equol exposure, serum concentrations of equol measured at lights on were 5.9 ± 0.7 and 8.1 ± 0.8 µM for the 500 and 1000 ppm groups, respectively (n = 5/group; Fig. 1). Neither injection nor feeding controls had measurable serum equol (n = 5/group).
|
Equol aglycone concentrations showed dose-dependent increases in the injected mice (Fig. 2). The concentration of the aglycone form of equol in the serum was approximately 20% that of total equol in the injected mice. This was 10-fold higher than the relative percentage of equol present as the aglycone following dietary equol exposure at 1000 ppm, which was only 2% of the total serum equol (Fig. 2).
|
Equol added in the diet did not affect feed consumption in the mice over the study period. Total average food consumption was 33.8 ± 3.5 g (control), 33.3 ± 2.4 g (500 ppm equol), and 32.4 ± 1.7 g (1000 ppm equol) over the 12-day experimental period. Similarly, body weights were not significantly different in any of the injected or dietary equol groups compared with controls (data not shown).
Uterotropic and Vaginal Response
Equol was uterotropic at high doses. Uterine weights at 12 and 20 mg (kg BW)–1 day–1 were 2- and 3.5-fold that of the controls, respectively (Fig. 3). The uterine weights even with the highest dose of injected equol were less than that seen in the E2-treated group, which functioned as a positive control. Although equol injected at 8 mg (kg BW)–1 day–1 produced a trend toward increased uterine weights, this did not reach statistical significance. Uterine weight was not significantly increased at either the 500 or 1000 ppm equol feeding doses, although again there was a trend toward an increase (P = 0.132 and 0.063 compared with control, respectively).
|
There was a dose-dependent increase in uterine epithelial proliferation with equol injections (Fig. 4), which was significantly different from control at 8 mg (kg BW)–1 day–1 and higher. Proliferation was lower following dietary equol treatments compared with the groups given the higher injected doses of equol (Fig. 4A), although dietary equol produced a significant increase in labeling index compared with the control at 1000 ppm (Fig. 4B). This observation was consistent with the increases in uterine weights seen following equol treatment, where even the highest dietary equol concentration (1000 ppm) produced less pronounced responses than the higher dose injections.
|
Equol also produced dose-dependent increases in vaginal epithelial height consistent with an estrogenic response (Fig. 5A). Vaginal epithelial height increased from 7.3 ± 0.5 µm in the control to 8.5 ± 0.6, 20.3 ± 1.1, 27.7 ± 2.4, and 39.3 ± 4.1 µm with equol injections of 4, 8, 12, and 20 mg (kg BW)–1 day–1, respectively, and to 21.5 ± 2.8 and 30.1 ± 2.4 µm with dietary equol at 500 and 1000 ppm, respectively (Fig. 5B). This increase was significant for the 8, 12, and 20 mg (kg BW)–1 day–1 equol injections and at both dietary equol concentrations.
|
Adipose Tissue and Thymus
Both adipose tissue and thymus express ER and ERß, and administration of phytoestrogens such as genistein produces thymic atrophy, immune suppression, and a decrease in adipose tissue [24, 28, 29]. However, neither the parametrial nor inguinal fat pads were significantly decreased in weight at any of the injected or dietary doses of equol (data not shown). Similarly, a decrease in thymic weight was not observed with any equol treatment (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Equol is optically active and exists as R and S enantiomers. Equol produced during intestinal digestion in humans and other animals is the S-form, which has greater ER binding affinity and increased ERß binding compared with the R-form [16]. However, overall ER and ERß binding of a racemic mixture produced by the new synthesis procedure is comparable with that of the naturally occurring S enantiomer [16], and this racemic mixture was used for all of our studies.
Increases in uterine weight and epithelial proliferation are classical estrogen effects mediated by ER, and this effect is used to measure estrogenicity of environmental estrogens [30–32]. Equol was initially reported to be biologically inactive [33], but in vitro experiments have shown that equol binds both ER and ERß [6, 16]. In addition, previous work has indicated that injected equol is uterotropic in mice and sheep [22, 34], a finding confirmed by our present results.
Equol injections produced greater increases in uterine weight and epithelial proliferation than dietary equol, although even injections with the highest equol dose did not increase uterine weight to the degree seen in the positive controls, which received E2. Dietary equol at 500–1000 ppm produced a weak estrogenic response with moderate increases in uterine weight that did not reach significance and a low Ki-67 epithelial labeling index that was significantly greater than control only at 1000 ppm. Mice fed commercial soy-based diets have high isoflavone intake and total plasma isoflavone levels ranging from 4.4 to 8.5 µM have been reported [20]. Rodents efficiently convert daidzein to equol, and up to 90% of circulating isoflavone in rodents fed commercial chow is equol, emphasizing the importance of equol in isoflavone action and its potential ability to alter processes regulated by estrogens [19–21]. Our study demonstrates the first estrogenic effects of dietary equol, and our results show that equol is weakly uterotropic at serum concentrations comparable with those in rodents fed soy-based diets.
Induction of vaginal epithelial proliferation and cornification is a classical estrogenic effect involving both stromal and epithelial ER [35]. Without estrogen exposure, vaginal epithelium is atrophic, consisting of two to three layers of flattened squamous epithelial cells. Estrogenic stimulation causes epithelial proliferation, stratification, and apical cornification. We observed dose-dependent increases in vaginal epithelial thickness with both dietary and injected equol. Vaginal epithelial thickness was the most sensitive estrogenic endpoint examined. Dietary doses of equol that produced slight uterotropic effects caused clear increases in vaginal epithelial height and stratification, although even at the highest equol doses vaginal stratification was less than seen with E2 treatment.
Adipose tissue expresses ER and is highly estrogen-responsive. Increases in adipose tissue occur in ovariectomized animals and postmenopausal women, and these increases are reversed with estrogen [36, 37]. Inhibitory effects of estrogens on adipose tissue are mediated by ER, as shown by comparable increases in adiposity in ER knockout mice [35] and aromatase-deficient mice [38]. Similarly, the thymus expresses ER, and estrogen treatment of young animals induces thymic atrophy. Genistein administered by injection or in the diet produces inhibitory thymic and adipose effects [24, 29]. The lack of effects on thymic or adipose weights following equol administration by injection or in the diet reflects the lower estrogenic potency of equol relative to genistein. Despite clear vaginal and uterine responses seen even with dietary equol, neither the injected nor dietary equol doses appear to reach a sufficient threshold for an estrogenic response in adipose tissue or thymus.
These findings with equol parallel our previous results with genistein. Although genistein had greater effects on all reproductive and non-reproductive endpoints than equol at similar doses [24, 29], higher genistein exposures were necessary to produce effects on adipose and thymus compared with uterus. For example, genistein produces uterotropic, adipose, and thymic effects at dietary concentrations of 300, 500, and 1000 ppm, respectively [24, 29]. These dietary genistein levels result in serum genistein concentrations of 1.0–2.5 µM. However, serum equol concentrations in this study of up to 8.1 µM in mice fed 1000 ppm equol had no effect on adipose tissue or thymus, had minimal effects on uterine epithelial proliferation, and did not produce the uterine weight increases seen with dietary genistein at 300 ppm or above and serum concentrations of 1 µM or greater.
Human epidemiological studies have correlated increased soy and phytoestrogen intake with decreased risk of certain diseases [39–41]. However, clinical effects of dietary phytoestrogens on parameters such as bone loss or hot flashes have been variable [42]. Equol producers, the subset of humans who convert daidzein to equol when exposed to dietary soy, have highly elevated serum and urinary levels of equol [43]. Steady-state total serum equol concentrations in these individuals are as high as 1 µM in Japanese men [44], and urinary equol levels are 1000-fold higher than endogenous estrogens [41, 43]. Setchell et al. [2] recently suggested that health benefits of isoflavone supplements in postmenopausal women were correlated with equol production, with equol producers having the highest response to isoflavones. In contrast, rodents fed 500–1000 ppm of equol had limited estrogenic responses despite total circulating equol concentrations (up to 8.1 µM) that were several-fold greater than the approximately 1 µM concentrations in humans who are equol producers and consume high-soy diets [2, 17, 18]. Based on this, equol concentrations normally seen in humans would not be expected to have significant estrogenicity on the ER-mediated parameters examined here, which is not consistent with a primary role for equol in the effectiveness of soy [2].
Based on feed intake, mice in the 500 and 1000 ppm dietary equol groups were exposed to approximately 60 or 120 (mg equol) (kg BW)–1 day–1, respectively, which was more on a weight basis than any of the injected equol doses. The comparable serum levels in the dietary vs. injected groups despite the higher overall exposure in the former likely reflects the relatively small portion of equol that is absorbed in the gut, as reported for genistein [41]. Although serum equol concentrations 2 h after injection provide some information about the magnitude of serum equol concentrations resulting from injection, these values also may not represent peak concentrations [25]. The relatively constant exposure to dietary equol throughout the day compared with one bolus injection would also contribute to the comparable serum equol levels obtained with each mode of administration, despite greater total exposure with dietary equol.
The relative proportion of free aglycone was much less with dietary equol compared with injection. Higher aglycone proportions following genistein injection compared with animals given dietary genistein has been reported [25] and may be the result of a significant portion of the aglycone being conjugated during absorption through the enteric epithelium. Because the free aglycone is considered the biologically active moiety, the increased proportion of circulating aglycone following equol injection could contribute to increased activity of injected vs. dietary equol on parameters such as uterine weight and Ki-67 labeling, and indeed our data indicate that the aglycone concentrations present with a given dietary or injected equol dose correlate well with the biological response. This may be critical in explaining the greater estrogenic effects of the injections despite our data indicating that the serum equol levels produced by the injections are comparable with those obtained with dietary administration. These results emphasize the important differences between injecting equol or other phytoestrogens compared with delivering them dietarily and indicate that results obtained from phytoestrogen injection studies must be extrapolated with extreme caution to understand effects resulting from normal dietary exposure.
Equol acts as a weak ER agonist in vivo in the present study, but a complete understanding of its biological effects in humans or animals must take into account the gamut of its actions and the complexity of the in vivo environment where other phytoestrogens, as well as various other environmental estrogens, may be present. For example, equol could have additive effects in vivo with genistein and other phytoestrogens, and therefore would contribute to the overall estrogenicity of circulating phytoestrogens. Likewise, there could also be equol effects through its non–ER-mediated antioxidant [45, 46] and antiproliferative properties [47, 48]; these types of effects might be more apparent with long-term exposure, rather than the relatively short-term exposure utilized in this study. Many phytoestrogens, including equol, bind with a higher affinity to ERß than to ER. ERß-mediated responses were not examined in this study, and the possibility that equol could have important biological effects through these pathways remains untested. Further, the potential role of equol in nongenotropic signaling mediated by a putative membrane ER, which may be responsible for reversal of bone loss [49], remains to be investigated.
|
FOOTNOTES |
|---|
2 Correspondence: William Helferich, Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61802. FAX: 217 244 2455; helferic{at}uiuc.edu. Paul S. Cooke, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, University of Illinois, Urbana, Illinois 61802. FAX: 217 244 1652; p-cooke{at}uiuc.edu
Received: 15 March 2004.
First decision: 10 April 2004.
Accepted: 13 May 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. E. Wood, S. E. Appt, T. B. Clarkson, A. A. Franke, C. J. Lees, D. R. Doerge, and J. M. Cline Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys Biol Reprod, September 1, 2006; 75(3): 477 - 486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. H. Ju, J. Fultz, K. F. Allred, D. R. Doerge, and W. G. Helferich Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice Carcinogenesis, April 1, 2006; 27(4): 856 - 863. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Cooke, V. Selvaraj, and S. Yellayi Genistein, Estrogen Receptors, and the Acquired Immune Response J. Nutr., March 1, 2006; 136(3): 704 - 708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Totta, F. Acconcia, F. Virgili, A. Cassidy, P. D. Weinberg, G. Rimbach, and M. Marino Daidzein-Sulfate Metabolites Affect Transcriptional and Antiproliferative Activities of Estrogen Receptor-{beta} in Cultured Human Cancer Cells J. Nutr., November 1, 2005; 135(11): 2687 - 2693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Atkinson, C. L. Frankenfeld, and J. W. Lampe Gut Bacterial Metabolism of the Soy Isoflavone Daidzein: Exploring the Relevance to Human Health Experimental Biology and Medicine, March 1, 2005; 230(3): 155 - 170. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
