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Equol Is a Novel Anti-Androgen that Inhibits Prostate Growth and Hormone Feedback¹

Department of Biomedical Sciences,³ Colorado State University, Fort Collins, Colorado 80524 Clinical Mass Spectrometry,⁴ Children's Hospital Medical Center, Cincinnati, Ohio 45229 The Neuroscience Center and Department of Physiology and Developmental Biology,⁵ Brigham Young University, Provo, Utah 84602

	ABSTRACT

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Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4–7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) ß, and no affinity for ER. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.

androgen receptor, epididymis, prostate, steroid hormones, testosterone

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In recent years phytoestrogens have received increased investigative attention due to their potential protective effects against age-related diseases (e.g., cardiovascular disease and osteoporosis) and hormone-dependent cancers (i.e., breast and prostate cancer [1–7]. There are three main classifications of phytoestrogens: 1) isoflavones (derived principally from soybeans), 2) lignans (found in flaxseed in large quantities), and coumestans (derived from sprouting plants like alfalfa) [1, 3, 7–9]. Of these three main classifications, human consumption of isoflavones has the largest impact due to its availability and variety in food products containing soy. Of the isoflavones, genistein and daidzein are thought to exert the most potent estrogenic hormone activity, and thus most attention has been directed toward these molecules [1, 3, 7, 9]. However, these isoflavone molecules do not exist at high levels in their biologically active form in soy foods, but rather are at high abundance in a precursor form [3]. For example, genistin, the precursor of genistein, is the glycosidic form that contains a carbohydrate portion of the molecule. Genistin is metabolized in the gastrointestinal tract by intestinal bacteria, which hydrolyze the carbohydrate moiety, to the biologically active phytoestrogen, genistein [8, 10]. The same metabolic step occurs for daidzein (the aglycone), which is converted from daidzin (the glycosidic form). Daidzein is then further metabolized in the intestine to equol [8]. Thereafter, genistein, daidzein, and equol circulate in the blood stream at very high concentrations [11–14].

The phenolic ring structures of isoflavones enable these compounds to bind estrogen receptors (ER) and mimic estrogen (E₂). Although genistein and daidzein bind ER, it is with a lower affinity when compared with estradiol [15–17], and greater affinity for ERß [17] than to ER [15–17]. Additionally, phytoestrogens have been reported to act like natural selective ER modulators (SERMs) at various tissue sites throughout the body [10–14, 17–19]. In some tissues, there is evidence that phytoestrogens act as E₂ agonists, whereas in others, they display antagonistic characteristics comparable with that of tamoxifen or especially raloxifene where SERM activity appears to be sex hormone and gender dependent [10–14, 16–19].

Relative to the commercially available plant-derived isoflavones, a paucity of data exists regarding the biological activity of equol. Equol was originally reported as having little or no estrogenic activity [20–23]. However, more recent investigations examining uterine weight [23, 24] and utilizing competition-binding assays [25] suggest that equol may possess some weak estrogenic properties. Additionally, equol has been shown to bind sex hormone binding globulin [26, 27] and -fetoprotein [28], although the clinical significance of these interactions is uncertain. Finally, equol has been shown to have antioxidant activity [29–31] and inhibits the growth of prostate cells in vitro [32].

Previous observations have shown that prostate weight is significantly decreased in male rats fed a diet high in phytoestrogens in comparison with those fed a diet low in phytoestrogens [11–14]. Close examination of reported plasma phytoestrogen levels in these publications [11–13] suggests that equol represents the predominant circulating phytoestrogen in phytoestrogen-fed animals. In the following studies, we first sought to determine if the decrease in prostate weight observed in consuming a phytoestrogen-rich diet was due to equol. Furthermore, we asked whether the effects of equol on prostate size are due solely to equol's estrogenic properties. In performing these studies, we discovered that equol had only a modest affinity for ERß and little or no affinity for ER; however, the results from our studies indicate that equol can act as an anti-androgen. The anti-androgenic properties of equol are unique in that equol does not bind the androgen receptor (AR) but specifically binds 5-dihydrotestosterone (DHT) with high affinity, and thereby prevents DHT from binding the AR.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals

Male Sprague-Dawley rats (400–500 g) were obtained from Charles Rivers Laboratories (Wilmington, MA). These animals were caged in pairs and housed in the Colorado State University vivarium and maintained on a 12D:12L schedule (lights on at 0700 h) with ad libitum access to food and water. The Animal Care and Use Committee (IACUC) at Colorado State University approved all animal protocols.

In vivo experiment 1 One week following arrival, animals were given daily s.c. injections for 4 days of either dimethylsulfoxide (DMSO; vehicle control) or equol (0.25 mg/kg) in a total volume of 0.3 ml. Eighteen hours after the final injection, animals were killed via decapitation, and trunk blood and ventral prostate were weighed and collected for later analysis. Plasma was removed and stored at -20°C for later analysis by radioimmunoassay (RIA).

In vivo experiment 2 One week following arrival, animals were gonadectomized (GDX) under isoflurane anesthesia and allowed to recover for 7 days. Following recovery, animals were assigned to the following groups: 1) DMSO (vehicle control); 2) DHT propionate (DHTP; 2 mg/kg); 3) equol (0.25 mg/kg); or 4) both DHTP and equol. Injections were given s.c. once daily for 4 days. Following treatments, animals were weighed and killed via decapitation. Trunk blood was collected and plasma saved for later analysis. The ventral prostates were weighed and frozen for later analysis.

In vivo experiment 3 One week following arrival, animals were GDX under isoflurane anesthesia and allowed to recover for 7 days. Following recovery, animals were assigned to the following groups: 1) DMSO (vehicle control); 2) testosterone propionate (TP; 2 mg/kg); 3) equol (0.25 mg/kg); or 4) both TP and equol. Injections were given s.c. once daily for 4 days. Following treatments, animals were weighed and killed via decapitation. Trunk blood was collected and plasma saved for later analysis. Ventral prostates were weighed and frozen for later analysis.

In vivo experiment 4 One week following arrival in our facility, intact males were given s.c. injections of either DMSO (control) or equol (0.5 mg/kg) once per day for 7 days (total volume of injections 0.3 mL). Following treatments, animals were weighed and then killed via decapitation. Trunk blood was collected and plasma saved at -20°C for later analysis. The ventral prostate, testes, epididymis, and pituitary were also collected, weighed, and frozen.

Plasma Hormones

At death, trunk blood was collected into tubes containing 0.5 M EDTA (200 µl). Blood was centrifuged and plasma was removed and stored at -20°C until assayed for LH, testosterone, and DHT by RIA.

Plasma levels of LH were measured using reagents provided by the National Hormone and Peptide Program. Rat LH-RP3 was used as a standard curve. Tracer LH was iodinated using the chloramine-T method by the Colorado State University RIA core. The inter- and intraassay coefficients of variation for this assay are 6.5% and 13%, respectively.

Serum testosterone levels were determined via coated-tube RIA kit (Diagnostic Systems Laboratories, Webster, TX) using a sensitive and specific antibody. Cross-reactivity for this testosterone antibody includes a small number of androgens (DHT = 5.8%, 11-oxotestosterone = 4.2%, androstenedione = 2.3%, and ethisterone = 1.9%) and nondetectable cross-reactivity with E₂, dihydroepiandrosterone (DHEA), corticosterone, or other glucocorticoids.

Serum DHT levels were determined via coated-tube RIA kit (Diagnostic Systems) utilizing a sample oxidation (with a potassium permanganate solution)/extraction (with n-hexane and ethanol) procedure that removes testosterone, coupled with a specific antibody for DHT. Cross-reactivity for this immunoassay includes 1.9% androstenedione, 1.4% estradiol, 0.02% testosterone (after extraction), and nondetectable cross-reactivity with glucocorticoids or progesterone.

Both immunoassays were run with internal control standards (high and low levels), and all control values were within normal range(s). The testosterone levels were expressed in nanograms per milliliter, whereas the DHT levels were expressed in picograms per milliliter. All samples were run in the same assay in duplicate, and the intraassay coefficients of variation were 5% for testosterone and 8% for DHT.

Histology

At death, ventral prostate, testes, and epididymis were removed from the animal, dissected free of fat and connective tissue, weighed, fixed by immersion in 4% paraformaldehyde, and then sectioned at 15 µm on a cryostat. Tissue sections were mounted on charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) prewarmed to 23°C and stained with hematoxylin and eosin (H&E), dehydrated in ascending alcohol, and cleared with xylene. Permount was used to coverslip the stained sections. Sections were examined with a Zeiss MC100 microscope (Carl Zeiss, Thornwood, NY), and micrographs were taken with a Zeiss CCD digital camera (Carl Zeiss).

Binding Studies

Synthesis of hormone receptor proteins Full-length rat ER expression vector (pcDNA-ER; R.H. Price, University of California, San Francisco, CA) and ERß expression vector (pcDNA-ERß; T.A. Brown, Pfizer, Groton, CT) were synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega, Madison, WI) with T7-RNA polymerase during a 90-min reaction at 30°C. Translation reaction mixtures were stored at -80°C until further use.

Saturation isotherms In order to calculate and establish the binding affinity of equol for ER, 100-µl aliquots of reticulocyte lysate supernatant were incubated at optimal time and temperature: 90 min at room temperature (ERß), and 18 h at 4°C (ER) with increasing (0.01–50 nM) concentrations of [³H]E₂. These times were determined empirically and represent optimal binding of the receptor with estrogen. Nonspecific binding was assessed using a 200-fold excess of the ER agonist, diethylstilbestrol, in parallel tubes. Following incubation, bound and unbound [³H]E₂ were separated by passing the incubation reaction through a 1-ml lipophilic Sephadex LH-20 (Sigma-Aldrich Co., St. Louis, MO) column. Columns were constructed by packing a disposable pipette tip (1 ml; Labcraft, Curtin Matheson Scientific, Inc., Houston, TX) with TEGMD (10 mM Tris-Cl, 1.5 mM EDTA, 10% glycerol, 25 mM molybdate, and 1 mM dithiothreitol, pH 7.4)-saturated Sephadex according to previously published protocols [33–35]. For chromatography, the columns were re-equilibrated with TEGMD (100 µl), and the incubation reactions were added individually to each column and allowed to incubate on the column for an additional 30 min. Following this incubation, 600 µl of TEGMD was added to each column, flow-through was collected, 4 ml scintillation fluid was added, and samples were counted (5 min each) in a 2900 TR Packard scintillation counter (Packard Bioscience, Meriden, CT).

Preparations of tissue extracts Prostate tissue (taken from male Sprague-Dawley rats) was minced and homogenized with glass homogenizers (Dounce Co., Vineland, NJ) in ice-cold TEGMD buffer. Homogenates were centrifuged at 100 000 x g in an ultracentrifuge (Beckman Coulter, Inc., Palo Alto, CA) with a fixed angle rotor (Sorvall TI 60, Dupont-Sorvall, Wilmington, DE) for 15 min at 4°C to separate the nuclear and cytosolic fractions.

Sephadex LH-20 column chromatography In order to further profile the binding of equol, we employed slight modifications of the above protocol such that bound and unbound [³H]E₂, [³H]DHT, [³H]testosterone, or [³H]DHEA were separated and collected as 200 µl elution fractions (for a total of 12 ml) from 10-ml serological pipettes (VWR International, West Chester, PA) packed to a height of 30 cm with Sephadex LH-20. Equol was combined with a number of [³H]-labeled compounds (E₂, DHT, testosterone, DHEA, corticosterone, and promegestone) in both the presence and absence of hormone receptor or prostate cytosols.

Competition studies For competition studies, recombinant receptors or prostate cytosols were incubated with 1 nM [³H]E₂ or [³H]DHT (for ER and AR, respectively) in the presence of increasing concentrations of equol, daidzein, or genistein (0.1–10 nM).

All steroids (DHT, testosterone, E₂, etc.) and phytoestrogens (genistein, daidzein, and equol) utilized in these studies were obtained from Sigma-Aldrich. Steroids and phytoestrogens utilized in the binding studies were dissolved in 95% EtOH to a final stock concentration of 600 µM and stored at 4°C until future dilution and use.

Statistics and Analysis

Where appropriate, data were analyzed by ANOVA followed by Newman-Keuls post hoc tests. Significance was set at P < 0.05. Curve fitting, scientific graphing, and analysis were completed using GraphPad Software (GraphPad Prism 3.0, San Diego, CA).

	RESULTS

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In Vivo Experiment 1: Effects of Equol on Ventral Prostate Size and Hormone Secretion

As shown in Figure 1A, a significant reduction in prostate weight was observed in intact males after four daily injections of equol when compared with intact control males (F_1,8 = 7.02, P < 0.05). Additionally, in these same animals, LH was significantly increased in the equol-treated group when compared with controls (F_1,8 = 5.2, P < 0.05; Fig. 1B).

View larger version (9K): [in this window] [in a new window]   FIG. 1. A) Equol reduces wet prostate weight in intact male rats. Animals were s.c. injected (1/day for 4 days) with DMSO (vehicle control) or equol (0.25 mg/kg) total volume 0.3 ml. B) Equol increases LH plasma levels in intact male rats. Animals were s.c. injected (daily for 4 days) with DMSO or equol (0.25 mg/kg). Each bar represents mean (±SEM). *Significantly different (P < 0.05) from control group (n = 5/treatment group)

Does equol bind estrogen and androgen receptors? Competition binding studies were used to begin to assess equol's estrogenic and androgenic properties. Based on the ability of equol to compete with [³H]E₂ for ER binding, equol's affinity for in vitro-translated ER was shown to be very low, with greatest affinity for ERß (K_i [nM] = 6.3 ± 1.2), whereas equol's affinity for ER was below 25 nM. Using prostate cytosol as a source of androgen receptor, equol was unable to compete with [³H]DHT binding.

Does equol directly bind DHT? In our initial binding competition studies to determine and establish equol's binding affinity for AR, we repetitively observed that the apparent binding of [³H]DHT was greater in the presence of equol than in its absence. Modifications in the protocol where prostate cytosol was omitted from the incubation tube (leaving only [³H]DHT and equol) resulted in the elution of [³H]DHT into the column eluate, which would normally contain the [³H]DHT-AR complex. To further investigate this phenomenon, we utilized a 30-cm long Sephadex LH-20 column to characterize the elution profile. As can be clearly seen in Figure 2, a peak of [³H]DHT is apparent in the elution fractions between 5 and 9 ml when the [³H]DHT + equol incubate is applied. This peak is not present when [³H]DHT alone is applied to the column. Furthermore, when DHT or DHT + equol (equol added at the same time as DHT) are incubated with prostate cytosol and then passed through the 30-cm column (Fig. 3A), two distinct binding peaks are identifiable. The first peak of [³H]DHT represents that bound to the AR in prostate. This is found in the elution fractions between 4 and 5 ml. In addition, there is a later peak (between 5 and 9 ml), which again suggests the binding of [³H]DHT to equol. However, it is interesting to note that in a separate study when [³H]DHT was allowed to incubate with the prostate until equilibrium (30 h) and then equol was introduced, there was no apparent [³H]DHT-equol complex formed (Fig. 3B). In this case, the column elution profile shows a single elution peak between 4 and 5 ml, which is identical to the profile of [³H]DHT + prostate cytosol alone (cold DHT did, however, effectively compete with bound [³H]DHT; Fig. 3B). This suggests that equol does not compete with DHT for the AR nor does it bind [³H]DHT, which is already bound to the receptor. However, when the DHT-equol complex, isolated by Sephadex chromatography, was incubated with prostate cytosol prior to passage through the 30-cm column, the [³H]DHT remained bound to equol and did not transfer to AR (data not shown). Furthermore, it is also interesting to note that equol binding is specific to DHT. Saturation analysis of equol binding to [³H]DHT shows an apparent K_d calculated at 1.32 ± 0.4 nM (Fig. 4). Similar competition and binding studies were conducted using [³H]E₂, [³H]testosterone, [³H]DHEA, [³H]corticosterone, [³H]progesterone, and [³H]promegestone without any evidence that equol binds these compounds (data not shown).

View larger version (13K): [in this window] [in a new window]   FIG. 2. Sephadex column chromatography identifies a peak corresponding to [3H]DHT-equol binding. A distinct elution peak is identified when [3H]DHT + equol, but not [3H]DHT alone, is added to the column. Following incubation of [3H]DHT with equol in vitro, the incubate was run through a 30-cm long Sephadex LH-20 column and 200 µl elution fractions were collected. DHT + equol (solid black) shows a peak between 5 and 9 ml; this peak is shifted from the elution profile by [3H]DHT alone (gray)

View larger version (14K): [in this window] [in a new window]   FIG. 3. A) Column chromatography showing the presence of three elution peaks corresponding to receptor-bound [3H]DHT, equol-bound [3H]DHT, and free [3H]DHT. Two distinct peaks are evident in [3H]DHT + equol incubated with prostate cytosols, whereas only a single peak is present in [3H]DHT incubated with prostate cytosols. Following incubation, bound was separated from free by passing cytosol through a 30-cm-long Sephadex LH-20 column. Two hundred microliter elution fractions were collected. When [3H]DHT and [3H]DHT + equol were incubated with cytosol, there was an elution peak between 4 and 5 ml, which reflects presumably the binding of DHT to prostate. Incubation of prostate cytosol with [3H]DHT + equol showed a second peak between 5 and 9 ml, which is presumably the binding of DHT to equol. The peak at 9 ml is presumably free [3H]DHT. B) In a separate study, when [3H]DHT is incubated to equilibrium with prostate cytosol (30 h) prior to the introduction of equol, there is only one elution peak presumably reflecting [3H]DHT bound to AR. In this study, equol was added 30 h following the beginning of incubation of [3H]DHT with prostate (cold DHT added 30 h following incubation did, however, effectively compete with bound [3H]DHT)

View larger version (19K): [in this window] [in a new window]   FIG. 4. Saturation analysis of equol binding to [3H]DHT shows an apparent Kd calculated at 1.32 ± 0.4 nM. Extracts were prepared, and ligand-binding affinity for tritiated DHT was determined as described in Materials and Methods. The inset shows linear transformation of the data by Scatchard analysis for illustrative purposes

In Vivo Experiment 2: Can Equol Affect DHT Action?

The studies described above indicate that equol could compete with DHT in vitro and could potentially sequester DHT from the AR. Consequently, we examined the effects of equol on DHT-treated males. GDX males treated with DHTP for 4 days showed a significant increase in ventral prostate weight compared with GDX controls. Concomitant treatment with equol (DHTP + equol) blocked the effects of DHTP, whereas equol alone had no effect on prostate size (F_3,16 = 7.84, P < 0.05). These results are presented in Figure 5A. Equol also blocked DHT's negative feedback effects on LH. In GDX males, LH was significantly decreased by DHTP treatment compared with males treated with vehicle. Treatment with equol in combination with DHTP blocked the negative feedback effects of DHTP on LH secretion. Equol alone had no effect on LH levels (F_3,16 = 11.67, P < 0.05). These results are presented graphically in Figure 5B.

View larger version (19K): [in this window] [in a new window]   FIG. 5. A) Ventral prostate weight in GDX male rats s.c. injected (1/day for 4 days) with DMSO (vehicle control), DHTP (2 mg/kg), equol (0.25 mg/kg), or both DHTP and equol. B) Equol blocks negative feedback effects of DHT on plasma LH levels in GDX male rats. Animals were injected (1/day for 4 days) with DMSO (vehicle control), DHTP (2 mg/kg), equol (0.25 mg/kg), or both DHTP and equol. Each bar represents mean (±SEM) of five animals. *Significantly different from all other groups

As expected, there were significant elevations of plasma DHT in animals treated with DHT (GDX + DHT, GDX + equol + DHT groups). Plasma DHT was further elevated, although not significantly (P = 0.07), by cotreatment with equol. These results are presented in Figure 6.

View larger version (35K): [in this window] [in a new window]   FIG. 6. Plasma DHT in GDX male rats s.c. injected (1/day for 4 days) with DMSO (vehicle control), DHTP (2 mg/kg), equol (0.25 mg/kg), or both DHTP and equol. Each bar represents mean (±SEM) of five animals. *Plasma DHT was significantly elevated in GDX males treated with DHT (P < 0.05). Plasma DHT was further elevated (arrow = approached significance, P = 0.07) by cotreatment with equol (n = 5/treatment group)

In Vivo Experiment 3: Does Equol Effect Testosterone Action?

Although prostate weight was significantly reduced with concomitant treatment of equol and DHT, this may not have relevance in normal animals, where there is little circulating DHT, but high levels of testosterone. Therefore, we examined the effects of equol on testosterone-treated males. GDX males treated with TP for 4 days showed a significant increase in ventral prostate weight compared with GDX control. Concomitant treatment with equol (TP + equol) blocked the effects of TP (although not completely, as the prostates weight of TP + equol-treated males were increased compared with controls). Equol alone had no effect on prostate size (F_3,16 = 14.27, P < 0.05). These results are presented in Figure 7. Taken together, the findings from in vivo experiments 2 and 3 suggest that equol can inhibit prostatic size by binding circulating and intracellular DHT.

View larger version (35K): [in this window] [in a new window]   FIG. 7. Ventral prostate weight in GDX male rats s.c. injected (1/day for 4 days) with DMSO (vehicle control), TP (2 mg/kg), equol (0.25 mg/kg), or both TP and equol. Each bar represents mean (±SEM) of five animals. *Significantly different from all other groups. **Significantly different from control (DMSO) group

In Vivo Experiment 4: Does Equol Act on Other Tissues?

In addition to equol's effects on prostate (Figures 1A and 5A), equol was found to block the effects of DHT on other tissues. The results from in vivo experiment 4 confirmed our previous findings in that intact males treated with equol showed a significant decrease in ventral prostate weight compared with control animals (F_1,8 = 6.9, P < 0.05). Furthermore, a significant decrease in epididymal weight was observed in equol-treated males compared with controls (F_1,8 = 7.9, P < 0.05). However, this effect of equol was not found in testes weight (F_1,8 = 0.09, P > 0.05) or pituitary weight (F_1,8 = 0.51, P > 0.05). Body weight was also not different between treatments (F_1,8 = 1.4, P > 0.05). These results are presented in Table 1.

Tissue Histology

H&E-stained prostates reflect a change due to both GDX and treatments. The prostate glands of gonadectomized control, equol, and DHT plus equol-treated groups show similar histology (Fig. 8, A, B, and D). In these animals, prostates are characterized by very small atrophic glands with little volume in the gland lumen. In DHT-treated animals (Fig. 8C), the glands show signs of cell proliferation. Lumen size is increased compared with GDX animals; the epithelium is of a tall columnar type (Fig. 8C). In comparison to intact control animals (Fig. 8E), the prostate of equol-treated males show involution and consist of more closely spaced, atrophic glands (Fig. 8F).

View larger version (161K): [in this window] [in a new window]   FIG. 8. Histological effects of equol in the ventral prostate gland of gonadectomized (A–D) and intact (E and F) animals. Male Sprague-Dawley rats (400–500 g) were treated s.c. (1/day for 4 days) with either DMSO (vehicle control), DHTP (2 mg/kg), equol (0.25 mg/kg), or both DHTP and equol. Control (A) and equol (B) prostates show very small atrophic glands; (C) DHT, the glands show proliferation; (D) DHT plus equol, the glands show a bit of proliferation in comparison to DHT, but not control or equol-treated animals. In comparison with intact control (E), the prostate of equol-treated animals (F) show involution and consist of more closely spaced, atrophic glands. Original magnification x50

In comparison with control males, the epididymis of equol-treated intact males show overall smaller ducts depicted by shrunken lumen (Fig. 9).

View larger version (74K): [in this window] [in a new window]   FIG. 9. Histological effects of equol on the epididymis of intact male Sprague-Dawley rats (400–500 g). Animals were treated s.c. (1/day for 7 days) with DMSO (vehicle control) or equol (0.50 mg/kg). In comparison to control (A), the epididymis of equol-treated animals (B) show overall smaller glands depicted by shrunken lumen with proliferation of the epithelium. Original magnification x50

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, it was suggested that equol is an important isoflavone in humans [36]. Specifically, the protective effects of soy protein in cardiovascular, bone, and menopausal health appear to be a function of the biotransformation of soy isoflavones to equol, a compound that displays more potent physiologic actions compared to its precursor molecules [36]. As shown by our studies, one aspect of equol's mechanism of action is apparently its ability to specifically bind DHT and prevent DHT's biological actions in physiological processes.

Our studies demonstrate that equol, a known metabolite of daidzein, effects prostate and epididymis weights and LH levels in male rats. Equol acts by preventing the hormonal action of 5-DHT in vivo. Equol's anti-androgenic action is due to its unique ability to specifically bind DHT without binding the androgen receptor. As a result, equol can sequester DHT from binding the androgen receptor. It is known that soy-derived phytoestrogens (isoflavones) have positive benefits in protecting against hormone-dependent (breast and prostate cancer) and age-related diseases (such as cardiovascular disease and osteoporosis) [1–6]. In this regard, isoflavones (e.g., daidzein and genistein) are thought to modulate estrogen action via the ER [14–16], and our data show that this would involve ERß binding, rather than ER. Although prior reports suggest that equol has the ability to bind the estrogen receptor in tissue homogenates [25], this is in contrast to the data we report herein. Using binding studies on preparations of recombinant steroid receptors or on prostatic cytosols, we have shown that equol does not bind ER or the AR. However, it did display a modest affinity for ERß at approximately 200-fold less than that of 17ß-estradiol (equol, K_i = 6.3 nM vs. 17ß-estradiol, K_i = 0.03 nM). These results are in contrast to previous reports showing trophic effects of equol in uterine tissue, a tissue that predominately expresses ER [24]. Based on these results, we suggest that the uterotrophic actions of equol are either due to the high doses used in previous studies or another property of equol that is presently unknown.

Growth of some male reproductive organs such as the prostate and epididymis are known to be under androgenic control [37, 38]. More specifically, the epididymis and prostate gland depend on androgen receptor stimulation for development and growth. However, testosterone is not the major androgen responsible for growth of the prostate and epididymis; rather, it is the intracellular conversion of testosterone to DHT by the enzyme 5-reductase that is primarily responsible for both prostate and epididymal development. This is not the case, however, with either testes or pituitary. Neither the testis nor pituitary requires the conversion of testosterone to DHT via 5-reductase. Our previous studies showed that rats fed a diet containing high levels of soy-derived isoflavones showed no change in prostate weights when measured before puberty when circulating androgen levels are very low [11]. However, after puberty when androgen levels increase, prostate weights were significantly decreased in rats fed a phytoestrogen-rich diet compared with animals fed a phytoestrogen-free diet [11]. In animals fed a phytoestrogen-rich diet, 70%–90% of the total circulating isoflavone is accounted for by equol [11–13]. Such results are consistent with our present findings that equol-treated intact male rats displayed significant decreases in prostate and epididymal weights without alterations in testes, pituitary, or body weights. Notably, if the prostate and epididymal values are standardized to body weight, the ratios are still significantly different between equol-treated vs. control values. Since DHT is produced from testosterone within prostate cells, this also suggests that equol is capable of binding intracellular DHT to prevent it from acting.

Consistent with these data, equol also blocked circulating DHT's androgenic trophic influence on the prostate and epididymis without significantly altering circulating DHT levels. Furthermore, histological analysis of equol's influence on prostate and epididymis suggest that equol acts as an anti-androgen in these tissues. In both the ventral prostate and epididymis, equol-treated animals showed tissue characteristics typical of animals with decreased androgen levels.

Given the high consumption of phytoestrogens in Asian populations, our findings correlate with the low incidence rates of benign prostate hyperplasia/prostate cancer in Asian men [1–4] compared with Western populations and suggest a mechanism for such epidemiology. It is interesting, in light of our observations, to note that the beneficial effects of soy in relation to prostate health may be due to equol's unique anti-androgenic properties, rather than its estrogenic properties. In fact, recent clinical research has identified that equol nonproducers are at higher risk for prostate cancer than are equol producers [39]. We suspect that this may be due to equol's anti-androgenic action. Thus, our data provide a potential avenue for the therapeutic use of equol in the treatment of androgen-mediated pathologies.

Our in vivo experiments also support the possibility that equol somewhat blocks the negative feedback effects of DHT on pituitary LH regulation in intact or in GDX-treated male rats [40, 41]. For instance, in intact animals, equol significantly increased LH levels vs. control values, indicating a slight blockade of negative feedback. Similarly, when GDX males were treated with equol, it completely reversed the inhibitory action of DHT on plasma LH levels, where DHT plus equol-treated male rats displayed LH levels similar to that of control values. Taken together, these data suggest that equol also has the ability to block the negative feedback action of this potent androgenic molecule. In these studies, equol's inhibitory effects occurred without changing plasma DHT levels. In fact, the opposite was found. Males injected with DHT in combination with equol had plasma DHT levels that were elevated above those animals treated with DHT alone. This observation further elucidates equol's ability to bind DHT and sequester DHT from AR, resulting in increased plasma DHT levels and making it unlikely that equol acts by decreasing androgen synthesis.

In order to determine whether equol specifically binds DHT, in vitro binding studies were performed using purified DHT and/or equol (with or without rat prostate tissue homogenates). Using lipophilic Sephadex column chromatography purified equol was shown to shift the [³H]DHT elution profile, but not the profile of other steroid molecules. Such data suggest that equol's binding is specific for DHT; however, the biochemical mechanism by which DHT and equol interact is still under study.

Analysis using rat prostate cytosols as a source of androgen receptors showed that when equol and DHT were concomitantly added to the cytosol, less [³H]DHT was bound to the prostate androgen receptor. However, if [³H]DHT was added to the prostate homogenate and binding allowed to equilibrate for 30 h prior to adding equol to the mixture, then equol was unable to displace [³H]DHT from the AR in the prostate tissue homogenate. This suggests the possibility that equol cannot compete with the DHT for binding to AR and cannot compete with AR for binding to DHT.

The possibility exists that equol can have anti-androgenic actions on a number of tissues throughout the body. In particular, blockade of androgen action could be beneficial not only for preventing growth of reproductive tissues with DHT dependency such as prostate and epididymis, other androgen-responsive tissues may also benefit from equol therapy. Such conditions include female- and male-pattern baldness [42–44]; facial and body hair growth [42–44]; skin health (acne, anti-aging, and anti-photo aging) [42, 45]; skin integrity (collagen and elastin robustness) [42, 45]; and emotional and mental health issues, such as, mood, depression, anxiety, learning, and memory [46, 47].

Taken together, the above results provide evidence for a novel mechanism of action of equol. It appears that equol is able to specifically bind DHT and that this interaction is in part responsible for the observed reduction in prostate and epididymal weights and alterations in LH levels following equol treatment. To our knowledge, this type of interaction is unique among all other phytoestrogens and xenoestrogens. These data may suggest a shift in our thinking of how environmental products such as equol can alter hormone-dependent physiological functions.

	ACKNOWLEDGMENTS

 
The NIDDK National Hormone and Peptide Program generously supplied some of the reagents used. The authors would also like to thank LiHong Bu for her technical assistance with the DHT RIA.

	FOOTNOTES

 
¹ Supported in part by NIH grants AA12693 and NS39951 (R.J.H.) and USDA grant 2002-00798 (E.D.L.).

² Correspondence: Trent D. Lund, Department of Biomedical Science, Colorado State University, Anatomy W103, 1617 Campus Delivery, Ft. Collins, CO 80523-1670. FAX: 970 491 7907; tlund{at}colostate.edu

Received: 27 September 2003.

First decision: 19 October 2003.

Accepted: 10 December 2003.

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