Life Sciences 128 (2015) 1–7

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The influence of equol on the hypothalamic–pituitary–thyroid axis and hepatic lipid metabolic parameters in adult male rats Panida Loutchanwoot a,⁎, Prayook Srivilai a, Hubertus Jarry b a b

Department of Biology Faculty of Science, Mahasarakham University, Khamriang Sub-district, Kantharawichai District, Mahasarakham Province 44150 Thailand Department of Endocrinology, University Medical Center Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany

a r t i c l e

i n f o

Article history: Received 13 October 2014 Received in revised form 15 November 2014 Accepted 2 February 2015 Available online 2 March 2015 Keywords: Equol Thyrotropin-releasing hormone Thyrotropin secretion Thyroid hormones Serum lipids Prostate

a b s t r a c t Aims: Equol, the principal active metabolite of soy-derived phytoestrogen daidzein, has well-known estrogenic actions. Results of several studies indicate that equol may also have anti-androgenic activities. However, mechanisms of action of equol on hypothalamic–pituitary–thyroid axis (HPTA) and hepatic lipid metabolism in adult male rats have not been determined yet. Main methods: Equol at two doses of 100 and 250 mg/kg body weight (BW)/day was orally gavaged for 5 days to groups of 4-month-old male rats. As a positive anti-androgenic control group, animals received 100 mg of pure anti-androgenic drug flutamide/kg BW/day. Circulating concentrations of thyroid hormones and lipids, and expression levels of genes underlying HPTA function were determined by radioimmunoassay and TaqMan® real-time reverse transcription polymerase chain reaction, respectively. Key findings: Flutamide significantly decreased relative prostate weight, whereas equol did not. Both equol and flutamide caused a significant increase in relative liver weights, and decreases in plasma levels of total tetraiodothyronine (T4) and triiodothyronine (T3), whereas free T4 and T3 concentrations were not reduced. Equol caused the marked down-regulation of hypothalamic thyrotropin-releasing hormone mRNA expression, whereas flutamide did not. Equol as well as flutamide significantly down-regulated the expression levels of pituitary thyrotropin beta-subunit mRNA, without altering thyrotropin secretion. Equol caused reductions in plasma levels of total cholesterol, high- and low-density lipoproteins and triglycerides, whereas flutamide exerted opposite effects. Significance: This is the first study to reveal that in male rats equol did not affect HPTA function and liver lipid metabolism through the anti-androgenic pathway, however, the intrinsic estrogenic actions of equol were observed. © 2015 Elsevier Inc. All rights reserved.

Introduction Equol is the major endocrine active metabolite of the biotransformation of soy-derived isoflavone daidzein by gut microflora [4,6]. Equol has the molecular structure similar to 17β-estradiol [12,36] that potently binds to both estrogen receptor subtypes beta (ERβ) and alpha (ERα), and exerts more estrogenic actions than the parent compound [21,25, 34]. In addition to its well-characterized estrogenic actions, there is increasing evidence that equol has potential as an androgen receptor (AR) antagonist because in vitro it has been demonstrated to bind with high affinity to AR [5], and inhibit proliferation of benign and malignant human prostatic cells and cell lines [10], prostate growth and in vivo feedback effects of 5α-dihydrotestosterone [21,22], which till date has attracted intensive interests from many physicians and offers insights into prevention of androgen-dependent prostate disorders [10,21,22]. ⁎ Corresponding author. Tel./fax: +66 43 754245. E-mail address: [email protected], [email protected] (P. Loutchanwoot).

http://dx.doi.org/10.1016/j.lfs.2015.02.002 0024-3205/© 2015 Elsevier Inc. All rights reserved.

In addition to steroid biosynthesis inhibitory effects, it has been recognized that several endocrine disruptors, including soy isoflavones genistein and daidzein, cause some profound effects on thyroid disruption [7,9,28], as well as having a positive effect on lowering serum lipid profiles [4,18,39]. As our previously published results have shown, administering equol orally to male rats induced clear-cut estrogenic effects on hypothalamic–pituitary–gonadal axis (HPGA) [19,20]. However, as HPGA is impacted by the thyroid system [9] and TSH or thyroid hormones may modulate hepatic lipid parameters [8,37,41], their interactions should be considered in detail. The main aim of this research was therefore to investigate mechanisms of action of equol on hypothalamic–pituitary–thyroid axis (HPTA) function and hepatic lipid metabolism. The measures employed were mRNA expression levels of thyrotropin-releasing hormone (TRH) and beta-subunit of thyroidstimulating hormone (TSHβ) genes, plasma levels of TSH, and total and free tetraiodothyronine (T4) and triiodothyronine (T3). In addition to the investigation of parameters of HPTA, wet weights of peripheral steroid-regulated organs prostate as the reference androgen-sensitive organ and liver as the primary site of lipid and xenobiotic metabolisms,

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as well as circulating concentrations of total cholesterol (TC), high- and low-density lipoprotein cholesterols (HDL-C and LDL-C) and triglycerides (TG) were also included. It is noteworthy that no animal studies that reported the anti-androgenic properties of equol have included a positive anti-androgenic control group. In contrast, this work used the pure AR antagonist flutamide that exerts neither AR agonistic [17,33] nor ER agonistic/antagonistic action [27,28] as reference substance. Furthermore, a diet that did not include any phytoestrogens but did contain excess iodine was used, and this would ensure that any data obtained from the present work would only be due to equol. Materials and methods Test compounds The chemicals were purchased from the following suppliers: equol [3,4-dihydro-3-(4-hydroxyphenyl)-2H-1-benzopyran-7-ol, CAS number 531-95-3, purity 98%], Changzhou Dahua Import and Export (Group) Corp. Ltd. (Changzhou, Jiangsu, China), flutamide [2-methylN-(4-nitro-3-(trifluoromethyl) phenyl) propanamide, CAS number 13,311-84-7, purity ≥ 99%], Sigma-Aldrich Chemie GmBH (Steinheim, Germany), and olive oil (Caesar&Loretz GmBH, Hilden, Germany). Animals and treatments Animals and treatments were performed according to the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (No. 123 , Council of Europe) and the Guide and Ethical Principles for the care and use of animals in scientific research (National Research Council of Thailand). 8-weekold male Sprague–Dawley rats were purchased (Winkelmann GmBH, Borchen, Germany), housed in Makrolon® type IV cage (six animals per cage), and given ad libitum access to filtered tap water and special soy-free rodent diet that contained excess iodine (Ssniff Spezialdiäten, Soest, Germany). They were exposed to specific environmental conditions (illumination from 06:00 a.m. until 06:00 p.m., relative humidity of 50–55%, temperature 23–25 °C, air changes per hour 16 h− 1) for the whole study time. The rats were kept in the laboratory for 2 months before the experiments were started so that they would be acclimatized. Experimental design Approval for the study design was obtained from the local Ethics Committee for Animal Care and Use at Mahasarakham University (Number 0019/2554) and University Medical Center Göttingen, in accordance with German animal welfare regulations with a permit issued in Braunschweig, Germany (Number 509.42502/01-36.03). The body weight (BW) of 48 male rats was recorded and they were randomly separated into four experimental groups of vehicle control, equol low dose, equol high dose, and flutamide with 12 individuals in each. Before treatments, the means of group BW were statistically the same (379.3 g). Rats were orally gavaged for 5 consecutive days with olive oil (1 ml/rat/day) as the vehicle control group, equol low dose (100 mg/kg BW/day), equol high dose (250 mg/kg BW/day), and flutamide (100 mg/kg BW/day) as the anti-androgenic reference positive control group. The low dose equol was determined due to it being the level at which estrogenic effects were observed in ovariectomized rats, but that did not result in negative toxicity [31,32], and was also equivalent to the effective dose of flutamide. The high dose equol was chosen to investigate if the possible androgen antagonistic effects could be detected with a pharmacologically relevant dose. The flutamide dose was selected from studies that have been published previously as the level that in adult male rats exerts potent AR antagonistic effects [1,16,28]. Equol and flutamide were dissolved in olive oil and

applied for 1 ml per rat per day. Animals were weighed daily to adjust the oral gavage dosing.

Necropsy, serum and target organ collection, and organ weights Necropsy was conducted across four groups of rats to eliminate the effect of time variations. Rats were decapitated under CO2 euthanasia between 9.00–12.00 a.m. on the last day of application. Trunk blood was collected and stored at 4 °C for 3 h. Serum was then separated by centrifugation (3000 rpm, 20 min, room temperature) and stored at −20 °C for further hormonal analysis. The brain and anterior pituitary were dissected from the skull, and dry ice was used to freeze them immediately, before they were stored at − 80 °C until further gene expression analysis. The liver and ventral prostate were collected, had fat removed and their weight was recorded.

Analysis of thyroid hormones in the serum Radioimmunoassay (RIA) kits from Diagnostic Systems Laboratories, Inc. (DSL) (Sinsheim, Germany) were used to determine the circulating concentrations of total T4 and T3. Free T4 and T3 levels were analyzed with RIAZENcoFT4 and RIAZENcoFT3 kits, respectively (ZenTech, Angleur, Belgium). When using the kits, the manufacturer's instructions were followed. The method of Baur et al. [3] was used to perform the TSH radioimmunoassay using reagents provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Hormone Distribution Program. 125I was used to label the TSH tracer [11].

Analysis of lipid metabolic parameters in the serum Plasma levels of lipid metabolic parameters, i.e., TC, HDL-C, LDL-C and TG were determined by a Roche/Hitachi 902 automatic clinical analyzer (Boehringer, Mannheim, Germany) using commercially available kits (Roche, Mannheim, Germany). The instructions of the manufacturer were followed when using the kits.

Analysis of circulating concentrations of equol and flutamide Using the previously described methods of Loutchanwoot et al. [19, 20], the concentrations of equol and flutamide were measured after extraction and hydrolysis from the serum by high-performance liquid chromatography and ultraviolet detection. An NC 250 × 4.6 mm, Hypersil-ODS 5.0 μm column (Bischoff, Leonberg, Germany) and a precolumn 7.5 × 4.6 mm, SS Jour-Guard RP/C18 5.0 μm (Jasco GmbH, Gross-Umstadt, Germany) were used to analyze the serum samples. H2O with 0.085% H3PO4 added and acetonitrile were eluents A and B, respectively. The following gradient was applied at a flow rate of 1 ml/min: 0 min: 75% A, 25% B; 5 min: 75% A, 25% B; 10 min: 20% A, 80% B; 25 min: 100% B; 30 min: 100% B; and 32 min: 75% A, 25% B. A spectrophotometer (LC-95, Perkin-Elmer LAS GmbH, RodgauJügesheim, Germany) was used to determine the signals at wavelengths of 260 nm and 286 nm for equol and flutamide, respectively.

Medial basal hypothalamus/median eminence (MBH/ME) microdissection Frontal sections were sliced from the frozen brains (600 μm in depth) on a Frigomobile Reichert-Jung and freezing microtome model 1206 (Leica Microsystems Nussloch GmBH, Wetzlar, Germany) at − 10 °C. As a reference, the brain atlas of Pellegrino et al. [30] was consulted. Following the method of Arias et al. [2], a scalpel was used to make 2 mm deep cuts to dissect the MBH/ME between the mammillary bodies, hypothalamic sulci, and optic chiasm.

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Table 1 Sequences of the gene-specific TaqMan probes and primers used in the TaqMan® real-time PCR assay. (FAM: 6-carboxy-fluorescein; TAMRA: 6-carboxyl-tetramethyl-rhodamine). Gene

Probe (5′ FAM–3′ TAMRA)

Forward primer Reverse primer

PCR product size (bp)

TRH

5′-AGCACCCTGGCAGGCGATCCTTC-3′

103

M36317.1

TSHβ

5′-TCAACACACCATCTGCGCTGGG-3′

5′-TTCTGGATTCCTGGTTCTCAGATG-3′ 5′-GGATGTTGCCTCTTGGTGACA-3′ 5′-GATGTACGTGGACAGGAGAGAGTGT-3′ 5′-GACATCCTGAGAGAGTGCGTACTTG-3′

130

BC058488.1

RNA extraction from homogenized tissue The anterior pituitary was minced in 350 μl of lysis buffer from the RNeasy Mini Kit (Qiagen, Hilden, Germany) using a sharpened hypodermic needle, while the microdissected MBH/ME was mixed with 500 μl of lysis buffer using the same process. Further homogenization of samples was performed via 10 s of ultrasonication on ice (Sonifier® Cell disruptor B-12, Branson Sonic Power Company, Danburg, CT, USA). After the extraction of the total RNA using the RNeasy Mini Kit as directed by the manufacturer, the RNase-free DNase I Set (Qiagen, Hilden, Germany) was used to remove the DNA. Absorption measurements (Eppendorf BioPhotometer, Hamburg, Germany) were used to identify the total RNA concentrations in the MBH/ME and pituitary samples at wavelengths of 260 nm and 280 nm. Then a final concentration of 20 ng/μl was obtained via the addition of RNase-free water. Reverse transcription reaction A thermal cycler model T3 (Biometra GmBH, Germany) was used to undertake reverse transcription. Each reaction was performed in a solution (final volume of 20 μl). This solution was composed of 1× Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) reaction buffer (Promega, Madison, WI, USA); random hexamers, 100 ng, and dNTPs, 0.5 mM (Invitrogen™, Karlsruhe, Germany); M-MLV RT RNase H minus, 200 U, and recombinant RNasin® ribonuclease inhibitor, 4 U (Promega, Madison, WI, USA); and total RNA from tissue samples, 200 ng. Primer annealing occurred first for 10 min at 22 °C, then reverse transcription was performed for 50 min at 42 °C, and the final step was for the enzyme and RNA–cDNA hybrids to be denatured for 10 min at 95 °C.

GenBank accession number

these were then obtained from Eurogentec (Seraing, Belgium). Table 1 includes a summary of all the genes, including their oligonucleotide sequences, accession numbers, and sizes of PCR products. A total volume of 25 μl was used to perform the amplification reaction. The total volume was made up of 1 × TaqMan™ universal PCR master mix (Eurogentec, Seraing, Belgium); forward and reverse gene-specific primers, 50–900 nM each; TaqMan probe, 200–225 nM; and cDNA samples, 4 μl. The TaqMan PCR reaction was performed under the following conditions: DNA glycosylase treatment to remove possible carryover PCR products, 2 min at 50 °C (stage 1), and Ampli Taq Gold® DNA polymerase activation for 10 min at 95 °C (stage 2). After this, there were 40 cycles for sample amplification. Each of which was composed of denaturation for 15 s at 95 °C and then annealing/extension as an integrated step for 1 min at 60 °C (stage 3). In addition, each TaqMan PCR cycle run had eight duplicate cDNA samples of known concentrations so that a standard curve could be constructed; no-template controls were also included to determine contamination of the reagents used or the presence of primer dimers. Statistical analysis The data shown in this study are arithmetic means and the standard error of means (SEM). For the data of relative mRNA expression levels of the genes, the vehicle-control group's mean mRNA transcript count was fixed as 100%, and then this was used to set the values from all treatment groups as determined in the respective real-time PCR assay. One-way analysis of variance (ANOVA) with multiple comparisons undertaken using Dunnett's post hoc test (GraphPad Prism version 5, San Diego, CA, USA) was used to compare the differences between means form the vehicle control and treatment groups. Statistical significance was represented by a P value that was b0.05.

TaqMan® real-time polymerase chain reaction (PCR) assay

Results

Using a previously described method [19,20], TaqMan® real-time PCR assay was undertaken by the ABI Prism® 7700 sequence detector (TaqMan, PE Applied Biosystems, Foster City, CA, USA) using the qPCR core kit from Eurogentec (Seraing, Belgium). Primer Express software (PE Applied Biosystems, Weiterstadt, Germany) was used to design the TaqMan primer pairs and probes that were gene-specific, and

Food intake, body weight, and circulating levels of test compounds During the study period, there were no deaths from any treatment or control groups. There were slight, but still significant, reductions in the mean body weights at the end of treatment in the equol high dose and flutamide-treated groups (Fig. 1). Likewise, the average food intake

Fig. 1. Mean final body weight and average food consumption of adult male rats treated orally by gavage for 5 days with either equol low and high doses, or flutamide. Each bar represents mean ± SEM (n = 12/group). *P b 0.05; **P b 0.01 versus vehicle control group.

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Table 2 Effects of a 5-day oral administration of either equol low and high doses, or flutamide to adult male rats on the absolute and relative weights of ventral prostate and liver. Prostate weight (g) Treatment

Absolute weight (g)

Vehicle control Equol low dose Equol high dose Flutamide

0.79 ± 0.04 100.00 0.72 ± 0.02 91.14 0.67 ± 0.03 84.81 0.56 ± 0.03** 70.89

Prostate weight/BW ratio

Liver weight (g)

Absolute weight Absolute Relative weight Relative weight (% of vehicle weight (g/g BW × 100 g) (% of vehicle control) control) (g) 0.202 ± 0.012 0.191 ± 0.004 0.182 ± 0.009 0.147 ± 0.007**

100.00 94.55 90.10 72.77

11.44 ± 0.16 11.84 ± 0.30 12.78 ± 0.23* 14.16 ± 0.35*

Liver weight/BW ratio

Absolute weight Relative weight Relative weight (% of vehicle control) (g/g BW × 100 g) (% of vehicle-control) 100.00 103.50 111.71 123.78

2.93 ± 0.03 3.16 ± 0.07* 3.46 ± 0.05** 3.73 ± 0.07**

100.00 107.85 118.09 127.30

Note: Values are means ± SEM (n = 12 rats in each group). Asterisk (*) shows significant statistical differences when compared to the vehicle control as calculated with one-way ANOVA followed by Dunnett's post hoc test (*P b 0.05; **P b 0.01).

of rats treated with equol high dose or flutamide was significantly decreased (Fig. 1). For the rats given the low and high doses of equol treatment, the respective mean serum levels of equol were 11.2 ± 1.8 and 27.0 ± 3.0 μmol/l. For the rats given flutamide, the mean circulating concentration was 148.4 ± 8.4 μmol/l. In the vehicle control group there were no detectable levels of equol and flutamide. Relative weights of the prostate and liver Table 2 summarizes the effects of equol and flutamide on wet weights at autopsy of the male rat ventral prostate and liver. In this study, the prostate weights were not affected by the two equol doses, but flutamide markedly reduced both the absolute and relative prostate weights, i.e., 71% and 75% of vehicle control, respectively. Treatments with equol low and high doses as well as flutamide caused significant increases in the absolute and relative liver weights, with the highest increase in the flutamide-treated group (124% and 127% of vehicle control, respectively). Plasma levels of TSH, and total and free T4 and T3 Mean TSH concentrations in the serum were unaffected by subacute oral application with either equol, or flutamide at the doses tested in this study (Table 3). Both equol at low and high doses caused significant reductions in circulating concentrations of total T4 and T3, the same as flutamide did, whereas free T4 and T3 levels were not significantly affected (Table 3). When taking total T3 and T4 as well as free T3 and T4 to the ratios, the total T3/total T4 levels were significantly increased only after equol supplementation both at low and high doses (Table 3), whereas neither equol nor flutamide treatment had any significant effect on the free T3/free T4 ratio (Table 3). Circulating concentrations of TC, LDL-C, HDL-C, and TG In a dose-dependent manner, equol significantly lowered the mean circulating concentrations of LDL-C, HDL-C, and TC, and the ratios of TC/HDL-C and LDL-C/HDL-C, whereas flutamide statistically exerted opposite effects (Table 4). In addition, mean TG concentrations were reduced significantly only by the equol high dose treatment (Table 4).

mRNA expression levels of hypothalamic TRH and pituitary TSH β-subunit genes In the MBH/ME, gene expression analysis revealed that the TRH mRNA expression levels were considerably reduced by equol high dose, whereas flutamide did not (Fig. 2). In the pituitary, the expression of TSH β-subunit mRNA was down-regulated by equol and flutamide (Fig. 3).

Discussion It has been shown recently that in addition to being an estrogenic compound, equol could be a putative anti-androgen that has the possibly of being used to reduce the risk of androgen-dependent prostate cancer [10,21,22]. Nevertheless, the possible estrogenic or antiandrogenic actions of equol on HPTA axis function and hepatic lipid metabolism have not yet been elucidated. This work aimed to determine whether equol supplementation can be a new alternative without any potential risks associated with thyroid hormones homeostasis and lipid metabolism in male individuals. As the model of study, the intact adult male rat was employed. Decreased mean food intakes and final BW, and increased relative liver weights of the animals were observed upon both equol and flutamide treatments. While flutamide caused a marked decrease of prostate weights, but equol did not. Similar to a previous study, dietary equol administration attenuated weight gain in adult ovariectomized rats as estradiol did [31]. Consistent with previous studies utilizing the same dose of flutamide, the decrease in mean BW of flutamide-treated rats occurred at the same time as the average food consumption also decreased, and the reduction of prostate size was due to the approval of the anticipated peripheral AR antagonistic action of flutamide in this androgen-sensitive reference organ [1,27,28]. These results were, however, not severe, since for the treatment and control groups no deaths were observed. Serum equol concentrations recorded in rats, from the 100 mg equol/kg BW/day treatment, were similar, in the same range, as those that are typical for Asian men who have a traditional diet that is rich in soy [10,24]. Mean concentration of equol in the serum was recorded as at a pharmacological level in rats given the 250 mg equol/ kg BW/day treatment. The levels of serum flutamide from this work were similar to those previously published and in the range that has

Table 3 Mean serum concentrations of TSH and total and free T4 and T3 of adult male rats treated orally via gavage for 5 days with either equol low and high doses, or flutamide. Treatment

TSH (ng/ml)

Total T4 (ng/ml)

Total T3 (ng/ml)

Total T3/T4 ratio

Free T4 (ng/ml)

Free T3 (ng/ml)

Free T3/T4 ratio

Vehicle control Equol low dose Equol high dose Flutamide

5.75 ± 0.88 5.97 ± 0.57 5.42 ± 0.75 4.26 ± 0.88

47.95 ± 2.02 35.64 ± 1.28** 28.53 ± 1.71** 29.78 ± 1.30**

2.28 ± 0.07 1.92 ± 0.05** 1.72 ± 0.10** 1.47 ± 0.08**

0.0480 ± 0.0012 0.0544 ± 0.0018** 0.0621 ± 0.0043** 0.0500 ± 0.0025

0.0303 ± 0.0074 0.0281 ± 0.0012 0.0315 ± 0.0088 0.0356 ± 0.0112

0.0072 ± 0.0016 0.0067 ± 0.0003 0.0084 ± 0.0004 0.0052 ± 0.0008

0.2780 ± 0.0510 0.3194 ± 0.0131 0.3284 ± 0.0537 0.1949 ± 0.0568

Note: Values are means ± SEM (n = 12 rats in each group). Asterisk shows significant statistical differences when compared to the vehicle control as calculated with one-way ANOVA followed by Dunnett's post hoc test (**P b 0.01).

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Table 4 Mean serum levels of TC, HDL-C and LDL-C, and TG of adult male rats orally exposed to either equol low and high doses, or flutamide for 5 days. Treatment

TG (mg/dl)

TC (mg/dl)

HDL-C (mg/dl)

LDL-C (mg/dl)

TC/HDL-C ratio

LDL-C/HDL-C ratio

Vehicle control Equol low dose Equol high dose Flutamide

227.5 ± 26.47 210.1 ± 33.46 142.8 ± 16.47* 170.7 ± 16.91

116.80 ± 3.25 39.08 ± 2.99** 12.82 ± 1.93** 176.00 ± 3.36**

74.17 ± 2.38 30.49 ± 3.65** 9.92 ± 0.98** 113.70 ± 2.34**

24.83 ± 2.38 5.00 ± 1.05** 0.60 ± 0.43** 43.92 ± 2.46**

1.58 ± 0.03 1.33 ± 0.17 1.18 ± 0.22* 1.55 ± 0.04

0.33 ± 0.02 0.18 ± 0.03** 0.06 ± 0.05** 0.39 ± 0.02

Note: Values are means ± SEM (n = 12 rats in each group). Asterisk (*) shows significant statistical differences when compared to the vehicle control as calculated with one-way ANOVA followed by Dunnett's post hoc test (*P b 0.05; **P b 0.01).

been identified to exert both in vitro and in vivo potent anti-androgenic actions [14,26]. At the applied doses, flutamide and equol both reduced circulating concentrations of total T4 and T3 but did not cause significant differences in TSH levels, whereas all the groups experienced significant down-regulation in TSH β-subunit mRNA expression in the pituitary. However, equol caused the marked down-regulation of the hypothalamic TRH mRNA expression, whereas flutamide did not. As has been demonstrated [15,35] in male rats, the mRNA expression levels and biosynthesis of hypothalamic TRH and pituitary TSH β-subunit genes are negatively regulated by circulating T4 and T3. Our data therefore clearly revealed that both equol and flutamide did not directly affect the male rat thyroid gland toxicity due to the lack of typical hormonal profile, i.e., reduced levels of circulating thyroid hormones along with the increased TSH concentrations, which characteristically is induced by thyroid disruptors [27,28]. The lowered circulating levels of total T4 and T3 in the equol-treated rats could occur primarily due to the inhibitory effect of equol on the hypothalamic TRH neurons, resulting in the decreased TRH mRNA expression, which in turn down-regulated pituitary TSH β-subunit mRNA levels. According to the fact that the tissue concentrations of proteins do not causally relate with their secretion and the mRNA levels of secretory proteins as the indicator of alterations in the biosynthesis may serve as a more reliable indicator of long-term change in their secretion and release [15,35], therefore it is most likely that upon the short-term administration to adult male rats equol may inhibit pituitary thyrotrophs via interfering with TSH β-subunit gene expression only at the step of transcription but not the posttranslational modifications in such a way that low peripheral thyroid hormone levels cannot be compensated by the increase in the biosynthesis and secretion of TSH. There are also previous descriptions of this effect in adult rats upon the treatments with estradiol [38] as well as octylmethoxycinnamate, which is an endocrine disruptor [13]. In addition, the reductions in circulating total T4 and T3 concentrations were observed along with the increase in relative liver weights in the rats treated with flutamide. These results were similar to those from previous experiments using the same dose of flutamide applied in this study, and may be caused by a secondary mechanism associated with the increased liver weight in response to the induction of hepatic enzymes, resulting in the decreased total T4 and T3 concentrations due to the enhanced clearance of thyroid hormones by the liver [1,23,

27,28]. Equol at concentrations which are achieved in human serum upon ingestion of the significant quantities of soy isoflavones and flutamide at concentration that exerts potent anti-androgenic effect did not affect the serum levels of free T4 and T3, suggesting that direct suppression of serum total thyroid hormones levels under our experimental conditions is not the primary mechanism involved in both equol and flutamide actions. The enhanced thyroid hormone metabolism and clearance by the liver might be the most likely explanation for a potential indirect effect of equol on the reduction of serum total thyroid hormones, as has previously been demonstrated for various endocrine disruptors, including flutamide [1,27,28]. Lately, another possibility has been suggested that the primary soy isoflavones genistein and daidzein could also inhibit the activity of thyroperoxidase and there could be a decrease in T3 biosynthesis [7,9]. Based on our findings that the ratios of total T3 to total T4 were significantly increased and the free T3/free T4 levels were not significantly affected after equol treatment at either dose level, we can assume that equol is not able to inhibit thyroperoxidase and T3 formation, resulting in the unchanged separate thyroid hormone levels. As far as we know, this report is the first showing the neuroendocrine-disrupting effect of equol on the male rat HPTA, which mediated directly through different mechanisms from flutamide. Equol at high dose seems likely to be associated with mild central hypothyroidism as a result of the simultaneous down-regulation of the hypothalamic TRH and pituitary TSHβ mRNA levels. However, the serum concentrations of TSH were unaffected by equol treatment at either dose level, which is compatible with the fact that plasma levels of free T4 and T3 were not reduced, therefore we hereby suggest that the short-term application of equol as a supplement does not have a relationship with thyroid gland malfunction in subjects that consume iodine at suitable levels. To support this hypothesis, it has been recently demonstrated that when male adult rats developed a hypothyroid state it occurred at the same time as an increase in TC concentrations and a decrease in TG levels [8], which were not concomitantly evident in our experimental conditions (see discussion below). Recent studies have consistently discovered that in adult rats and mice the exposures to natural estrogens, or estrogenic chemicals caused the marked reductions in circulating levels of parameters associated with hepatic lipid metabolism, i.e., TC, TG, LDL-C, and HDL-C [4,18,29, 31,40]. These results suggest that soy isoflavones exert lowering effects on serum lipid parameters by their estrogenic properties. According to

Fig. 2. Relative mRNA expression levels of TRH gene in the MBH/ME of adult male rats treated by gavage with either equol low and high doses, or flutamide for 5 days. Data represent means ± SEM (n = 12/group). *P b 0.05 versus vehicle control group.

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Fig. 3. Relative mRNA expression levels of TSH β-subunit gene in the pituitary of adult male rats orally gavaged for 5 days with either equol low and high doses, or flutamide. Data are presented as means ± SEM (n = 12/group). *P b 0.05; **P b 0.01 versus vehicle control group.

these recent studies, equol at two doses applied clearly shows estrogenic actions in adult male rats by decreasing serum concentrations of cholesterols (TC, LDL-C, HDL-C), and additionally reducing the LDL-C/HDL-C and TC/HDL-C ratios. Furthermore, there were significant reductions in serum TG levels by equol high dose treatment, which was similar to previously published studies demonstrating that supplementation with either equol, or its precursor isoflavone, daidzein, has estrogenlike effects on TG concentrations in ovariectomized mice and rats [29,31], and adult male rats [18], thereby possibly contributing to explain the decreased terminal BW in equol high dose-treated rats. In contrast, exposure of male rats to flutamide resulted in dramatic increases in serum concentrations of TC, LDL-C, and HDL-C, without altering serum TG levels and ratios of LDL-C/HDL-C and TC/HDL-C. Similar to the current work, when flutamide was used at the same does as in our work the plasma concentrations of cholesterols in male rats were increased significantly [16,23], this may also have an effect on the increased weight of the liver. Hence, it has been proven by the data generated in this study that the influences on liver lipid metabolism via the regulation by equol and flutamide are caused by different mechanisms of action. Moreover, the status of TSH or thyroid hormones was unlikely to modulate the cholesterol-lowering effects of equol in adult male rats. Conclusion The present findings are the first data, to our knowledge, to clearly reveal that in adult male rats equol interferes with the HPTA and hepatic lipid metabolic parameters via separate modes of direct action from those of the AR antagonistic drug flutamide. The HPTA homeostasis and lipid metabolic disturbance in the liver are affected by equol through its well-known estrogenic property rather than anti-androgenic pathway. From these results it can be seen that the male rat thyroid does not suffer serious effects from equol. Furthermore, equol might have a putative role in the prevention of cardiovascular diseases via lowering the cardiometabolic risk biomarkers LDL-C, and LDL-C/HDL-C and TC/ HDL-C ratios. However, equol did not exert anti-androgenic action on the size of the prostate. Therefore, the therapeutic potential of equol as an alternative remedy to prevent and treat the androgen-dependent prostate cancer may be questionable and warrants further studies. Conflict of interest statement The authors do not have any conflicts of interest that relate to this manuscript and its contents.

Acknowledgments The authors are very grateful to Professor Dr. med. Wolfgang Wuttke and Professor Dr. med. Dana Seidlova′-Wuttke, University Medical Center, Göttingen, Germany for invaluable guidance and support in the laboratory work, Dr. Jolyon Dodgson, Faculty of Science, Mahasarakham

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