Effects of dietary equol administration on ovariectomy induced bone loss in Sprague–Dawley rats

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Maturitas 58 (2007) 308–315

Effects of dietary equol administration on ovariectomy induced bone loss in Sprague–Dawley rats Dominik Racho´n a,b,∗ , Dana Seidlov´a-Wuttke a , Tina Vortherms a , Wolfgang Wuttke a a

b

Department of Clinical and Experimental Endocrinology, University of G¨ottingen, Robert-Koch-Strasse 40, 37075 G¨ottingen, Germany Department of Immunology, Medical University of Gda´nsk, ul. D˛ebinki 1, 80-210 Gda´nsk, Poland

Received 13 February 2007; received in revised form 27 August 2007; accepted 9 September 2007

Abstract Oestrogen deficiency leads to a considerable bone loss, thus, osteopenia and osteoporosis are serious complications after menopause. Objectives: To evaluate the effects of a daidzein metabolite equol on bone mass density (BMD) and markers of bone remodelling in an ovariectomized (ovx) rat model of postmenopausal bone loss and compare them with the effects of 17␤-estradiol. Methods: Twenty-eight female Sprague–Dawley rats were ovx and fed soy-free chow only (control group, n = 8), or with the addition of oestradiol-3 benzoate (E2B) (10 mg/kg, n = 10) or equol (400 mg/kg, n = 10). At baseline and after 6-week treatment period, proximal tibia and lumbar spine BMD were measured using computer tomography. Animals were then sacrificed, blood was collected and uteri were removed. Results: Similarly to E2B, dietary equol decreased weight gain and showed mild uterotropic activity. E2B attenuated ovx induced BMD loss at proximal tibia whereas equol had no effect. At lumbar spine, however, equol not only attenuated trabecular bone loss but also increased its density. This effect was also apparent in animals treated with E2B. Cortical BMD at proximal tibia and lumbar spine were not very much influenced by ovx and treatment with E2B or equol did not induce significant changes at these sites. Plasma osteocalcin and type I collagen fragments (cross-laps) in equol treated animals did not differ from the controls whereas in E2B treated animals they were both significantly decreased. Conclusions: In spite of its mild uterotropic potential, dietary equol shows limited bone sparing effects in ovx rats. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Menopause; Osteopenia; Osteoporosis; BMD; Estradiol; Daidzein; Equol; Uterus; Phytoestrogens; Osteocalcin; Cross-laps; Sprague– Dawley rats

∗ Corresponding author at: Department of Immunology, Medical University of Gda´ nsk, ul. D˛ebinki 1, 80-210 Gda´nsk, Poland. Tel.: +48 58 349 1535; fax: +48 58 349 2503. E-mail address: [email protected] (D. Racho´n).

0378-5122/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2007.09.005

D. Racho´n et al. / Maturitas 58 (2007) 308–315

1. Introduction Oestrogen deficiency leads to a considerable bone loss, thus, osteopenia and osteoporosis are serious complications after menopause. Osteoporosis, a systemic disease characterized by a decreased bone mass density (BMD) and its increased fracture risk, affects one in four women over the age of 50 and is associated with a significant morbidity and mortality [1]. Hormone replacement therapy (HRT) has been shown to have protective effects against bone loss and osteoporosis development in postmenopausal women [2]. However, long-term oestrogen supplementation after menopause poses several risks [3] and is not anymore recommended as a prevention of chronic diseases [4–8]. Over the next decades, due to the prolongation of life expectancy, the number of postmenopausal women will increase dramatically [9] and osteoporosis will constitute a great socio-economic problem, with a negative impact both on women’s morbidity and mortality. Therefore, there is a strong need for new strategies attenuating postmenopausal bone loss. Equol [7-hydroxy-3-(4 -hydroxyphenyl)-chroman] is a heterocyclic phenol that was first isolated and identified from pregnant-mares’ urine [10]. In humans, equol is produced in the gastrointestinal tract by gut microflora from an isoflavone daidzein abundantly present in soy foods and supplements. It is structurally similar to 17␤-estradiol (E2) and it possesses estrogenic activity, having affinity for both oestrogen receptors (ER␣ and ER␤) [11,12]. Data from our previous experiments have shown that dietary equol exerts mild estrogenic effects in the pituitary, uterus, mammary gland, liver and fat tissue in an ovariectomized (ovx) rat model of menopause [13–16]. It is, however, still unclear, what are the effects of equol on bone tissue. Therefore, the aim of the present study was to evaluate the effects of dietary equol on BMD and markers of bone remodelling in an ovx rat model of postmenopausal bone loss and compare them with the effects of E2.

2. Materials and methods 2.1. Animals All animal experiments were approved by the Local Ethics Committee for Animal Care and Use at the

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Georg August University in G¨ottingen according to the German animal welfare regulations under permission given by the district authorities of Braunschweig, Germany (number 509.42502/01–36.03) and conformed with the FRAME’s guidelines. Twenty-eight virgin female Sprague–Dawley (SD) rats raised in the animal facility of the G¨ottingen’s University Hospital were used. Animals were kept in groups of four or five in Makrolon® cages (type 4) under a 12 h light, 12 h dark cycle, in room temperature of 22–24 ◦ C and relative humidity 50–55%, with free access to regular rodent chow and water. Two weeks before the start of the experiment they were put on soy-free food (Ssniff Spezialdi¨aten GmbH, Soest, Germany) in order to eliminate exposure to soy derived estrogenic compounds found in regular rodent chow. At the age of 3 months (mean body weight: 251 g), rats were bilaterally ovx under isoflurane (Abbott GmbH & Co. KG, Wiesbaden, Germany) anaesthesia. For postoperative analgesia caprophene (Rimadyl® , Pfizer GmbH, Karlsruhe, Germany) at the dose of 4 mg/kg BW was administered subcutaneously. After ovx animals were randomized, placed in groups of four or five per cage and divided into three groups. The control group (n = 8) received soy-free food only. E2B group (n = 10) received soyfree food with the addition of estradiol-3 benzoate (E2B) (98% purity, Sigma–Aldrich Chemie GmbH, St. Louis, USA) at the concentration of 10 mg/kg of chow. The equol group (n = 10) received soy-free food with the addition of equol (98% purity, Changzhou Dahua Imp. & Exp. Group, China) at the concentration of 400 mg/kg of chow. The rodent chow was provided by Ssniff special diets GmbH (Soest, Germany) and was prepared by mixing the studied substances with the soy-free formulation Ssniff SM R/M (10 mm pellets) to homogeneity before the process of pelleting. The rationale for the doses used in this experiment was based on the results of our previous studies which showed that E2B at the dose of 10 mg/kg of chow given orally to 3-month-old ovx SD rats raises their serum E2 levels to those observed in the proestrus of the intact animals and exhibits clear estrogenic effects in the uterus. Also, according to our previous data equol at a dose of 400 mg/kg of chow already shows estrogenic activity in the uterus whereas lower doses have no effect [16]. The food consumption and animal’s body mass were measured once a week and according to the records of animal food intake, the average intake of the tested

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substances per animal throughout the whole experiment period was calculated. The average consumption of E2B and equol were 0.175 and 6.870 mg per animal per day, respectively.

from final BMD values and are expressed in percentage (%).

2.2. BMD measurements

After 6 weeks of treatment, animals were decapitated under CO2 anaesthesia between 8:00 and 12:00 h. Blood was drained from the trunk into glass tubes containing EDTA (Becton Dickinson, Plymouth, UK) and kept at 4 ◦ C for 2–4 h. Plasma was then obtained after centrifugation of the samples at 2500 rpm for 30 min at the temperature of 4 ◦ C. Samples were aliquoted in quadruplets into 1.5 ml polypropylene tubes (Eppendorf AG, Hamburg, Germany) and stored at −20 ◦ C until further analyses. The abdominal cavity was opened with a longitudinal cut and uteri were removed. After trimming associated fat and expelling any internal fluid the organs were weighed.

Before ovx and after 6 weeks of treatment with the tested substances, BMD of the proximal tibia and lumbar spine were measured using quantitative computer tomography (CT) (XCT Research SA+, Stratec Medizintechnik, Pforzheim, Germany). Six-week treatment period was used because it has been well documented that in young adult rats ovx induced changes in trabecular bone mass are already clearly evident after 2 weeks and unequivocal after 4 weeks [17,18]. Also, we have chosen to study the changes in proximal tibia instead of proximal and distal femur because according to the data from our previous experiments this site is most profoundly influenced by hypoestrogenaemia and more representative of BMD changes in long bones. Animals were anesthetized with isoflurane, placed on their right side with their left hind limb passed through a polypropylene funnel. The claw was taped to an acrylic frame with the ankle joint at a 90◦ angle and the knee joint at 180◦ . The animal was then placed in a sliding platform so that the distal end of the femur and the proximal end of the tibia were in the scanning field. A two-dimensional scout view was run for a length of 8–10 mm and reference line was set at the proximal growth plate of the tibia. The scans were taken 3.75 and 4.25 mm distally from the reference line. After completing proximal tibia BMD measurements the animal was placed on its back so that the lumbar spine region was in the scanning field. Again a two-dimensional scout view was run for a length of 8–10 mm and a reference line was set in the middle of the L4 vertebra. The scans were taken at the reference line, 1 mm above and 1 mm below it. After scanning all the animals, the images were displayed on the monitor and the regions of interest were outlined. The trabecular bone region and the cortical bone region were defined as the inner 45 and 55% of the total cross-sectional area, respectively, and were both reported in mg/cm3 [19]. Means were then calculated from the two scans of the proximal tibia and three scans of the L4 vertebra. Final BMD are expressed in mg/mm3 . Changes in bone mass were evaluated by subtracting baseline

2.3. Plasma and tissue collection

2.4. Plasma analyses Plasma E2 levels were measured by commercially available rat RIA kit (DSL, Inc., Webster, Teras, USA). Plasma osteocalcin levels were measured on a Roche Elecsys 2010 immunoassay analyzer using N-MID Osteocalcin reagent kit (Roche Diagnostics GmbH, Mannheim). Plasma concentrations of type I collagen fragments (cross-laps) were measured by commercially available rat ELISA kit (RatLaps ELISA, Nordic Bioscience Diagnostics A/S, Denmark). Plasma equol concentrations were measured by high-pressure liquid chromatography (HPLC) and UV-detection at 260 nm. The HPLC conditions were gradient elution for 0 min: 70% A, 30% B; and 15 min: 25% A, 75% B (A = water containing 0.085% o-phosphoric acid and B = acetonitrile; 250 mm × 4 mm C18 reverse-phase column, flow rate 1 ml/min). 2.5. Statistical analysis Data are presented as arithmetic means ± S.E.M. To compare differences in body mass, uterus weight, and concentrations of plasma bone markers, equol and 17␤-estradiol between the studied groups, one-way ANOVA was performed. Differences in BMD between the groups were compared using ANCOVA with body weight (BW) as a confounding variable (covariate) and presented as BW adjusted means ± S.E.M. Post

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hoc comparisons were carried out using Dunnett’s test and P values < 0.05 were considered statistically significant. All analyses were performed using STATISTICA version 7.1 for Windows (StatSoft Inc., Tulsa, OK, USA, www.statsoft.com).

3. Results 3.1. Effects on food intake and body weight The average weekly food intake throughout the experiment, calculated in grams per kg of BW, did not differ between the studied groups. Mean amounts of rodent chow consumed by controls, E2B and equol treated animals were 444 ± 31, 504 ± 47 and 453 ± 16 g/kg BW/week, respectively (P > 0.05). Nevertheless, at the end of the experiment animals treated with dietary E2B and equol had significantly lower final BW compared to controls (254 ± 4 and 280 ± 5 g vs. 336 ± 8 g, respectively, P < 0.05). 3.2. Effects on uterine mass, serum E2 and equol levels As expected, uterine mass in animals treated with dietary E2B was significantly higher compared to control animals (399 ± 27 mg vs. 88 ± 3 mg, P < 0.05). Also, animals consuming equol containing chow had higher uterine weight compared to the controls (114 ± 6 mg vs. 88 ± 3 mg, P < 0.05), however, this difference was 3.5 times lower in magnitude compared to E2B treated animals. Accordingly, feeding our ovx rats with E2B containing chow raised their plasma E2 levels to 723 ± 121 pmol/l which was significantly higher compared to the controls where plasma E2 levels were only 48 ± 11 pmol/l (P < 0.05). Plasma E2 levels in equol treated animals did not differ significantly from the control group (71 ± 23 pmol/l vs. 48 ± 11 pmol/l, P > 0.05). Plasma equol concentrations in animals fed with equol containing chow were 2460 ± 343 nmol/l. Equol was undetectable in plasma of the controls and E2B treated animals. 3.3. Effects on proximal tibia and lumbar spine BMD Body mass is a major determinant of BMD and weight gain is generally accompanied by an increase

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in bone density and strength [20–22]. Since animals in our control group gained significantly more weight throughout the experiment compared to E2B and equol treated groups, we adjusted all BMD data to BW. As expected, dietary E2B treatment almost completely prevented trabecular bone loss at the proximal tibia. Compared to baseline values, animals treated with dietary E2B lost only 1.3 ± 5.0% of proximal tibia trabecular BMD whereas animals from the control group lost 27.9 ± 6.4% (P < 0.05). Trabecular bone loss at the proximal tibia in equol treated animals was comparable to the control group (−23.2 ± 3.2% vs. −27.9 ± 6.4%, P > 0.05). Accordingly, animals treated with E2B had significantly higher final proximal tibia trabecular BMD compared to control animals (290 ± 15 mg/mm3 vs. 221 ± 19 mg/mm3 , P < 0.05) and the trabecular BMD at the proximal tibia in animals treated with dietary equol did not differ from the controls (233 ± 9 mg/mm3 vs. 221 ± 19 mg/mm3 , P > 0.05). Ovx did not induce significant cortical bone loss of the proximal tibia. Nevertheless, in animals treated with dietary E2B and equol there was a slight increase in BMD gain at this site compared to controls, but this difference was not statistically significant (13.3 ± 1.6 and 11.5 ± 0.9% vs. 6.9 ± 1.8%, respectively, P > 0.05). There were also no statistically significant differences in the final cortical BMD at the proximal tibia in animals treated with E2B or equol compared to controls (1103 ± 11 and 1110 ± 7 mg/cm3 vs. 1104 ± 13 mg/cm3 , respectively, P > 0.05) (Fig. 1).

Fig. 1. Effects of dietary equol and E2B treatment on trabecular and cortical BMD at proximal tibia of ovx Sprague–Dawley rats. Compared to controls, trabecular BMD at proximal tibia was significantly increased only in E2B treated animals. There were no significant differences in cortical BMD. E2B, estradiol-3 benzoate, * P < 0.05.

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E2B treatment also attenuated trabecular bone loss at the lumbar spine. Compared to baseline values, animals treated with dietary E2B gained 5.1 ± 8.7% of the trabecular BMD at the L4 vertebra whereas animals from the control group lost 16.7 ± 11.1% (P < 0.05). Surprisingly, in animals treated with dietary equol there was also a significant trabecular bone gain at this site (15.5 ± 5.5% vs. −16.7 ± 11.1%, P < 0.05). Correspondingly, final trabecular BMD at the L4 vertebra in animals treated with E2B and equol was higher compared to controls (221 ± 12 and 230 ± 7 mg/cm3 vs. 186 ± 15 mg/cm3 , P < 0.05). There were no significant differences in changes of cortical BMD at the L4 vertebra between E2B or equol treated animals and the controls (4.0 ± 1.5 and 2.0 ± 0.9% vs. −2.0 ± 1.9%, respectively, P > 0.05) and final cortical BMD at this site also did not differ between these groups (857 ± 9 and 860 ± 6 mg/cm3 vs. 838 ± 11 mg/cm3 , respectively, P > 0.05) (Fig. 2).

Fig. 3. Effects of dietary equol and E2B treatment on plasma osteocalcin and cross-laps in ovx Sprague–Dawley rats. E2B treated animals had significantly lower plasma osteocalcin and cross-laps compared to controls. E2B, estradiol-3 benzoate, * P < 0.05.

fer in equol treated animals (25.4 ± 2.1 ng/ml vs. 22.6 ± 1.6 ng/ml, P > 0.05) (Fig. 3).

3.4. Effects on plasma osteocalcin and cross-laps 4. Discussion Animals treated with dietary E2B had significantly lower plasma osteocalcin levels compared to control animals (20.2 ± 2.1 ng/ml vs. 38.6 ± 1.6 ng/ml, P < 0.05). Plasma osteocalcin in equol treated animals did not differ from the controls (34.6 ± 1.7 ng/ml vs. 38.6 ± 1.6 ng/ml, P > 0.05). Similarly, plasma crosslaps concentrations were significantly lower in E2B treated animals compared to controls (16.6 ± 1.3 ng/ml vs. 22.6 ± 1.6 ng/ml, P < 0.05) and did not dif-

Fig. 2. Effects of dietary equol and E2B treatment on trabecular and cortical BMD at lumbar spine (L4 vertebra) of ovx Sprague–Dawley rats. Compared to controls, trabecular BMD was significantly increased in E2B and equol treated animals. There were no differences in cortical BMD. E2B, estradiol-3 benzoate, * P < 0.05.

In the present study, we have evaluated the effects of dietary equol administration on BMD and markers of bone remodelling in an ovx rat model of postmenopausal bone loss. In the first step of our experiment we have evaluated the uterotropic potential of equol. Our results showed that animals consuming equol containing chow had significantly higher uterine weights compared to the controls. This effect was, however, 3.5 times lower in magnitude compared to the effects of E2B. These findings are consistent with our previous data [16] and the results of others where equol has been shown to be uterotropic in mice and sheep [23,24]. In postmenopausal women, uterotropic effects of oestrogen therapy are undesirable since they stimulate endometrial hyperplasia and may increase the risk of endometrial cancer [25]. Hence, it is also possible that uncontrolled equol intake could endanger human endometrium to became hyperplasic. Further studies are, however, warranted to evaluate the actual risk. Dietary equol administration, also similarly to E2B, attenuated ovx induced BW gain. This is also consistent with our previous observations and the possible mechanisms of this effect have been already discussed by us elsewhere [15]. Nevertheless, body mass is a major determinant of BMD [20–22] and obesity-induced

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mechanical loading is certainly a contributing factor in the positive relationship between BW and bone mass [26]. Therefore, in our study it was necessary to adjust all the BMD data to animal’s BW. Our results have shown that there is a considerable loss of trabecular BMD at the proximal tibia after ovx and dietary E2B administration has a protective effect. In contrast, treatment with dietary equol does not attenuate trabecular bone loss at proximal tibia. Cortical BMD at proximal tibia was not very much influenced by ovx and treatment with E2B or equol did not induce significant changes at this site. Ovx also induced loss of trabecular BMD at the lumbar spine. In contrast to the effect at proximal tibia region, dietary equol administration not only protected from ovx loss of trabecular bone at the L4 vertebra but also increased its density. This effect was also apparent in animals treated with E2B. Cortical BMD at the lumbar spine was not significantly influenced by ovx and treatment with our studied substances did not induce significant changes at this site. There is a considerable amount of data from animal studies showing that soy isoflavones such as daidzein and genistein have a potential of inhibiting ovx induced bone loss [27–29]. However, to our knowledge this is the first report on the effects of dietary equol administration on ovx induced osteopenia in SD rats. Nevertheless, equol administered subcutaneously to mice for 4 weeks has been shown to attenuate ovx induced BMD loss in the lumbar spine and femur [30]. In that study, however, equol was administered s.c. and although the doses were comparable to the dose used in our experiment, it had no effects on final body and uterine weight. This shows that receptiveness of murine tissues to the estrogenic effects of equol may differ very much from those of SD rats. Nevertheless, in a double-blind placebo controlled trail of postmenopausal women treated with a red clover preparation which contained considerable amounts of another equol precursor-formononetin, BMD loss at the lumbar spine was significantly lower compared to the placebo group whereas femur BMD was not affected [31]. Also Setchell and LydekingOlsen [32] reported that BMD of the lumbar spine of equol producers increased by 2.4% compared with the control group after a 2-year intervention with isoflavones. Osteocalcin, the most important non-collagen calcium-binding protein of the bone matrix, is pro-

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duced by the osteoblasts during bone synthesis. Apart from being assimilated into the bone matrix it is also secreted into the blood stream, therefore, plasma osteocalcin levels reflect the rate of bone turnover. Type I collagen accounts for more than 90% of the organic bone matrix [33]. During bone remodelling, bone matrix is degraded by osteoclasts and fragments of type 1 collagen are released into the circulation. Thus, blood levels of these bone related degradation products (cross-laps) reflect the magnitude of osteoclastic bone resorption. In our experiment, plasma osteocalcin and cross-laps in animals treated dietary equol did not differ from the controls. In contrast, in E2B treated group they were both significantly decreased, which is in accordance with our previous studies [34,35] and points out that oestrogen attenuates ovx induced BMD loss due to the inhibition of bone resorption rather then stimulation of its formation. Although equol showed mild estrogenic effects in the uterus, it did not show evident bone sparing effects in our ovx rat model of menopause. This shows that receptiveness of uterine tissue to the estrogenic effects of equol may differ very much from those observed in the skeleton. The pleiotropic and tissue-specific effects of estrogens are mostly mediated by the differential expression of ER subtypes (ER␣ and ER␤) as well as their coregulators and show a great degree of complexity in the dynamics of ER-mediated transcription of estrogen target genes [36]. Apart from diverse ER binding potencies, there are also big differences in types of the corepressors and coactivators which are recruited by different estrogens to the promotor region of specific oestrogen regulated genes in certain tissues [37]. Therefore, we may speculate, that the differences in the estrogenic potential of equol in uterine and bone tissues which were observed in our experiment may be due to the different ER expression patterns in these tissues as well as activation of different ER-dependent pathways. Similarly, a greater increase in trabecular BMD of the lumbar spine compared to proximal tibia in animals treated with equol could also be caused by the activation of different ER-pathways in this site of the skeleton. These assumptions, however, warrant further studies. Summarising, in spite of its mild uterotropic potential, dietary equol shows limited bone sparing effects in rats after ovx. It only attenuates ovx induced trabecular bone loss at the lumbar spine and does not show

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any effects at the proximal tibia or plasma markers of bone remodelling. Therefore, we may speculate that equol may not be a good candidate for the prevention of postmenopausal bone loss.

[10]

[11]

Conflict of interest The authors have no conflicts of interest relevant to the contents of this manuscript.

[12]

[13]

Acknowledgements This work was founded by the European Commission Grants: EURISKED (contract no. EVK1-CT200200128) and CASCADE (contract no. FOOD-CT-2004506319).

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