Effects of different doses of soy isoflavones on bone tissue of ...

CLIMACTERIC 2014;17:393–401

Effects of different doses of soy isoflavones on bone tissue of ovariectomized rats M. A. Santos*, R. Florencio-Silva*, V. P. Medeiros†, H. B. Nader†, K. O. Nonaka‡, G. R. S. Sasso*, M. J. Simões* and R. D. Reginato* *Federal University of São Paulo, Morphology and Genetics, São Paulo; †Federal University of São Paulo, Biochemistry, São Paulo; ‡Federal University of São Carlos, Physiological Sciences, São Carlos, Brazil Key words: ISOFLAVONES, BONE, COLLAGEN, GLYCOSAMINOGLYCANS, RATS

ABSTRACT Aim Studies report that hormone replacement prevents osteoporosis, but there are doubts whether isoflavones are really efficient in this process. The aim of this study was to evaluate the effects of different doses of soy isoflavones on bone tissue of ovariectomized rats. Methods Forty female rats at the age of 6 months were ovariectomized and, after 3 months, the animals were divided into four groups: GI – Control (treated with drug vehicle); GII – treated with isoflavones (80 mg/kg per day); GIII – treated with isoflavones (200 mg/kg per day) and GIV – treated with isoflavones (350 mg/kg per day). Soy isoflavones were administered by gavage for 90 consecutive days. After treatment, the rats were euthanized and their distal femurs were removed for histological routine, histochemistry and biochemical study. Histological sections were stained with hematoxylin–eosin or subjected to picrosirius red and alcian blue methods. Shafts of femurs were submitted to biochemical assay and tibias were subjected to biophysical and biomechanical tests. Results In distal femurs, the trabecular bone volume was higher in the groups treated with isoflavones, being higher in GIV, while the cortical bone width and the presence of mature type I collagen fibers were higher in GII. At the trabecular bone region, the percentage of total glycosaminoglycans (GAGs) was higher in GII and the percentage of only sulfated GAGs was higher in GIII, while the higher content of chondroitin sulfate in shafts of femurs was seen in GIV. Biophysical and biomechanical tests in tibias did not differ among the groups. Conclusion Our data indicate that soy isoflavones improve bone quality in femurs of rats by increasing histomorphometric parameters, the content of GAGs and mature type I collagen fibers. These positive effects are dose-dependent and it was different in cortical and trabecular bone.

INTRODUCTION Osteoporosis is one of the major public health problems due to its association with a high incidence of fractures, which lead to increased patient morbidity and significant mortality1,2. It affects one in three postmenopausal women, and ten million American women were estimated to have osteoporosis, while 34 million its precursor, osteopenia3. The public health burden of osteoporotic fracture is likely to rise in future generations, due in part to an increase in life expectancy4,5. Hormone replacement therapy is considered to be the most effective therapy to reduce or prevent osteoporosis in

postmenopausal women6. However, estrogen is associated with a greater risk for stroke, and the estrogen–progestin combination is associated with a higher incidence of endometrial and breast cancers7–9. Thus, researchers have investigated alternative options for prevention of osteoporosis. Soy isoflavones have received considerable attention in the medical literature and they are being investigated as a potential alternative to hormone therapy10,11. It is suggested that soy isoflavones act as selective estrogen receptor modulators due to their preferential binding to estrogen receptor (ER) β12. Because of their structural similarities with the endogenous estrogens and their mode of action, soy isoflavones can bind

Correspondence: Dr R. D. Reginato, Universidade Federal de São Paulo, Morfologia e Genética, Rua Botucatu 740, Vila Clementino, São Paulo, São Paulo, 04021-001 Brazil; E-mail: [email protected] ORIGINAL ARTICLE © 2014 International Menopause Society DOI: 10.3109/13697137.2013.830606

Received 21-05-2013 Revised 27-07-2013 Accepted 28-07-2013

Soy isoflavone effects on bone tissue in ovariectomized rats to the ERα and ERβ and exert the same beneficial effects as estrogen therapy on menopausal symptoms12–14. Despite the related positive effects of soy isoflavones in diminishing menopausal symptoms such as hot flushes and cardiovascular diseases15,16, the effects of these compounds in counteracting postmenopausal bone loss have been an issue of controversy in clinical trials, because their positive effects have been observed in some17–19 but not other studies20–22. These inconsistent results have been attributed to the limited sample sizes, different populations and inferences from the individual studies and, possibly, differences in the doses applied22. After isoflavone ingestion at the dose level of mg, the blood levels appear at the concentration of ng/mg, due to the fact that these compounds are metabolized by bacteria in the gut flora and only the bioactive forms of isoflavones are absorbed by the intestine. It is known that the gut microflora are different among distinct human populations and animals, and several studies have shown that these differences influence the amount of isoflavones absorbed in the gut. It is suggested that the conflicting results concerning isoflavones seen in the literature are due, at least in part, to the differences in the gut microflora which in turn lead to distinct amounts of isoflavones absorbed by the intestine22. Inconsistent data have also been observed in some animal model studies regarding the effectiveness of different doses of isoflavones in counteracting bone loss23–25. However, the majority of these studies examined the effects of soy isoflavones on bone mass, bone turnover markers and bone mineral density22,26–28, while few studies have evaluated the effects of these compounds on bone collagen fibers and glycosaminoglycans (GAGs). Consequently, the effects of soy isoflavones on these bone matrix constituents are poorly understood. Despite that, soy isoflavones has been reported to reduce the quantity of GAGs in a mouse model of mucopolysaccharidosis type II29 and reduce it synthesis in osteosarcoma cell lines30. In addition, it was shown that topical application of estradiol and genistein (a soy isoflavone) was able to counteract the decrease of hyaluronic acid content in the facial skin of postmenopausal women31. Also, it was recently reported that a genistein-rich soy extract has no significant effects on collagen fibers in the bones of ovariectomized rats32, while, in another study, soy isoflavone extract has been shown to increase the presence of mature type I collagen fibers and the content of GAGs in bone tissue of rats33. Thus, there are no conclusive studies regarding the effects of different doses of soy isoflavones on bone tissue. In this perspective, the objective of this study was to evaluate the effects of different doses of soy isoflavones on bone tissue of ovariectomized rats.

METHODS This experimental study was conducted in the Laboratory Animal Care Committee of Federal University of São Paulo, in which the animals were maintained in accordance with the

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Santos et al. Guiding Principles for the Care and Use of Animals. The project was approved under the number 0702/09.

Drugs Soy isoflavone, a fermented soybean extract rich in the major aglycone forms genistein and daidzein (Novasoy-ADM Natural Health & Nutrition, Illinois, USA) were used in this experiment. This isoflavone extract contained approximately 36% of genistein, 62% of daidzein, and 2% of glycitein (including the isoforms of isoflavones).

Animals We used 40 virgin adult (6-month-old) rats (Rattus norvegicus albinus), of 270 g body weight. The animals were provided by the Center for Development of Experimental Models of Federal University of São Paulo. All animals were kept in plastic cages with controlled light and temperature and fed with a soy-free diet and water ad libitum. The animals were initially submitted to a colpocytological examination for 4 consecutive days for the normal estrous cycle evaluation and then 40 animals were subjected to bilateral ovariectomy. A postsurgical period of 90 days was observed to obtain significant depletion of the estrogen levels and subsequent bone loss. Ninety days after ovariectomy, the rats were divided into four groups as follows: group I (GI) of ovariectomized rats that received only drug vehicle (propylene glycol); group II (GII) of ovariectomized rats treated with concentrated soy isoflavone extract (80 mg/kg); group III (GIII) of ovariectomized rats treated with concentrated soy isoflavone extract (200 mg/ kg); and group IV (GIV) of ovariectomized rats treated with concentrated soy isoflavone extract (350 mg/kg)34. The isoflavones were dissolved in 0.5 ml of propylenoglycol and administered daily by gavage during the 90 days. Subsequently, the rats were euthanized by overdose with a mixture of cetamine (100 mg/kg) and xylazine (20 mg/kg) intraperitoneally; the femurs were removed and kept in fixative solution. Tibias were kept in 0.9% NaCl solution at 4°C for subsequent bone density assessment through biophysical analysis and biomechanical tests. The distal femurs were processed for histology and subsequently subjected to histomorphometry and histochemistry. Shafts of the femurs were destined to biochemical analysis for GAGs identification and quantification.

Tissue collection and processing The distal femurs were dissected and fixed in 4% formaldehyde (freshly derived from paraformaldehyde), buffered at pH 7.2 with 0.1 mol/l phosphate solution at room temperature, for 4 days. After decalcification for approximately 40 days in a 25% solution of formic acid at pH 2.0, the specimens were conserved in 70% alcohol solution and thereafter dehydrated

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Soy isoflavone effects on bone tissue in ovariectomized rats in graded alcohols, cleared in xylene and embedded in paraffin. Longitudinal sections (5 μm thick) were stained with hematoxylin and eosin (H&E) for histomorphometric analysis, subjected to the histochemical methods of alcian blue at pH of 2.5 or 0.5 for GAGs quantification and picrosirius red for collagen fiber evaluation (picrosirius red, alcian blue, H&E were obtained from Sigma, St. Louis, MO, USA). Sections were cut in a Minot type microtome (Leica, Model RM 2145) and the photomicrographs of the sections subjected to H&E staining and the alcian blue method were examined and documented using a light microscope (Axiolab Standard 2.0, Carl Zeiss) equipped with software and a highresolution videocamera (AxionCam, Carl Zeiss). The photomicrographs of the sections submitted to the picrosirius red method were documented and analyzed with polarized light microscopy (Axiolab Standard 2.0, Carl Zeiss).

Histomorphometry In semi-serial sections of distal femurs, stained with H&E, measurements were determined in the secondary spongiosa, starting 390 μm below the lowest point of the growth plate and 390 μm from the periosteum35, establishing a total area of 2 907 011.22 μm² where trabecular bone volume and cortical bone width were measured. The histomorphometric parameters were defined according to the report by the American Society for Bone and Mineral Research Committee36. Bone histomorphometry was performed using an AxionVision 4.2 REL image analysis software program (Carl Zeiss).

Histochemical methods Sections were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol and subjected to the alcian blue and picrosirius red methods. The slices destined for the alcian blue method were immersed in a 0.5 M HCl solution (alcian blue at pH 0.5) or immersed in a 3% acetic acid solution (alcian blue at pH 2.5) for 2 min and then treated in alcian blue solution for 10 min. The slices subjected to the picrosirius red method were immersed in a 0.2% phosphomolibdic acid solution for 10 min, washed in distilled water, treated with picrosirius red at 0.1% in saturated picric acid aqueous solution for 90 min and then washed in a 0.01 M HCl solution for 2 min. Afterwards, sections were washed in distilled water, dehydrated in increasing concentrations of ethanol, cleared in xylene and mounted in Entellan® medium. The slices treated with alcian blue were subjected to quantitative analysis of GAGs (ImageLab 2000®), while the ones treated with picrosirius red were semi-quantitatively analyzed by polarized light microscopy in both cortical and trabecular bone regions, for identification and birefringence pattern evaluation of collagen fibers against a black background.

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Extraction, characterization and measurement of sulfated GAGs Five shafts (2 mm) containing regions of trabecular and cortical bone from the distal femurs were collected from each group and the epiphyses were removed to exclude the growth plate from this analysis. Then, shafts were washed in PBS solution containing protease inhibitor for 2 h, decalcified in a 25% solution of formic acid, at pH 2.0, for 3 days, kept in PBS solution containing protease inhibitor, at a temperature of 4°C for 24 h and then submitted to proteolysis with papain, overnight at 60°C. The identification of each GAG was performed using the following standards: chondroitin-4-sulfate from whale cartilage, chondroitin-6-sulfate from shark cartilage, dermatan sulfate from bovine intestinal mucosa (Seikagaku Kogyo Co., Tokyo, Japan) and heparan sulfate from bovine lung37. To improve the identification of GAGs, controls were generated by enzymatic degradation of the sulfated GAGs by Flavobacterium heparinum-derived chondroitinases. Dermatan sulfate and chondroitin sulfate were characterized after incubation of the samples with chondroitinase AC and chondroitinase ABC. The incubation products were submitted to agarose gel electrophoresis, and the confirmation of the particular type of GAG was accomplished by the distinct band pattern in the agarose gel. The same standards were also used for quantitative determination of the samples by 525 nm densitometry. The results were expressed as μg of GAG per mg of tissue (mean ⫾ standard deviation). All samples were assayed in triplicate. The detection limit was 1 μg/μl of GAG sulfate.

Bone density and biomechanical tests in tibias Bone density was estimated by the Archimedes principle as follows. The wet weight (WW) was measured with the tibia suspended from a copper thread and submerged in water after removal of trapped air bubbles by vacuum centrifugation. The dry weight (DW) was obtained after dehydration in an oven at 100°C for 24 h. The difference between the DW and WW, divided by the density of distilled water, was the tibia’s volume, VO (VO ⫽ (DW ⫺ WW)/δH2O]38,39. Bone density was calculated based on the formula WW/VO (g/cm3). Biomechanical tests were performed by the three-point bending test, in an Instron universal testing machine (Instron Cop. model 4444) in which maximal load and fracture load were analyzed. All tests were performed by an investigator blinded to the treatment regimens.

Statistical analysis Data were expressed as mean ⫹ ⫺ standard deviation. The groups were compared using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to evaluate differences among groups. Statistical analyses were

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performed using the GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Significance was established at p ⬍ 0.05.

the lower intensity in the control group. The higher greenish birefringence intensity of collagen fibers in cortical and trabecular bones was seen in the control group (GI) (Figure 1).

RESULTS

Biochemical analysis of sulfated GAGs

Histomorphometry

Chondroitin sulfate was found in bone of all animal groups studied; no heparan or dermatan sulfate was identified in these samples. Biochemical determinations indicated that the chondroitin sulfate concentration was significant higher (p ⬍ 0.05) in the GIV group compared with all the other groups (Table 1 and Figure 2).

The trabecular bone volume was higher in GIV and GII and lower in GI (statistically not significant). On the other hand, the cortical bone width was significantly higher in GI and GII when compared with GIII (GI and GII ⬎ GIII; p ⬍ 0.05) (Table 1 and Figure 1).

Bone density and biomechanical tests in tibias Histochemical analysis The higher concentration (%) of total GAGs (sulfated and carboxylated) was seen in the group treated with the lowest dose and it was significant higher (p ⬍ 0.05) when compared with the control group (GII ⬎ GIII ⬎ GIV⬎ GI; p ⬍ 0.05), at the pH of 2.5 (Table 1 and Figure 1). The concentration (%) of sulfated GAGs (pH ⫽ 0.5) was significant higher (p ⬍ 0.05) in the group treated with the intermediate dose (GIII) compared with the control and GIV groups (GIII ⬎ GII ⬎ GIV ⬎ GI) (Table 1 and Figure 1).

Picrosirius red Semi-quantitative analysis through polarized light microscopy showed that, in the cortical and trabecular bones of the distal femurs, the collagen fibers showed the higher red birefringence intensity in the group treated with the lowest dose (GII) and

The results of the bone density and biomechanical tests in tibias did not differ significantly among the groups (Table 1).

DISCUSSION This study was designed to investigate whether soy isoflavones, at different doses, exert trophic effects on bone tissue of ovariectomized rats. It should be noted that the lowest dose that we used in this study (80 mg/kg) corresponds to the one normally reported in the literature. For these purposes, we used ovariectomized rats, a classical animal model of postmenopausal bone loss40. We found that soy isoflavones in all doses used, in our experimental condition, improved bone quality in femurs of rats by increasing histomorphometric parameters, the content of GAGs and mature type I collagen fibers. In addition, these positive effects were bone-site specific and depended on the dose applied.

Table 1 Histomorphometric parameters and glycosaminoglycans (GAGs) content in femurs and biophysical and biomechanical parameters in tibias of rats. Data are given as means ⫹ ⫺ standard deviations. Total GAGs ⫽ sulfated and carboxylated GAGs. Animal groups are as follows: GI ⫽ ovariectomized control group; GII ⫽ ovariectomized rats treated with isoflavones (80 mg/kg); GIII ⫽ ovariectomized rats treated with isoflavones (200 mg/kg); GIV ⫽ ovariectomized rats treated with isoflavones (350 mg/kg). Asterisks indicate statistically significant (p ⬍ 0.05) differences among the groups. Note the total GAGs of *GII ⬎ GI; observe the sulfated GAGs of *GIII ⬎ GI and GIV, and the concentration of chondroitin sulfate in *GIV ⬎ GI, GII and GIII Groups Parameters Femur Trabecular bone volume (%) Cortical bone width (μm) Total GAGs (%) Sulfated GAGs (%) Chondroitin sulfate (μg/mg of tissue) Tibia Bone density (g/cm3) Maximal load (kN) Fracture load (N)

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GI

GII

GIII

GIV

15.93 ⫹ ⫺ 1.93 378.8 ⫹ ⫺ 37.13* 0.810 ⫹ ⫺ 0.268 0.556 ⫹ 0.316 ⫺ 2.132 ⫹ ⫺ 0.376

17.37 ⫹ ⫺ 4.70 366.5 ⫹ ⫺ 39.45* 2.713 ⫹ ⫺ 1.040* 1.388 ⫹ ⫺ 0.526 3.550 ⫹ ⫺ 1.945

16.00 ⫹ ⫺ 3.84 289.4 ⫹ ⫺ 33.74 2.390 ⫹ ⫺ 1.978 2.089 ⫹ ⫺ 1.588* 2.032 ⫹ ⫺ 2.032

18.88 ⫹ ⫺ 1.45 335.6 ⫹ ⫺ 33.13 2.063 ⫹ ⫺ 2.074 0.767 ⫹ ⫺ 0.490 6.648 ⫹ ⫺ 1.770*

1.596 ⫹ ⫺ 0.078 0.048 ⫹ ⫺ 0.01 0.077 ⫹ ⫺ 0.01

1.610 ⫹ ⫺ 0.041 0.039 ⫹ ⫺ 0.01 0.062 ⫹ ⫺ 0.02

1.600 ⫹ ⫺ 0.021 0.054 ⫹ ⫺ 0.02 0.066 ⫹ ⫺ 0.01

1.607 ⫹ ⫺ 0.010 0.031 ⫹ ⫺ 0.01 0.076 ⫹ ⫺ 0.01

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Figure 1 Photomicrographs of bone tissue sections from distal femurs of ovariectomized rats (GI), ovariectomized rats treated with 80 mg/kg of soy isoflavones (GII); ovariectomized rats treated with 200 mg/kg soy isoflavones (GIII) and ovariectomized rats treated with 350 mg/kg of soy isoflavones (GIV). Note the higher trabecular bone (Tb) area in GII (B) compared with GI (A) and the higher cortical bone (Ct) width in GII (D) compared with GIII (C); hematoxylin & eosin staining. Note the higher presence of alcian blue-stained glycosaminoglycans (black arrows) in GII (I and J), GIII (M and N) and GIV (Q and R), compared with GI (E and F); see Table 1 for detailed quantitative data. Observe, in the cortical and trabecular bone stained by picrosirius red method and visualized in polarized light microscopy, the higher greenish birefringence intensity (type I immature collagen fibers) in the GI group, in cortical (G) and trabecular bone regions (H). Note the higher reddish birefringence intensity (type I mature collagen fibers) in cortical (K, O and S) and trabecular bone (L, P and T) region of the GII (K and L), GIII (O and P) and GIV (S and T)

Soy isoflavones belong to the group of phytoestrogens that are plant-derived, non-steroidal molecules that exhibit estrogen-like properties41. These compounds are structurally similar to mammalian estrogen, giving them the ability to bind to both known estrogen receptors (ERα and ERβ) and then mimic the biological effects of estrogens12. The preferential binding of isoflavones to ERβ indicates that they act as selective

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estrogen receptor modulators23. Thus, soy isoflavones have received considerable attention in the medical literature in recent years and are being investigated as a potential alternative to hormone therapy42–44. Soy isoflavones has been shown to counteract bone loss in ovariectomized rats45–48 and epidemiological studies indicate a positive relationship between the higher intake of isoflavones

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Figure 2 Typical electrophoretic pattern of sulfated glycosaminoglycans in femur shafts in each group. CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; Or, origin (at the negative pole). No HS and DS were identified in these samples; concentration of CS in GIV was significantly higher (p ⫽ 0.05) compared with all the other groups. See Table 1 for detailed data

and a higher bone mineral density in Asian women26,49–51. Despite that, a number of randomized, clinical trials have provided conflicting results, since the effects of soy isoflavones in counteracting bone loss have been observed in some17–19 but not in other studies20–22,52,53. The biological effects of a soy diet are dependent on many factors, including the diversity of isoflavones in soy, dose, duration of use, individual metabolism and endogenous estrogen level54. Factors possibly influencing the effects of isoflavones on bone health and, thus, the different results in the literature have been attributed to the characteristics of the studied population, study products and design, physical activity, differences in the dietary habits and in the dose applied22. The effects of phytoestrogens on bone have been proposed to be dose-dependent, possibly related to a balance between their interaction with estrogen and its receptors55. Soy isoflavones, which bind to ERα and ERβ, can also exert agonistic or antagonistic estrogenic effects depending on the target tissue and its estrogen receptor status12. In our study, we found that the group treated with 350 mg of isoflavones had the higher trabecular bone volume, while the group treated with 80 mg showed the higher cortical bone width among the isoflavone-treated groups. In addition to the different doses applied, these results could be explained by the fact that cortical and trabecular bone display different bone remodeling rates56 and, thus, could respond differently to such agents in different doses. Furthermore, it was demonstrated that cortical bone and trabecular bone show distinct

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Santos et al. patterns of expression for the two ER subtypes. ERα is predominantly expressed in cortical bone, whereas ERβ shows higher levels of expression in trabecular bone57. As soy isoflavones bind preferentially to ERβ, it is possible that the higher trabecular bone area that was observed in the group treated with the higher dose is due, at least in part, to the higher availability of its receptor in the trabecular bone, compared to cortical bone. It has been reported that in the estrogen deficiency condition there is an increase in cortical bone width in response to trabecular bone loss, which has been associated with an improvement of biomechanical resistances, as a compensative response. These data may explain, at least in part, why the ovariectomized control group (GI) showed the higher cortical bone width compared to all the other groups58. We did not find significant effects of soy isoflavones on the parameters of bone density and biomechanical tests on tibia. However, we cannot exclude the possibility that soy isoflavones act differentially depending on the bone site. In fact, Breitman and colleagues59 found that soy isoflavones did not have effects on biomechanical strength properties at the midshaft femur in ovariectomized rats, but did exert a positive effect at the vertebrae. These findings give rise to the possibility that soy isoflavones may act in a bone site-specific manner, which is also in accordance with the different results that we noticed in the trabecular and cortical bone regions of femurs. Numerous studies have examined the effects of soy isoflavones on bone mass, bone turnover markers and bone mineral density22,26–28. In contrast, few studies have analyzed the effects of these compounds on bone matrix constituents, such as collagen fibers and GAGs. Soy isoflavones have been shown to increase the mRNA level of type I collagen in orchidectomized rats60. Recently, no significant effects of soy extract enriched with 300 mg/kg of genistein were reported on the thickness of collagen fibers in the bones of ovariectomized rats32. On the other hand, the effects of soy isoflavones on GAGs are poorly understood. Soy isoflavones were shown to reduce the quantity of GAGs in a mouse model of mucopolysaccharidosis29, while its synthesis is reduced in osteosarcoma cell lines30. Also, it has recently been reported that topical application of estradiol and genistein (a soy isoflavone) was able to counteract the decrease of hyaluronic acid content in the facial skin of postmenopausal women31. In a recent work of our group, which used the same animal model as this study, soy isoflavones at a dose of 200 mg/body weight, improved bone quality by enhancing the presence of type I mature collagen fibers and the content of total and sulfated GAGs, compared with the control group33. In the present study, the higher level of type I mature collagen fibers in distal femurs was seen in all isoflavone-treated groups and the best result was found in the group treated at the dose of 80 mg/body weight. The higher content of total GAGs was seen in the 80 mg dose-treated group and the higher content of only sulfated GAGs in the 200 mg dosetreated group, while the higher content of chondroitin sulfate

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Soy isoflavone effects on bone tissue in ovariectomized rats was found in the 350 mg dose-treated group. This suggests that different GAGs may respond differently to soy isoflavones in a dose-dependent manner. GAGs are important components of the extracellular matrix in a high variety of different tissues61,62, as these molecules are related to cell recognition, migration, proliferation and differentiation63,64. In addition, these compounds have important roles in the initial process of bone mineralization65,66. It is suggested that the deficiency of, or abnormalities in, these molecules is associated with the development of osteopenia and osteoporosis67. GAGs are also related to the development and the structural consolidation of the collagen fibers in bone matrix68. Furthermore, it has been reported that a marked disorganization in shape and size of the collagen fibers occurs when there is a decrease in proteoglycans with chondroitin sulfate69. In our experiment, soy isoflavones were able to increase the presence of type I mature collagen fibers and the content of GAGs and, thus, may have protective effects on these components in an animal model of established bone loss. Moreover, these compounds showed positive effects on histomorphometric parameters, such as trabecular bone volume and cortical bone width. These positive effects were different for all the

Santos et al. doses applied and in the bone site analyzed; this may be related, at least in part, to the differences in the bone remodeling rate and ER status of the bone site. In conclusion, soy isoflavones improve bone quality in femurs of rats by increasing histomorphometric parameters, the content of GAGs and mature type I collagen fibers. These positive effects are dose-dependent and are different in cortical and trabecular bone.

ACKNOWLEDGEMENT The authors wish to thank Novasoy-ADM Natural Health & Nutrition, Illinois, USA, for kindly providing the soy isoflavones extract. Conflict of interest The authors report no confl ict of interest. The authors alone are responsible for the content and writing of this paper. Source of funding This research was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil.

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