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Soybean concentrated extract counteracts oxidative stress in the uterus of rats C. P. Teixeira, R. S. Simões*, M. A. Santos, M. L. Calió†, J. M. Soares Jr*, M. J. Simões, C. R. A. Bertoncini‡, E. M. S. Higa** and A. F. Carbonel Department of Morphology and Genetics, Federal University of São Paulo, São Paulo; *Department of Obstetrics and Gynecology, University of São Paulo, São Paulo; †Department of Molecular Biology, Federal University of São Paulo, São Paulo; ‡Center for Development of Experimental Models for Medicine and Biology, Federal University of São Paulo, São Paulo; **Department of Nephrology, Federal University of São Paulo, São Paulo, Brazil

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Key words: OXIDATIVE STRESS, ANTIOXIDANTS, UTERUS, ISOFLAVONES, ESTROGEN, LIPID OXIDATION

ABSTRACT Objective To evaluate the effects of soy isoflavone extract in the pro-oxidant/antioxidant balance in the uterus of ovariectomized rats. Methods Twenty 3-month-old adult female Wistar rats were divided into four equal groups: GI: shamoperated (estrous phase); GII: control ovariectomized rats; GIII: ovariectomized rats treated with genistein (50 μg/kg/day) by gavage; GIV: ovariectomized rats subcutaneously treated with estrogen (10 μg/kg/day). After 30 consecutive days of treatment, the rats were euthanized and the uterus removed. The distal thirds of the uterine horns were processed for histomorphometric analyses of endometrial and myometrial thicknesses and glandular area. Other regions of the uteri were kept in liquid nitrogen and subsequently processed for analysis of reactive species quantification (DCF), total antioxidant capacity (TAC) and lipid oxidation status (TBARS). Data were statistically analyzed by one-way ANOVA, complemented by the Tukey–Kramer test (p  0.05). Results GII and GIII exhibited lower endometrial thickness, glandular area and myometrial thickness than GI and GIV, while a higher myometrial thickness was observed in GIV compared with the other groups. Moreover, the isoflavone-treated group showed lower DCF and TBARS compared to GII, and also an improvement of TAC compared to GI and GIV. Despite the significant decrease in TBARS, no significant difference in DCF nor a decrease in TAC were observed in GIV when compared to GII. Conclusion Our data show that isoflavones improve antioxidant status and counteract oxidative stress, without promoting a trophic effect in the uterus of rats.

INTRODUCTION Reactive species (RS), which comprise the reactive oxygen species (ROS) and the reactive nitrogen species (RNS), are continuously generated by metabolic processes and external factors1. Physiological levels of RS are necessary for cells to live, since they are involved in the regulation of cell growth, intercellular and intracellular signaling pathways, immunological defense and in the synthesis of important substances2–4. On the other hand, antioxidant defense should also be present, since the perfect pro-oxidant/antioxidant balance

is important for maintenance of cell integrity and function. An imbalance in this process could promote an increase in oxidative stress, which favors the occurrence of lesions in cell membrane lipids, proteins, DNA, RNA and other tissue components that undergo oxidation by the action of RS5,6. Estrogen has been shown to have antioxidant effects, due to its ability to scavenge free radicals, as well as by an indirect upregulation of antioxidant enzymes and chelation of redoxactive metal ions. These properties of estrogen prevent the deleterious effects of oxidative stress and protect against cellular degeneration7,8.

Correspondence: Dr A. Ferraz Carbonel, Federal Universty of São Paulo, Morphology and Genetics, Street Botucat 940, São Paulo, 04023900 Brazil; E-mail: [email protected] ORIGINAL ARTICLE © 2014 International Menopause Society DOI: 10.3109/13697137.2013.856402

Received 19-09-2013 Accepted 14-10-2013

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Soybean concentrated extract counteracts oxidative stress During menopause, when the levels of estrogen decrease, there is an increase in RS generation and a decrease in antioxidant activity, which enhance oxidative damage, contributing significantly to the emergence of cardiovascular and neurodegenerative diseases, genitourinary system disorders, bone loss, as well as carcinogenic and others diseases related to the aging process9–11. Hormone replacement therapy (HRT) with estrogen could provide many benefits for women in the menopausal transition. However, in studies estrogen was related to the induction of cell proliferation in the uterus and mammary gland, increasing the risk of cancer development in these organs, at least after prolonged treatment12,13. These adverse effects, such as thromboembolism and even stroke, were reported in other studies14,15. Non-steroidal, estrogen-like plant compounds called phytoestrogens, particularly the soy isoflavones, are presently being investigated as alternatives to HRT for the prevention and treatment of menopausal symptoms16. The antioxidant activity of the isoflavone aglycone and structurally related compounds was suggested as only having the weak ability to scavenge free radicals; nevertheless, they have the ability to reduce DNA susceptibility to oxidative stress and boost the activity and expression of antioxidant enzymes17. Despite several studies to test the efficacy of soy food and its products, the results are still controversial, and most were probably produced by variables throughout the studies18. Thus, the aim of this research was to investigate the hypothesis that soy isoflavone extract improves antioxidant defense and avoids oxidative stress in the absence of estrogen, without producing cell proliferation in the uterus of rats19.

MATERIALS AND METHODS Animals and experimental design Twenty female, 3-month-old virgin Wistar rats (Rattus norvegicus albinus), average weight   231 g, were housed in 45  30  15 cm3 cages (five rats per cage) under a 12/12 h light/dark cycle at 22°C, with diet and water ad libitum. The procedures were approved by the ethics committee of the Federal University of São Paulo (0023/12) and all the residues generated in this study were discarded in accordance with guidelines from the Federal University of Sao Paulo, Brazil. The soy isoflavone extract (Novasoy, Archer Daniels Midland, Decatur, IL, USA) used in this study contains 40% of total isoflavones in the ratio of 1.3 : 1 : 0.3 of genistein: daidzein : glycitein, 7–12% protein, 4% ash, and 6% moisture, and the remaining 41% was comprised of other natural soy phytocomponents. The estrogen used was diethylstilbestrol (Sigma-Aldrich Chemicals, Oakville, ON, Canada). After a period of 7 days of adaptation to the new environment, the rats were anesthetized with ketamine–xylazine (ketamine HCl 75 mg/kg body weight; xylazine 10 mg/kg body weight) and subjected to bilateral ovariectomy or shamoperated (the uterine horns and the ovaries were exteriorized

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Teixeira et al. and then reintroduced intact into the abdominal cavity). The animals were then divided into four equal groups: GI: shamoperated; GII: ovariectomized rats that received vehicle solution (propylene glycol); GIII: ovariectomized rats treated with 50 μg genistein/g body weight/day; GIV: ovariectomized rats treated with 10 μg/kg/day diethylstilbestrol. Diethylstilbestrol was dissolved in 0.1 ml corn oil and was administered via subcutaneous injection behind the neck cuff. Soy isoflavone extract was dissolved in 0.1 ml propylene glycol and was administered by gavage. The treatments started immediately after ovariectomy and lasted for 30 consecutive days. Body weights were recorded weekly. After treatments, the rats were decapitated to collect the blood samples in the estrous phase in GI. The uterine fragments were removed for biochemical assays and some uterine horns were separated and processed for paraffin embedding.

Estradiol assay Blood samples (1 ml) were collected in serum tubes (BD Vacutainer®), centrifuged at 3000 rpm during 20 min at 4°C and serum was immediately drawn off and stored at 20°C until the assay. Serum estrogen determination was carried out by electrochemiluminescence immunoassays and the measurements were conducted using an automatic counter Cobas e601 (Roche Diagnostic Co, USA), which was calibrated automatically. The assay sensitivity was 5 pg/ml, and the intra-assay coefficient of variation was 3.3%. Values below the detection limit are reported as  5 pg/ml.

Uterine sample processing and histomorphometric analysis The second third of the uterine horn was separated and fixed in phosphate-buffered formaldehyde solution for 12 h. The samples were then dehydrated in graded concentrations of ethanol, cleared in xylene and embedded in paraffin according to standard techniques. Sections (4 μm) were cut in a Minot-type microtome (Leica, Model RM 2145) and then stained with Masson’s trichrome for histomorphometric analysis, which was performed using an image capturing system and then analyzed in the software Axiovision 4.8 REL (Carl Zeiss). Three sections were obtained for each animal, at a minimum interval of 80 μm between each section. Measurements were carried out under the objective lenses at 2.5  for analysis of endometrium and myometrial thickness and at 4  for the glandular area assessment. The myometrial thickness was measured from the inner margin of the circular muscle layer to the outer margin of the longitudinal muscle layer, while the endometrial thickness was measured from the uterus lumen to the upper margin of the myometrium. Measurements were obtained in eight different regions for each layer. From the eight regions measured, an average was obtained for each parameter analyzed, expressed in μm. For the glandular area analysis, all spaces occupied by the

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Soybean concentrated extract counteracts oxidative stress uterine glands in the endometrium were delimited and then a summation of the areas was obtained, expressed in mm2.

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Preparation of homogenates The remaining uterus fragments were dipped in liquid nitrogen and kept at 70°C. To perform the biochemical assays, the uterine fragments were weighed and then immersed in a solution of phosphate buffer (0.1 mol/l, pH 7.4) at a ratio of 0.5 ml buffer per 100 mg tissue, where the samples were ground with the aid of Polytron PT1200CL® (Kinematica). A homogenate of each uterus sample was obtained and centrifuged at 3000 g for 15 min at 4°C; the supernatant was separated for measurements of lipid oxidation, total antioxidant capacity and reactive species. The tissue protein concentration was measured using the Quick Start Bradford Protein Assay (Bio-Rad Laboratories Inc, USA). Dosages were made according to the datasheet instructions and bovine serum albumin (BSA) was used as standard.

Formation of 2,7-dichlorofluorescein assay The quantification of reactive species is determined by the direct fluorescent method, using the Cellular Reactive Oxygen Species Detection Assay Kit (Abcam Biochemical, USA). After passing through the plasma membrane, the non-fluorescent substance 2,7-dichlorodihydrofluorescein diacetate is oxidized by the reactive species present in the sample to the fluorescent compound 2,7-dichlorofluorescein (DCF). The formation of DCF was detected at wavelengths of excitation and emission of 485 and 538 nm, respectively, using the fluorescence spectrophotometer 3.0 Flex Station (Molecular Devices). Samples were assayed in duplicate and a positive control (tert-butyl hydroperoxide) was used. The results were expressed as μmol DCF produced/mg protein.

Total antioxidant capacity assay We used the QuantiChromTM Antioxidant Assay Kit (BioAssay Systems, USA) which is capable of detecting three types of antioxidant species: the enzyme system (superoxide dismutase, catalase, glutathione peroxidase and reductase), macromolecules (albumin, ceruloplasmin, ferritin and transferrin) and an array of small molecules (ascorbic acid, α-tocopherol, β-carotene, glutathione, uric acid and bilirubin). This assay, the total antioxidant capacity assay (TAC), is based on the ability of antioxidants to reduce Cu2 to Cu through the electron transfer mechanism. After reduction, the ion Cu reacts with a chromogenic reagent that produces a color reaction detected by spectrophotometry at 570 nm. A standard curve was prepared using Trolox 1 mmol/l as standard. The standard and each sample were assayed in duplicate. The results were expressed as mmol/l Trolox equivalent/mg protein.

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Lipid oxidation status Oxidative stress can be assessed by quantification of byproducts released as a result of lipid oxidation, through the method of thiobarbituric acid-reactive substances (TBARS). This assay is based on the ability of aldehydes and other compounds, especially malondialdehyde (MDA), an end-product of lipid oxidation, to form a red-colored complex with thiobarbituric acid (Sigma-Aldrich Chemicals, CA, USA), detectable by spectrophotometry at 532 nm. Results were expressed as nmol of MDA formed/mg protein.

Statistical analysis Results were analyzed using Prism 5.0 software (GraphPad, San Diego, CA, USA). The values were expressed as mean   standard deviation. The groups were compared using one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test. A p value  0.05 was considered statistically significant.

RESULTS Estradiol assay The estradiol serum levels in GIV were significantly higher when compared with the sham group (GIV: 281.5   9.9 pg/ml  GI: 76.7  6.9 pg/ml). Estradiol levels in GII and GIII  were significantly lower and were undetectable by the method used ( 5 pg/ml), which indicates the success of the bilateral ovariectomy.

Histomorphometry of uterus The endometrial thickness and the glandular area were higher in GI and GIV compared with GII and GIII (GI  GIV  GII  GIII, p  0.05), while the myometrial thickness was higher in GIV compared to the other groups and also in GI than in GII and GIII (GIV  GI  GII  GIII, p  0.05) (Table 1 and Figure 1).

Biochemical assays As seen in Figure 2, levels of reactive species (DCF) were increased in GII and GIV when compared with GI and GIII (GII  GIV  GI  GIII, p  0.05). We observed a greater increase in the TAC in GII and GIII than in GI and GIV (GII  GIII  GI  GIV, p  0.05). In addition, GII exhibited a significant increase in the lipid oxidation status (TBARS) when compared to the other groups (GII  GI  GIII  GIV, p  0.05).

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Table 1 Endometrial thickness, myometrial thickness and glandular area in the different groups. GI, sham-operated rats; GII, ovariectomized (OVX) rats; GIII, OVX rats treated with soy isoflavones concentration; GIV, OVX rats treated with estrogen. Asterisks indicate statistical differences (p  0.05): endometrial thickness: *GI and GIV  GII and GIII; myometrial thickness: **GIV  *GI  GII and GIII; glandular area: *GI and GIV  GII and GIII

Endometrial thickness (μm) Myometrial thickness (μm) Glandular area (mm2)

GI

GII

GIII

GIV

* 654.1   96.3 * 281.1  30.8  * 72.5   28.4

400.7   33.8 171.1   24.8 26.6   3.8

380.3   21.4 201.8   12.2 24.9   6.8

* 584.7   63.9 ** 323.8  20.7  * 69.4   23.0

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DISCUSSION In this study, we aimed to investigate whether treatment with soy isoflavones would exert antioxidant effects in the uterus of ovariectomized rats, which is a much used animal model to study postmenopausal disorders19, and whether these compounds exert trophic effects in the uteri of rats20. Soy isoflavones have been receiving increased clinical interest due to their related beneficial effects on menopausal symptoms in several organs such as the breast, uterus and bone, as well as on the cardiovascular system and cognitive functions21. It has been reported that soy isoflavones act as selective estrogen receptor modulators due to their preferential binding to the estrogen receptor β22. Because of their structural similarities with the endogenous estrogens and

their mode of action, these compounds have the ability to bind to estrogen receptors α and β and to exert the same beneficial effects as estrogen therapy on menopausal symptoms, with the advantage of not showing the undesirable side-effects observed in classical estrogen replacement therapy22,23 such as thromboembolism, endometrial and breast cancers24,25. Soy isoflavones have been reported by some authors to have uterotropic activity, but not in other studies, and, thus, this has become a subject of increasing debate. Bahr and colleagues18 revealed that there was no significant difference in uterine wet weights or histomorphometry between control ovariectomized rats and ovariectomized groups treated with soy protein or isoflavone extract18. On the other hand, Salleh and colleagues reported that a high genistein intake could

Figure 1 Photomicrographs of uterine sections from the different groups: GI: sham-operated rats; GII: ovariectomized (OVX) rats; GIII: OVX rats treated with soy isofl avone extract; GIV: OVX rats treated with estrogen. Asterisks indicate statistically significant differences (p  0.05). *, Note that endometrium (E) and myometrium (M) in GI and GIV are clearly higher when compared with GII and GIII; **, GIV showed an increase in myometrial thickness compared to the other groups. Sections were stained with Masson’s trichrome; magnification  40

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Figure 2 The graphs show the data from the biochemical assays on the uterus of rats in the groups. GI: sham-operated rats; GII: ovariectomized (OVX) rats; GIII: OVX rats treated with soy isoflavone extract; GIV: OVX rats treated with estrogen. (a) Reactive species quantification (DCF); (b) total antioxidant capacity (TAC); (c) lipid oxidation status (TBARS). Data are shown as mean  standard deviation. Asterisks indicate statistically significant differences (p  0.05); DCF: *GII and GIV  GI and GIII; TAC: *GII and GIII  GI and GIV; TBARS: *GII  GI, GIII and GIV. Note that DCF in GII and GIV were significantly higher than in GI and GIII, while TAC was statistically higher in GII and GIII; and TBARS was significantly higher only in GII

potentially induce hyperplastic and hypertrophic changes in the uterus of rats26. In humans, a pilot study demonstrated no effect of phytoestrogen supplementation during a 6-month period on endometrial histology27, whereas a large 5-year study with phytoestrogen supplementation found increased rates of endometrial hyperplasia26. Recently, Quaas and colleagues reported that a 3-year isoflavone soy protein supplementation had no effect on endometrial thickness or on the rates of endometrial hyperplasia and cancer in postmenopausal women28. Soy extract was administered in this study as the source of isoflavones. The dose was determined based on a previous study where, using the same extract in rats, the dose–effect curve regarding the colpocytologic examination and weight of the uterus was determined. The doses selected were based on the following parameters: the maximum dose that did not change the weight of the uterus nor the results of the colpocytologic examination (50 μg genistein/g body weight/day)29. In our study, we noted that treatment with soy isoflavone extract did not cause any significant histological changes in the endometrial thickness, myometrial thickness or glandular

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area compared to control ovariectomized rats. All these parameters, as well as the myometrial thickness, were higher in the estrogen-treated group, suggesting uterotropic effects of the estrogen treatment, which is in agreement with previous findings30,31, but not higher than with isoflavone administration. It has been suggested that the different populations analyzed, the animal model used, the differences in the dose and route of administration, as well as the duration of treatment and form of soy supplements (soy protein, isoflavone extract or individual isoflavones like genistein and daidzein) may be the causes of disparity observed in the literature data18. The pro-oxidant/antioxidant balance and detoxification of potentially damaging ROS are crucial for cellular homeostasis32,33. The modulation of antioxidant activities after menopause could be important to prevent the oxidative status and pathologies associated with menopause-related oxidative stress34,35. Chen and Kotani even suggested in their research that the use of antioxidant supplements should maintain a high antioxidative potential and may reduce menopausal symptoms in women during the menopausal transition36.

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Soybean concentrated extract counteracts oxidative stress It is known that estrogen exerts antioxidant effects in different organs. Indeed, studies led by Chen and Kotani showed that the severity of menopausal symptoms was inversely linked to serum levels of antioxidant potential in women at menopause, leading to the suspicion that decreased levels of female sex hormones could cause an oxidative–antioxidative imbalance36. In prior research, free radical-induced lipid oxidation has been proposed as an etiological factor in aging after menopause and in various age-related diseases, including atherosclerosis, neurodegenerative diseases and cancer37,38. Significant decreases in oxidative stress and significant increases in antioxidant status were observed after treatment with estradiol in the brain34 and myocardium39 of ovariectomized rats, exerting protective effects in these organs. In this study, we observed that DCF and TBARS were higher in the ovariectomized control group, compared to the sham group, probably due to the loss of estrogen. On the other hand, the TAC was also higher in the ovariectomized control group, which could be explained by the excessive activation of the natural antioxidant system, suggesting a possible adaptive response due to the increased production of RS40. However, this compensatory response was not enough to counteract oxidative stress since TBARS was increased in the ovariectomized group compared to the other groups41. In the estrogen-treated group, we observed a decrease in the oxidative damage to lipids as the TBARS was revealed to decrease, despite TAC not being significantly increased and DCF not significantly diminished. Escalante-Gómez and Quesada concluded in their research that HRT with conjugated equine estrogen decreases oxidative damage to both DNA and lipids in the serum of postmenopausal women, inasmuch as no statistical difference was found for total antioxidant status compared to the group of women who did not receive HT42. Mobley and Bruegggemeler even showed that treatment of normal human breast epithelial cells in culture with estradiol presented a decrease in the cellular catalase activity, an important antioxidant enzyme43. Pajovic and Saicic related that antioxidant enzyme activity is dependent upon the concentration of progesterone and estrogen and, after a certain time period, bilateral ovariectomy results in a significant increase in catalase activity, while hormonal treatments have no effect on the activity of this enzyme44. Therapy with estrogen plus low-dose progesterone was associated with decreased TBARS levels and oxidative status, as well as increased TAC levels in the uterus of OVX rats, and may prevent pathologies caused by RS44. These studies are in accordance with our findings, since the estrogen treatment exerted antioxidant effects in the uterus and reduced oxidative damage. Indeed, it is generally believed that many of the beneficial effects of isoflavones might be related, at least in part, to their

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Teixeira et al. antioxidant activity45. For instance, Erba and colleagues showed a positive effect of soy isoflavone supplementation on oxidative stress in women, thus suggesting that the healthy benefits ascribed to soy consumption may be partially related to the antioxidant potential of soy isoflavones, as demonstrated by the decrease in DNA oxidative damage after isoflavone supplementation17. Moreover, Azadbakht and colleagues46 reported that soy consumption reduces plasma TBARS and increases plasma TAC levels in postmenopausal women with metabolic syndrome47. On other hand, some studies have demonstrated little or no effect of soya-derived isoflavones on the biomarkers of oxidative stress46. In this case, some authors have attributed the absence of isoflavone effects to the low quality of the soy derivatives used in the studies48. Although many studies have reported the effects of isoflavones on oxidative stress-related diseases, there are not much data on the protective effects of isoflavones in the uterus or reproductive organs, interestingly as was previously observed with estrogen supplementation49. We observed in our experiments that soy isoflavones exerted antioxidant effects in the uterus. The treatment with soy isoflavone extract in ovariectomized rats revealed an increase in TAC followed by a decrease in DCF, leading to a decrease in the oxidative stress status (Figure 2). In addition, the lower levels of TBARS (Figure 2) strongly suggest an antioxidant effect of the soy isoflavones. In conclusion, the present study demonstrates that treatment with soy isoflavone extract in ovariectomized rats improves antioxidant capacity and decreases oxidative stress, without producing histological changes and a tropic effect in the uterus of rats. Therefore, isoflavones could be useful to improve the antioxidant/pro-oxidant balance in the uterus, thus preventing the deleterious damages caused by the inefficiency of antioxidant defenses against oxygen and nitrogen reactive species in the absence of estrogen in menopause.

ACKNOWLEDGEMENTS We would like to express our gratitude to postgraduate students Gisela Rodrigues da Silva Sasso and Rinaldo Florencio da Silva, Department of Morphology and Genetics, Federal University of São Paulo. 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 The authors are grateful for the fi nancial support provided by FAPESP 2011/1898-0 and 2011/11900-0.

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Soybean concentrated extract counteracts oxidative stress in the uterus of rats.

To evaluate the effects of soy isoflavone extract in the pro-oxidant/antioxidant balance in the uterus of ovariectomized rats...
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