Ulisvaldo Brunno de Oliveira Macedo, Rand Randall Martins, Francisco Paulo Freire Neto, Yonara Monique da Costa Oliveira, Aldo da Cunha Medeiros, José Brandão-Neto, Adriana Augusto de Rezende, and Maria das Graças Almeida
Abstract:Oxidative stress is associated with postmenopause and is also responsible for various metabolic alterations. The redox imbalance observed during ovarian decline can be induced experimentally by bilateral ovariectomy in rats. In addition to hormone replacement, regular moderate physical exercise is indicated to prevent several common postmeno- pausal diseases. This study aimed to assess the effect of daily swimming on the antioxidant defense system of oophorec- tomized Wistar rats. Control and oophorectomized groups were submitted to 1 h of daily swimming for 90 days. Levels of lipid peroxidation and glutathione content and the activities of superoxide dismutase enzyme and glutathione peroxidase in erythrocytes, liver, and brain were assessed every 30 days. The control group exhibited lower lipoperoxidation that was associated with a significant increase in superoxide dismutase enzyme activity, glutathione peroxidase activity, and glutathione content in erythrocytes and liver; however, swimming did not cause changes in antioxidant parameters in the brain over time. The oophorectomized group showed no antioxidant adaptation to daily swimming and had greater oxidative damage in the liver and blood. Our results suggest that ovariectomy hinders antioxidant adaptation in Wistar rats submitted to daily swimming.
Key words: oophorectomy, menopause, oxidative stress, physical exercise.
Résumé :Le stress oxydatif est associée a` la post-ménopause et est également responsable de diverses altérations métaboliques. Le déséquilibre redox observé pendant le déclin ovarien peut être obtenu expérimentalement grâce a` une ovariectomie bilaté- rale chez des rattes. En plus de l'apport hormonal, l'exercice physique régulier et modéré est conseillé dans la prévention de différentes maladies communes lors de la post-ménopause. Cette étude visait a` évaluer l'effet de la natation tous les jours sur le système de défense antioxydant de rats Wistar ovariectomisés. Les groupes de contrôle et de rats ovariectomisés ont été soumis a` 1 h de natation quotidienne pendant 90 jours. Niveaux de la peroxydation lipidique et le GSH et les activités de la SOD et la GPx dans les globules rouges, le foie et le cerveau ont été évalués tous les 30 jours. Les animaux contrôles ont présenté une moindre lipoperoxydation associée a` une augmentation significative de la concentration de GSH et de l'activité de la SOD et GPX dans les érythrocytes et le foie. Mais la période de natation n'a pas provoqué de changements quant aux paramètres antioxydants du cerveau. Les animaux oophorectomisés n'ont pas montré d'adaptation antioxydante lors de la natation quotidienne, et a eu plus de dégâts oxydatifs dans le foie et le sang. Nos résultats suggèrent que l'ovariectomie entrave l'adaptation antioxydant chez les rats Wistar soumis a` la natation tous les jours.
Mots-clés : ovariectomie, la ménopause, le stress oxydatif, l'exercice physique.
Introduction
Reactive oxygen species (ROS) are products of aerobic metabolism and are important in signaling, apoptosis, and phagocytosis (Radak
et al. 2008). Increased ROS production is involved in the physiopa-
thology of several chronic degenerative diseases such as Alzheimer
(Ansari and Scheff 2010), hypertension (Campese 2010), diabetes mel-
litus (Pacal et al. 2011), and osteoporosis (Baek et al. 2010) and is associated with postmenopause (Behr et al. 2011). Redox imbalance, known as oxidative stress (OS), participates in disease physiopathol- ogy because it causes significant alterations in functionally impor- tant biomolecules (Halliwell 2007).
The increase in ROS production in postmenopausal women sug- gests that the decrease in sex hormones causes a predisposition to the condition of oxidative stress (Signorelli et al. 2006;Unfer et al. 2006). One of the mechanisms that explain the antioxidant action
of estrogens is related to the connections to their cellular surface receptors, which promote an increase in antioxidant enzyme ac- tion (Mann et al. 2007). Another mechanism is the direct action of the estrogen molecule in the elimination of ROS (Prokai-Tatrai
et al. 2008). During this period of life, regular physical exercise can
be used as an adjuvant in the treatment and prevention of chronic degenerative diseases (Agil et al. 2010).
Paradoxically, physical activity increases the production of ROS due to the increased volume of O2inhaled, alterations in intracellu- lar Ca++homeostasis, vasomotor variations, and ischemia–reperfu-
sion (Jackson 2000;Radak et al. 2008). In healthy individuals, ROS generated in physical exercise signals an adaptive response of the antioxidant system. This response improves its protective capacity, thereby explaining some of the positive outcomes of physical exer- cise (Gomez-Cabrera et al. 2008;Karolkiewicz et al. 2009).
Received 27 March 2012. Accepted 31 July 2012.
U.B.O. Macedo, F.P. Freire Neto, and Y.M.C. Oliveira.Postgraduate Pharmaceutical Sciences Program, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil.
R.R. Martins.Education and Health Center, Federal University of Campina Grande (UFCG), Cuité, Brazil.
A.C. Medeiros.Department of Surgery, UFRN, Natal, Brazil.
J. Brandão-Neto.Department of Clinical Medicine, UFRN, Natal, Brazil.
A.A. Rezende and M.G. Almeida.*Department of Clinical and Toxicological Analysis, UFRN, Natal, Brazil.
Corresponding author:Maria das Graças Almeida (e-mail:[email protected]).
*Present address: Laboratório Multidisciplinar, Centro de Ciências da Saúde, UFRN, Rua Gal. Gustavo Cordeiro de Farias, s/n, Petrópolis, CEP: 59012-570 - Natal, RN, Brazil. 148
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Models that involve oxidative stress, physical exercises, and hypoestrogenism in animals show similarity to the results obtained in humans (Vina et al. 2000). Biomarkers such as thiobarbituric acid reactive substances (TBARS), glutathione (GSH) content, and superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPx; EC 1.6.4.2) enzyme activities are commonly used to evaluate alterations of the antioxidant system in humans and the animal model exercised (Jammes et al. 2004;
Kaczor et al. 2007).
In light of the benefits of regular physical exercise (Gomez-Cabrera
et al. 2008) and the participation of estrogen in antioxidant pro-
tection, the present study assessed the influence of oophorectomy on antioxidant response in regularly exercised Wistar rats.
Materials and methods
Animals
Forty female 60-day-old Wistar rats (215 ± 35 g) were fed standard rations (Purina, São Paulo-SP, Brazil) with the following characteris- tics: maximum humidity, 13%; minimum protein, 23%; ether extract; fiber materials (maximum), 8%; mineral material (maximum), 10%; calcium (maximum), 1.5%; phosphorus (minimum), 0.8%; and water provided ad libitum during the study. Animals were housed at a con- stant temperature (23 ± 2 °C) and were maintained on a 12 h light − 12 h dark cycle.
Study groups
Rats were separated into two groups of 20 animals each: a control group (CG), consisting of sham-operated animals, and an oophorectomized group (OG). Each group was subdivided into four subgroups (0, 30, 60, and 90 days of exercise) contain-
ing five animals each. The experimental protocol was conducted according to Brazilian College of Animal Experimentation (COBEA) guidelines, and the project was approved by the Re- search Ethics Committee of Onofre Lopes University Hospital (CEP-HUOL).
Physical exercise protocol
In the morning at 0900 hours, the animals were given daily 1 h swimming sessions in a tank (160 cm long, 80 cm wide, and 50 cm deep) containing water at a controlled temperature (27 to 29 °C). No more than 20 animals were exercised in the tank simultane- ously. During the first week, exercise time was gradually in- creased by 10 min/day up to a maximum of 60 min. During this period of adaptation, the performance differences in training were considered normal given the biological individuality of the animals . Five animals in each group were sacrificed at the end of 0, 30, 60, and 90 days of the exercise protocol. The animals were sacrificed 30 min after exercise.
Ovariectomy and sham procedure
Oophorectomies were carried out via flank incisions under so- dium pentobarbital anesthesia (60 mg/kg, IP). The skin on both sides of the body was shaved from the hip to the lowest rib. Bilat- eral oophorectomies were performed using an incision 1.5 cm below the palpated rib cage. Ovaries and surrounding fat tissue were removed, and the incision was closed by suturing the mus- cles and skin. Animals were submitted to the exercise protocol 50 days after surgery. The sham-operated control group (CG) un- derwent the same surgical procedure, except that the ovaries were not removed.
Fig. 1. Influence of physical exercise on antioxidant biomarkers (GSH, SOD, and GPx) and lipid peroxidation (TBARS) on the blood of CG () and OG (Œ). Significant differences: *, among groups (ANOVA followed by the Tukey–Kramer test, p < 0.05); #, correlation between time (days) and parameter evaluated (Pearson correlation test, p < 0.05).
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Samples
After euthanasia by cervical dislocation, the brain and liver were removed, and 5 mL of blood was collected by cardiac punc- ture. Liver and brain samples were homogenized in phosphate buffer (pH 7.0) at 4 °C with a Potter homogenizer MA-099 (Piracicaba-SP, Brazil) for analysis.
Lipid peroxidation
Lipid peroxidation in tissues was determined by measuring the concentration of TBARS (Yagi 1982), which was assessed colori- metrically (Shimadzu UV-1650 PC, Kyoto, Japan) using the ab- sorbance at 532 nm after the reaction of lipoperoxidation subproducts with thiobarbituric acid. Results are expressed in mol/L.
Antioxidant biomarkers
Glutathione (GSH) content was analyzed using the technique described byBeutler et al. (1963). GPx and SOD activities were determined according to Sies et al. (1979) and McCord and
Fridovich (1969), respectively. GSH, GPx, and SOD were assessed
colorimetrically (Shimadzu UV-1650 PC). In blood, results were expressed in mmol/L, U/mg hemoglobin, and U/mg hemoglobin, respectively. In liver and brain, the results were expressed in mmol/L, U/mg protein, and U/mg protein, respectively.
Statistics
Results are expressed as the mean ± SEM. Intergroup differ- ences were analyzed using two-way ANOVA followed by the
Student–Newman–Keuls multiple comparisons test. Correla- tion analysis was performed by determining Pearson's coeffi- cient. Differences were considered significant at p < 0.05.
Results
Blood
There was a positive correlation between antioxidant bio- marker concentrations (GSH, SOD, and GPx) and swimming time in the CG (Figs. 1A,1B, and1C), which is characteristic of the antioxidant adaptation process. The OG exhibited no cor- relation between any of the parameters and swimming time.
Intergroup comparison within the same period showed that OG animals obtained relatively high TBARS concentrations at 30 and 60 days of swimming, whereas GSH content was comparatively lower at the start and end of the experimental period (Figs. 1Aand 1D). SOD and GPx enzyme activity in the CG were comparatively higher during the entire swimming period (Figs. 1Band1C).
Liver
Positive correlations were observed between SOD and GPX en- zyme activities and swimming time (Figs. 2Band2C) in the liver homogenate of the CG, indicating an adaptive process. However, this group exhibited a negative correlation with respect to GSH content (Fig. 2A). The oophorectomized group showed no antiox- idant adaptation.
An elevated concentration of TBARS was recorded in OG when compared with the CG at 30, 60, and 90 days of exercise
(Fig. 2D). GSH content and SOD and GPx activities were signif-
Fig. 2. Influence of physical exercise on antioxidant biomarkers (GSH, SOD, and GPx) and lipid peroxidation (TBARS) on the liver of CG () and OG (Œ). Significant differences: *, between groups (ANOVA followed by the Tukey–Kramer test, p < 0.05); #, correlation between time (days) and biological parameters evaluated (Pearson correlation test, p < 0.05).
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icantly lower in the OG compared with the CG in at least three periods (Figs. 2A,2B, and2C).
Brain
The brain showed no correlation between the parameters assessed and swimming time (Figs. 3A,3B,3C, and3D), indicating a lack of adaptation. However, intergroup comparisons demonstrate that SOD activity was significantly lower in the OG when compared with the CG (Fig. 3B) for all time periods. Additionally, GPx activity at 60 and 90 days was significantly lower in the OG (Fig. 3C).
Discussion
Animals submitted to swimming improved their antioxidant defense system in blood and liver cells but not in the brain. How- ever, the oophorectomized animals do not show the adaptive ca- pacity of the antioxidant defenses in the tissues studied.
Blood is one of the tissues most used for the evaluation of the biomarkers of antioxidant stress, as these reflect the antioxidant activity of the skeletal and cardiac muscles (Veskoukis et al. 2009). The erythrocyte cells exhibit a number of physiological and bio- chemical peculiarities, particularly in relation to redox metabo- lism. Due to high O2 pressure and iron content in heme,
erythrocytes are continuously submitted to high levels of ROS and require their own antioxidant system (Cimen 2008). However, the antioxidant efficiency of erythrocytes decreases in senescent cells
(Petibois and Deleris 2005).
In the CG, the adaptation observed in antioxidant enzymes and GSH content could be attributed to erythrocyte turnover. Chemi-
cal stress caused by regular physical exercise generally stimulates premature removal of senescent erythrocytes and increases new cell synthesis via erythropoietin production (Petibois and Deleris 2005). Thus, the growth in the population of young erythrocytes with greater antioxidant potential is likely responsible for the improved antioxidant defense in the CG. In contrast to the CG, the OG showed an absence of antioxidant adaptation. Low estrogen concentration decreases antioxidant protection, which could ex- plain the diminished antioxidant adaptation observed in OG and the greater lipoperoxidation in this group. According toUlas and
Cay (2011), estrogens could have a beneficial effect on the blood
antioxidant defense system by stimulating antioxidant enzyme activity.
The absence of adaption observed in the OG is consistent with the literature, which points to the influence of estrogen as a me- diator in the activity of antioxidant enzymes.Massafra et al. (1998) showed that hormone reposition in women with amenorrhea re- stored the erythrocytes antioxidant activity by raising the GPx activity, thus improving the antioxidant potential of these pa- tients. Other studies report the reduction of SOD erythrocyte ac- tivity in postmenopausal women (Krstevska et al. 2001).
In the present study, besides the lower antioxidant activity at the end of the period, the oophorectomized animals also showed lower sensitivity to the adaptive process promoted by exercise. Oophorectomized rats had higher levels of nuclear factor B (NF-B) receptors and other inflammatory regulators, including VCAM-1, TNF, and RANTES (Evans et al. 2001). In hepatocytes, estrogen inhibits activation of the NF-B pathway, attenuating oxidative
Fig. 3. Influence of physical exercise on antioxidant biomarkers (GSH, GPx, and SOD) and lipid peroxidation (TBARS) on the brain of CG () and OG (Œ). Significant differences: *, between groups (ANOVA followed by the Tukey–Kramer test, p < 0.05); #, correlation between time (days) and biological parameters evaluated (Pearson correlation test, p < 0.05).
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bursts and lactate dehydrogenase levels during exercise (Straub 2007). The findings indicate that the low levels of estrogens may slow antioxidant adaptation in OG animals.
Xanthine oxidase activation and the ischemia–reperfusion pro- cess during physical exercise are responsible for ROS production in the liver (Radak et al. 2008). CG showed antioxidant adaptation that was promoted by swimming, suggesting a role of ROS in improving the antioxidant system. ROS act in important intracel- lular signaling pathways that are sensitive to oxidative stress such as NF-B and mitogen-activated protein kinase (MAPK) pathways. These pathways are capable of promoting the expression of anti- oxidant enzyme-related genes such as SOD and GPx and thereby maintain intracellular redox equilibrium (Ji et al. 2004). Although GSH in the CG decreased with swimming time, this observation may be attributed to the rise in GPx activity, which uses GSH as a cofactor.
As was observed in blood tissue, the liver in the OG exhibited no antioxidant adaptation. According toOmoya et al. (2001), estrogen has a hepatoprotective role given that it interferes in the NF-B signaling pathway in a manner that favors antioxidant enzyme activity. Our results show diminished antioxidant adaptive capac- ity in oophorectomized animals. The lower lipid peroxidation in the CG was likely due to the higher antioxidant activities in that group. These results are in accordance with studies that demon- strate a hepatoprotective effect of estrogens, as estrogens have a high capacity to combat ROS and thereby avoid tissue necrosis and apoptosis (Omoya et al. 2001;Liang et al. 2011).
The brain exhibits a metabolic demand of 240 kcal/kg body weight per day and receives a relatively constant flow of oxygen during physical exercise (Radak et al. 2008). No antioxidant adap- tation was recorded in the brain in any of the groups studied. The constant flow of oxygen, even during daily physical exercise, may not have caused oxidative damage capable of inducing antioxi- dant adaptation (Devi and Kiran 2004). It is important to empha- size that the response of antioxidant enzyme activity to physical exercise differs according to the region of the brain and method- ology used (Somani and Rybak 1996;Devi and Kiran 2004).
Despite the absence of adaptation in both groups, SOD and GPx antioxidant activities in the brain showed a significant decrease in the OG relative to controls, which indicates the neuroprotective effect of estrogens acting via the elevated expression of these enzymes (Razmara et al. 2007;Prokai-Tatrai et al. 2008).
In summary, regular swimming promoted antioxidant adapta- tion in the control animals, which was primarily characterized by increased SOD and GPx enzyme activities in erythrocytes and the liver. Oophorectomy impeded the increase in antioxidant protec- tion induced by swimming. In addition, oophorectomy resulted in increased lipid peroxidation and diminished overall antioxidant enzyme activity in the tissues analyzed. Thus, estrogen produc- tion plays an important role in obtaining antioxidant benefits from physical activity. Although physical exercise in women pre- vents pathological processes that are associated with oxidative stress, low estrogen concentration, as occurs in postmenopausal women, may make this activity less effective. Therefore, clinical studies will be necessary in order to establish the real effects of exercise on antioxidant adaptation in postmenopausal women, as physical exercises are largely prescribed in this group of patients and the literature is still poor in this subject.
Acknowledgments
This study was supported by CNPq grant no. 4811/2007.2 and FAPERN (1st Edital-PRONEX). We thank Naira Josele Neves de Brito for her invaluable technical assistance.
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