1

Oophorectomy hinders antioxidant adaptation promoted by swimming in Wistar rats Ulisvaldo Brunno de Oliveira Macedo1, Rand Randal Martins 2,Francisco Paulo Freire Neto1,Yonara Monique da Costa Oliveira1Aldo da Cunha Medeiros 3, Jos Brando Neto4, Adriana Augusto de Rezende5,Maria das Graas Almeida 5 Running title: Oophorectomy inhibits swimming-induced antioxidant adaptation. 1. Graduate Pharmaceutical Sciences Program, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil. 2. Center for Education and Health, Federal University of Campina Grande (UFCG), Cuit, Brazil. 3. Department of Surgery, UFRN, Natal, Brazil. 4. Department of Clinical Medicine, UFRN, Natal, Brazil. 5. Department of Clinical and Toxicologic Analyses,UFRN, Natal, Brazil.

Correspondence to:

Maria das Graas Almeida, M.D. Laboratrio Multidisciplinar, Centro de Cincias da Sade Universidade Federal do Rio Grande do Norte (UFRN) Rua Gal. Gustavo Cordeiro de Farias, S/N, Petrpolis CEP: 59012 570 - Natal (RN) Brazil Phone: 55-84-3342 9824 Fax: 55-84- 3342 9833 E-mail: mgalmeida@digi.com.br

Abstract Oxidative stress is involved in a number of chronic-degenerative diseases. These include cardiac disorders, type II diabetes, rheumatoid arthritis, Alzheimers and Parkinsons and other conditions such as post menopause, which is responsible for various metabolic alterations. The redox imbalance observed during ovarian decline can be obtained experimentally by bilateral ovariectomy in rats. In addition to hormone replacement, regular moderate physical exercise is indicated to prevent several common postmenopausal diseases. This study aimed to assess the effect of daily swimming on the antioxidant defense system of ovariectomized Wistar rats. Ovariectomized control rats were submitted to one hour of daily swimming for 90 days. Levels of lipid
1

peroxidation, GSH and SOD and GPx activity in erythrocytes, liver and brain were assessed every 30 days. Although control animals exhibited lower lipoperoxidation associated to a significant increase in GSH, SOD and GPx concentrations in erythrocytes and liver, the swimming period did not cause changes in antioxidant parameters in the brain. Oophorectomized animals showed no antioxidant adaptation to daily swimming, in addition to greater oxidative damage in all tissues assessed. Therefore, our results suggest that ovariectomy hinders antioxidant adaptation in Wistar rats submitted to daily swimming. Keywords: ovariectomy, menopause, oxidative stress and physical exercise.

1. Introduction Reactive oxygen species (ROS) are products of the aerobic metabolism in signaling, apoptosis and phagocytosis processes (Radaket al. 2008). Increased ROS production is involved in the physiopathology of several chronic-degenerative diseases such as Alzheimers (Ansari and Scheff 2010), hypertension (Campese 2010), osteoporosis (Baeket al. 2010), diabetes mellitus (Pacal et al. 2011) and in menopause (Behr et al. 2011). Redox imbalance, known as oxidative stress (OS), participates in disease physiopathology since it causes significant alterations in functionally important biomolecules (Halliwell 2007). Frequent OS in postmenopausal women suggests that a decrease in sex hormones predisposes them to this condition (Signorelli et al. 2006). 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 raises the production of reactive species (RS) due to the volume of O2 inhaled, alterations in intracellular Ca++ homeostasis, vasomotor variations and ischemia/reperfusion (Radaket al. 2008). In healthy individuals, RS generated in physical exercise signals an adaptive response of the antioxidant system, improving its protective capacity (Karolkiewiczet al. 2009; GomezCabrera et al. 2008), and explaining some of the positive outcomes of physical exercise. In light of the benefits promoted by regular physical exercise (Gomez-Cabrera et al. 2008) and the participation of estrogen in antioxidant protection, the present study

assessed the influence of ovariectomy on antioxidant response in regularly exercised Wistar rats.

2. Materials and Methods Animals Forty female, 60 day-old Wistar rats (215 35 g) fed standard ration (Purina, So Paulo-SP, Brazil) and water ad libitum were used in the study. Animals were housed at constant temperature (23 2C) and a 12:12h light-dark cycle.

Study groups Rats were separated into two groups of 20 animals each: controls (CG) and an oophorectomized group (OG). Each group was subdivided into 4 subgroups containing 5 animals each (0, 30, 60 and 90 days of exercise). The experimental protocol was conducted according to Brazilian College of Animal Experimentation (COBEA) guidelines and the project was approved by the Research Ethics Committee of Onofre Lopes University Hospital (CEP-HUOL).

Physical exercise protocol Animals were submitted to daily 1-hour swimming sessions in a tank (160 long, 80 cm wide and 50 cm deep) containing water at controlled temperature (27 to 29 C). A limit of 20 animals swimming simultaneously was established. In the first week, exercise time was gradually increased by 10 minutes a day up to a maximum of 60 minutes. Each group was composed of 20 animals and five animals at a time were sacrificed at the end of day 0, 30, 60 and 90 of the exercise protocol.

Ovariectomy and sham procedure. Ovariectomies were carried out under sodium pentobarbital anesthesia (60 mg/kg, ip) through flank incisions. The fur on both sides of the body was shaved from the hip to the lowest rib. Bilateral ovariectomies 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 muscles and skin. Animals were submitted to the protocol fifty days after surgery. The sham-operated group (CG) underwent the same surgical procedure except for ovary removal.
3

Samples After sacrifice, the brain and liver were removed and 5mL of blood was by collected by cardiac puncture. Liver and brain samples were homogenized in PotterElvehjem homogenizer in phosphate buffer, pH 7.0 at 4C for analysis.

Lipid peroxidation Lipid peroxidation in tissues was determined by concentrations of thiobarbituric acid reactive substances (TBARS) (Yagi 1982) and colorimetrically assessed using the measure of absorbance at 532 nm, after the reaction of lipoperoxidation subproducts with thiobarbituric acid (TBA). Results were expressed in mol/L.

Antioxidant biomarkers Reduced glutathione (GSH) was analyzed with the technique described by Beutler (1963). Glutathione peroxidase (GPx) (EC 1.6.4.2) and SOD (EC 1.15.1.1) activities were determined according to Sies (1979) and McCord and Fridovich (1969) respectively.

Statistics Results were expressed as mean SEM. Intergroup differences were analyzed using 2-way ANOVA, followed by the Student-Newman-Keuls multiple comparisons test. Correlation analysis was performed by determining Pearsons coefficient. Differences were considered significant for p <0.05.

3. Results

All parameters were compared between the control (CG) and ovariectomized (OG) group in the same period, in order to observe the influence of a decrease in estrogens on OG parameters. The existence of antioxidant adaptation was determined by the positive correlation between the parameter analyzed and exercise time.

Blood

Blood tissue showed a positive correlation between antioxidant biomarkers (GSH, SOD and GPx) in the CG and swimming time (fig1. B, C and D), characterizing the antioxidant adaptation process. The OG exhibited no correlation between any of the parameters and swimming time. Intergroup comparison in a same period showed that OG animals obtained high TBARS concentrations at 30 and 60 days of swimming, whereas GSH content was lower at the start and end of the experimental period (fig1. A and B). Superoxidase (SOD) and GPx enzyme activity in the CG was higher during the entire swimming period (fig1. C and D).

Liver A positive correlation was observed between SOD and GPX enzyme activity and swimming time (fig2. C and D) in the liver homogenate of the CG, indicating an adaptive process. However, this group exhibited a negative correlation with respect to GSH content (fig2. B). Ophorectomized animals showed no antioxidant adaptation. An increase in TBARS was recorded in OG animals when compared to the CG at 30, 60 and 90 days of exercise (fig2. A). Glutathione (GSH) content and SOD and GPx activity decreased significantly in the OG compared to the CG in at least three periods (fig2. B, C and D).

Brain The brain showed no correlation between the parameters assessed and swimming time (fig3. A, B, C and D), indicating absence of adaptation. However, intergroup comparisons demonstrate that SOD activity decreased significantly in all periods in the OG when compared to the CG (fig.3 C), while GPx activity fell at 60 and 90 days in the OG (fig.3 D).

5. Discussion

Animals submitted to swimming improved their antioxidant defense system in blood and liver cells, except for the brain. On the other hand, oophorectomized animals showed less adaptive capacity to swimming in the tissues under study. Erythrocyte cells exhibit a number of physiological and biochemical peculiarities, mainly in relation to the redox metabolism. They are continuously
5

submitted to RS, due to high O2 pressure and iron content in the heme component, requiring their own antioxidant system (Cimen 2008). However, the antioxidant efficiency of erythrocytes decreases in senescent cells (Petibois and Deleris 2005). In the CG, adaptation observed in antioxidant enzymes and GSH content can be attributed to erythrocyte turnover. Chemical 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 improved antioxidant defense in the CG. In contrast to the CG, the OG showed less erythrocyte renewal, suggesting absence of antioxidant adaptation. Low estrogen concentration decreases antioxidant protection, justifying the lower antioxidant adaptation observed in oophorectomized animals, in addition to greater lipoperoxidation in this group. According to Ulas (2011), estrogens could have a beneficial effect on the blood antioxidant defense system, stimulating antioxidant enzyme activity. Xanthine oxidase activation and the ischemia-reperfusion process during physical exercise are responsible for RS production in the liver (Radaket al. 2008). Control group animals showed antioxidant adaptation promoted by the swimming period, suggesting a role of RS in improving the antioxidant system. Reactive species (RS) act in important intracellular signaling pathways sensitive to oxidative stress, such as nuclear factor B(NF- B) and mitogen-activated protein kinase (MAPK). These

pathways are capable of promoting the expression of enzyme-related genes, such as SOD and GPx, maintaining intracellular redox equilibrium (Ji et al. 2004). Although GSH in the CG decreased with swimming time, this behavior may be attributed to the rise in GPx activity, which it uses as a cofactor. The liver of ovariectomized animals exhibited no antioxidant adaptation, as was observed in blood tissue. According to Omoya et al. (2001), estrogen has a hepatoprotective role given that it interferes in the NF-Kb signaling pathway, favoring antioxidant enzyme activity. Our results show diminished antioxidant adaptive capacity in oophorectomized animals. The lower lipid peroxidation in the CG was due to the improvement in the other antioxidant parameters assessed. These results are in accordance with studies confirming the hepatoprotective effect of estrogens, which have greater capacity in combating RS, thereby avoiding tissue necrosis and apoptosis (Omoyaet al. 2001; Liang et al. 2011).
Comment [M1]: que a utiliza como co-fator No ficou clarou o que a representa (GSh ou GPx). Favor esclarecer e eu ajeito

The brain exhibits metabolic demand of 240 kcal/kg body weight/ day, with a relatively constant flow of oxygen during physical exercise (Radaket al. 2008). No antioxidant adaptation was recorded in the brain in any of the groups evaluated. The constant flow of oxygen, even during daily physical exercise, may not have caused oxidative damage capable of inducing antioxidant adaptation (Devi and Kiran 2004). It is important to underscore that the response of antioxidant enzyme activity to physical exercise differs according to the region of the brain and methodology used (Devi and Kiran 2004; Somani and Rybak 1996). Despite the absence of adaptation in both groups, SOD and GPx antioxidant activity in the brain showed a significant decrease in oophorectomized animals, indicating the neuroprotective effect of estrogens through the elevated expression of these enzymes (Razmara et al. 2007; Prokai-Tatrai et al. 2008). In summary, regular swimming promoted antioxidant adaptation in the control animals, mainly in regard to SOD and GPx enzyme activity in erythrocytes and the liver. Ovariectomy impeded an increase in antioxidant protection induced by swimming, in addition to enhancing lipid peroxidation and diminishing antioxidant enzyme activity in the tissues analyzed. Thus, estrogen production plays an important role in obtaining antioxidant benefits from physical activity. Although physical exercise in women prevents pathological processes associated to oxidative stress, low estrogen concentration, such as in post-menopause, may make this activity less effective. Therefore, further research is needed on the impact of physical exercise on antioxidant adaptation in postmenopausal women in order to clarify this issue.

Acknowledgements This study was supported by CNPq grant nr4811/2007.2 and FAPERN (1 Edital-PRONEX). We would like to thank Naira Josele Neves de Brito for her invaluable technical assistance.
o

REFERENCES Ansari MA, Scheff SW. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. 2010. J Neuropathol Exp Neurol. 69(2):155-67.
8

Agil A, Abike F, Daskapan A, Alaca R, Tuzun H. Short-term exercise approaches on menopausal symptoms, psychological health, and quality of life in postmenopausal women. 2010. Obstet Gynecol Int. Baek KH, Oh KW, Lee WY, Lee SS, Kim MK, Kwon HS, et al. Association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. 2010. Calcif Tissue Int. 87(3):226-35. Behr GA, Schnorr CE, Moreira JC. Increased blood oxidative stress in experimental menopause rat model: the effects of vitamin A low-dose supplementation upon antioxidant status in bilateral ovariectomized rats. 2011. Fundam Clin Pharmacol. 13. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. 1963. The Journal of laboratory and clinical medicine. 61:882-8. Campese VM. Oxidative stress and sympathetic activity in hypertension. 2010. Am J Hypertens. 23(5):456. Cimen MY. Free radical metabolism in human erythrocytes. 2008. Clin Chim Acta. 390(1-2):1-11. Devi SA, Kiran TR. Regional responses in antioxidant system to exercise training and dietary vitamin E in aging rat brain. 2004. Neurobiol Aging. 25(4):501-8. Gomez-Cabrera MC, Domenech E, Vina J. Moderate exercise is an antioxidant: upregulation of antioxidant genes by training. 2008. Free Radic Biol Med. 15;44(2):126-31. Halliwell B. Biochemistry of oxidative stress. 2007. Biochemical Society transactions. 035(5):1147-50. Ji LL, Gomez-Cabrera MC, Steinhafel N, Vina J. Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. 2004. FASEB J. 18(13):1499506. Karolkiewicz J, Michalak E, Pospieszna B, Deskur-Smielecka E, Nowak A, Pilaczynska-Szczesniak L. Response of oxidative stress markers and antioxidant parameters to an 8-week aerobic physical activity program in healthy, postmenopausal women. 2009. Arch Gerontol Geriatr. 49(1):e67-71. Liang Q, Sheng Y, Jiang P, Ji L, Xia Y, Min Y, et al. The gender-dependent difference of liver GSH antioxidant system in mice and its influence on isoline-induced liver injury. 2011. Toxicology. 280(1-2):61-9.

10

McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). 1969. J Biol Chem. 244(22):6049-55. Omoya T, Shimizu I, Zhou Y, Okamura Y, Inoue H, Lu G, et al. Effects of idoxifene and estradiol on NF-kappaB activation in cultured rat hepatocytes undergoing oxidative stress. 2001. Liver. 21(3):183-91. Pacal L, Varvarovska J, Rusavy Z, Lacigova S, Stetina R, Racek J, et al. 2011. Parameters of oxidative stress, DNA damage and DNA repair in type 1 and type 2 diabetes mellitus. Arch Physiol Biochem. 22. Petibois C, Deleris G. Erythrocyte adaptation to oxidative stress in endurance training. 2005. Arch Med Res. 36(5):524-31. Prokai-Tatrai K, Perjesi P, Rivera-Portalatin NM, Simpkins JW, Prokai L. Mechanistic investigations on the antioxidant action of a neuroprotective estrogen derivative. 2008. Steroids. 73(3):280-8. Radak Z, Chung HY, Goto S. Systemic adaptation to oxidative challenge induced by regular exercise. 2008. Free Radic Biol Med. 15;44(2):153-9. Razmara A, Duckles SP, Krause DN, Procaccio V. Estrogen suppresses brain mitochondrial oxidative stress in female and male rats. 2007. Brain Research. 1176:7181. Signorelli SS, Neri S, Sciacchitano S, Pino LD, Costa MP, Marchese G, et al. Behaviour of some indicators of oxidative stress in postmenopausal and fertile women. 2006. Maturitas. 10;53(1):77-82. Sies H, Koch OR, Martino E, Boveris A. Increased biliary glutathione disulfide release in chronically ethanol-treated rats. 1979. FEBS Lett. 15;103(2):287-90. Somani SM, Rybak LP. Comparative effects of exercise training on transcription of antioxidant enzyme and the activity in old rat heart. 1996. Indian J Physiol Pharmacol. 40(3):205-12. Ulas M, Cay M. Effects of 17beta-estradiol and vitamin E treatments on blood trace element and antioxidant enzyme levels in ovariectomized rats. 2011. Biol Trace Elem Res. 139(3):347-55. Yagi K. Lipid peroxides in biology and medicine. 1982. Academic Press;.

10

11

Figure legends Fig. 1. Influence of physical exercise on lipid peroxidation (TBARS) and antioxidant biomarkers (GSH, GPx and SOD) on the blood of control (__
__

) and oophorectomized

animals (--- ---). Significant differences: * between groups (ANOVA followed by the Tukey-Kramer test, p<0.05) and # correlation between time (days) and parameter evaluated (Pearsons correlation test, p<0.05).

Fig. 2. Influence of physical exercise on lipid peroxidation (TBARS) and antioxidant biomarkers (GSH, GPx and SOD) on the liver of control (__
__

) and oophorectomized

animals (--- ---). Significant differences: * between groups (ANOVA followed by the Tukey-Kramer test, p<0.05) and # correlation between time (days) and parameter evaluated (Pearsons correlation test, p<0.05).

Fig. 3. Influence of physical exercise on lipid peroxidation (TBARS) and antioxidant biomarkers (GSH, GPx and SOD) on the brain of control (__
__

) and oophorectomized

animals (--- ---). Significant differences: * between groups (ANOVA followed by the Tukey-Kramer test, p<0.05) and # correlation between time (days) and parameter evaluated (Pearsons correlation test, p<0.05).

11

12

Figure 1

75

A
*
GSH (mmol/L)

2.0 1.5 1.0 0.5 0.0

B
*
#

TBARS (Qmol/L)

50

25

0
SOD (U/mg hemoglobin)

30

60

90
GPx (U/mg Hemoglobin)

0 400 300

30

60

90

400 300 200 100 0

C
* *

*
#

D
* * * *
#

200 100 0

30

Day

60

90

30

Day

60

90

12

13

Figure 2

75

A
*
GSH (mmol/L)

* * *
6 4 2 0

B
* * *
#

TBARS (Q mol/L)

50

25

0 0 300
SOD (U/mg protein)

30

60

90 250

30

60

90

C
*

*
GPx (U/mg protein)

D
* *

*
#

200 150 100 50 0

200

100

0 0 30
Day

60

90

30
Day

60

90

13

14

Figure 3

75 70
TBARS (Q mol/L)

A
GSH (mmol/L)

2.5 2.0 1.5 1.0 0.5 0.0

* *

65 60 55 50 45 0 30 60 90

0 6

30

60

90

40
SOD (U/mg protein)

*
GPx (U/mg Protein)

D
*

30 20 10 0 0 30
Day

0 60 90 0 30
Day

60

90

14