cgSpringer
Molecular and Cellular Biochemistry 280: 135–138, 2005.
2005
The effects of vitamin C supplementation on oxidative stress and antioxidant content in the brains of chronically exercised rats S¸ule Co¸skun,1 Bilge G¨on¨ul,2 Nevin Atalay G¨uzel3 and Barbaros Balabanlı1 1
Department of Biology, Art and Science Faculty, Gazi University; 2 Department of Physiology, Faculty of Medicine, Gazi University; 3 Sport and Exercise Faculty, Gazi University, Besevler, Ankara, Turkey
Received 4 May 2005; accepted 6 June 2005
Abstract The purpose of this study was to ascertain whether vitamin C supplementation during chronic exercise training alters rat brain antioxidant content. Female Wistar albino rats were exercised on a treadmill for 30 min/day for 6.5 weeks and were administered daily intraperitoneal injections of vitamin C (20 mg/kg). After the training period, chronically exercised rats showed no significant changes in total brain thiobarbituric acid reactive substances (TBARS) levels. In contrast, rats supplemented with vitamin C during the training period showed significantly elevated brain TBARS levels. If such results were extrapolated to man, where vitamin supplementation is a common practice, this would indicate that vitamin C supplementation may not protect brain tissue against exercise-induced oxidative damage, in such circumstances, this water-soluble antioxidant behaves as a pro-oxidant. (Mol Cell Biochem 280: 135–138, 2005) Key words: antioxidant, brain, chronic exercise training, lipid peroxidation, vitamin C supplementation
Introduction Exercise increases the generation of oxygen-free radicals and lipid peroxidation within cells. During physical exercise, the aerobic metabolic rate may increase up to 10-fold and the oxygen uptake of skeletal muscles may rise by up to 100– 200 times [1], enhancing leakage of superoxide from the mitochondria to the cytosol [2]. Such a rise in free radical concentration could exceed the protective capacity of cell antioxidant defense systems [3]. Enzymatic and non-enzymatic antioxidant defense systems are present in cells to protect the membranes and other cell organelles from the damaging effects of free radicals [4]. Glutathione (GSH) plays a central role in co-ordinating cellular antioxidant defense processes and is present in high concentration (1–2 mM) in all cells [5].
Brain also has high levels of vitamin C (ascorbic acid = AA) which play an important role as a reducing agent [6], by acting as an electron donor to vitamin E radicals generated in the cell membrane during oxidative stress [7]. AA is water soluble, present in the cytosolic compartment of the cell, and is able to rapidly traverse the blood brain barrier, its concentration in brain exceeding that of blood by 10-fold [8]. It is concentrated in tissues and fluids with a high potential for radical generation, e.g. eye and brain [9]. Some studies have reported that supplementation with antioxidants such as vitamin C can reduce symptoms or indicators of oxidative stress as a result of exercise [10]. Physical exercise initiates oxidative stress in neuronal system and increases lipid peroxidation [11, 12] by altering neuronal membrane permeability [13]. Lipid peroxidation is
Address for offprints: S¸. Co¸skun, Department of Biology, Science and Art Faculty, Gazi University, 06500 Be¸sevler, Ankara, Turkey (E-mail:
[email protected])
136 the most common index of oxidative stress and TBARS, the last product of lipid peroxidation, and is routinely used as a marker of this process. Some studies showed that ascorbate acts as an antioxidant at physiological levels; while high doses may act as a pro-oxidant [14, 15]. Since brain is susceptible to oxidative damage, in part due to its high oxygen consumption and in part due to its high levels of polyunsaturated fatty acids [11, 12] the high level of ascorbic acid and glutathione may play a major role in protecting tissues against oxidative stress [6]. In this study, we examined the effects of vitamin C supplementation on brain TBARS, glutathione and ascorbic acid in chronically exercised rats.
Material and methods
to storage at −30 ◦ C. The rats in the chronically exercised groups were killed 24 h after the last exercise session. Lipid peroxidation was quantified by measuring the formation of TBARS. Total brain TBARS [16], GSH [17] and vitamin C [18] levels were assayed by spectrophotometric methods. Statistical analysis Data are presented as the mean ± S.D. There were four groups with 10 animals in each group, the results were compared statistically by the Anova Variance Analysis and Mann Whitney U test using the Statview program. Significance was set at p < 0.05.
Animals
Results
Forty female Wistar albino rats (The Military Medicine Academy of Gulhane Laboratories, Ankara, Turkey), 200 ± 20 g were randomly divided into four equal groups:
Brain TBARS levels
1. Controls (n = 10). 2. Chronic exercise training: The rats were run on a treadmill for 6.5 weeks (n = 10). 3. Vitamin C supplementation (Ascorbic Acid = AA. Sigma A-5960). The rats were supplemented with vitamin C for 6.5 weeks (n = 10). 4. Chronic exercise training+vitamin C supplementation. The rats were supplemented daily with vitamin C supplementation (20 mg/kg i.p. per day) and run each day on a treadmill for 6.5 weeks (n = 10). Rats were maintained on a 12:12 h light–dark cycle and received food and water ad libitum. All procedures using these animals adhered to the guiding principles of the Gazi University Council on Animal Care.
Mean brain TBARS levels increased significantly in chronically exercised rats with+vitamin C ( p < 0.05) (Table 1) by comparison to controls. In addition, brain TBARS levels increased in the rats which had been administered vitamin C alone by comparison to control (Table 1 and Fig. 1).
Brain GSH and Vitamin C levels Brain GSH levels increased after vitamin C supplementation ± chronic exercise regime although this was not significant (Table 1 and Fig. 2). Brain ascorbic acid concentration decreased after chronic exercise, as well as in the both AA-supplementation and chronic exercise with AAsupplementation groups, although the changes just failed to reach significance (Table 1 and Fig. 3).
Training program The animals in the chronic exercise groups were initially acclimatized to the training program of treadmill exercise over a 1-week period. During this period of habituatation (1 wk), rats exercised for 5–10 min/day at a speed of 20 m/min up a 10% gradient. The rats were then chronically exercised on a treadmill for 30 min/day at 27 m/min, 15% gradient during a 6.5 weeks period on a motor-driven treadmill. Tissue preparation and assays At the end of 6.5 weeks, the rats were killed, the brains removed immediately and placed in liquid nitrogen prior
Fig. 1. The effect of chronic exercise and vitamin C supplementation on brain TBARS levels. ∗ Significantly different from control group ( p < 0.05). C: control, Ch E: chronic exercise, AA suppl: ascorbic acid supplementation, Ch E+AA suppl: chronic exercise+ascorbic acid supplementation.
137 Table 1. The effects of chronic exercise and vitamin C supplementation on brain TBARS, GSH and AA levels
Groups (n)
Brain TBARS levels (nmol/g tissue)
Brain GSH levels (µmol/g tissue)
Brain AA levels (mg/g tissue)
Control (10)
26.5 ± 7.6
4.8 ± 1.2
14.4 ± 4.1
Chronic (10) exercise
28.3 ± 10.2
4.7 ± 1.5
12.6 ± 2.4
AA suppl. (10)
29.8 ± 13.5
5.4 ± 0.8
12.3 ± 3.0
Chronic (10) exercise+AA suppl.
30.5 ± 8.4∗
5.8 ± 1.7
12.4 ± 3.7
Note. Values are expressed as mean ± S.D. ∗ Significantly different from control group ( p < 0.05).
Fig. 2. The effect of chronic exercise and vitamin C supplementation on brain GSH levels. C: control, Ch E: chronic exercise, AA suppl: ascorbic acid supplementation, Ch E+AA suppl: chronic exercise+ascorbic acid supplementation.
Fig. 3. The effect of chronic exercise and vitamin C supplementation on brain AA levels. C: control, Ch E: chronic exercise, AA suppl: ascorbic acid supplementation, Ch E+AA suppl: chronic exercise+ascorbic acid supplementation.
Discussion Since exercise increases the generation of oxygen-free radicals and incidence of lipid peroxidation [4] changes may occur in the balance between pro-oxidants and antioxidants [7]. There are conflicting results as to whether oxidative damage occurs in the brain after exercise, some studies have identified significant decreases in MDA after treadmill training for either 8 weeks [19] or 6.5 weeks [20], while others [21–23] showed no significant alterations. Another study indicated that regular exercise improves cognitive function and decreases oxidative damage in rat brain [24].
In our study, the brain TBARS levels increased in the brains of chronically exercised rats when compared to the control group as well as in the chronically exercised group which had been supplemented with vitamin C. However, no corresponding decrease in glutathione was observed. Somani et al. [11] pointed out that different brain regions contained different activities of antioxidant enzymes, as well as GSH and GSSG levels, which were preferentially altered as a result of exercise training in order to cope with oxidative stress. Somani and Husain [20] found increased glutathione peroxidase activity in some brain regions, while glutathione reductase activity increased in brain regions after chronic exercise training (6.5 weeks treadmill running). Consistent with our results, Liu et al. [19] reported that there were no significant differences between control and chronic exercise groups’ brain GSH levels while brain GSH/GSSG ratio was also unaffected by either exercise or indolamine administration [23]. In our experimental protocol, brain GSH levels did not change in the chronically exercised group when compared to control, but ascorbic acid supplementation did increase brain GSH levels±chronic exercise although this did not reach statistical significance. Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol [25]. Acute ascorbic acid supplementation prevented exercise-induced oxidative stress in healthy subjects [26]. Liu et al. [19] reported that chronic exercise training increased rat brain AA levels, although this was not evident in this present study that may indicate a negative-feedback control in the synthesis of vitamin C by the rat. Some studies have suggested that training enhances antioxidant capacity but different training regimes have been used in the subjects, who were of varying degrees of fitness. Antioxidant capacity is largely a measure of vitamin C contents, the latter did not alter in our present study of rat brains after 6.5 weeks of chronic exercise. Further studies should investigate whether antioxidant capacities are altered between different brain regions. In conclusion, our study of the effects of chronic exercise on brain TBARS, GSH and AA levels after 6.5 weeks of chronic exercise ± vitamin C supplementation has indicated no beneficial effects of such supplementation and furthermore
138 suggested that such supplementation may in fact initiate lipid peroxidation within the brain. Further studies are clearly warranted to ascertain whether cellular dysfunction may be initiated by chronic exercise.
References 1. Guyton AC, Hall, EH: Textbook of Medical Physiology,16th edn. WB Saunders Company, Philadelphia, 1996 2. Davies KJ, Quintanilha AT, Brooks GA, Packer L: Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 107: 1198–1205, 1982 3. Duthie GG, Robertson JD, Maughan RJ, Morrice PC: Blood antioxidant status and erythrocyte lipid peroxidation following distance running. Arch Biochem Biophys 282: 78–83, 1990 4. Evans WJ: Vitamin E, vitamin C, and exercise. Am J Clin Nutr 72: 647S–652S, 2000 5. Raps SP, Lai JC, Hertz L, Cooper AL: Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons. Brain Res 493: 398–401, 1989 6. Rose RC: Cerebral metabolism of oxidized ascorbate. Brain Res 628: 49–55, 1993 7. Ji LL: Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222: 283–292, 1999 8. Agus DB, Gambhir SS, Pardridge WM, Spielholz C, Baselga J, Vera JC, Golde WV: Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters. J Clin Invest 100: 2842–2848, 1997 9. Jacob RA, Burri BJ: Oxidative damage and defense. Am J Clin Nutr 63: 985S–990S, 1996 10. Clarkson PM, Thompson HS: Antioxidants: what role do they play in physical activity and health? Am J Clin Nutr 72: 637S–646S, 2000 11. Somani SM, Ravi R, Rybak LP: Effect of exercise training on antioxidant system in brain regions of rat. Pharmacol Biochem Behav 50: 635–639, 1995 12. Somani SM, Husain K, Diaz-Phillips L, Lanzotti DJ, Kareti KR, Trammell GL: Interaction of exercise and ethanol on antioxidant enzymes in brain regions of the rat. Alcohol 13: 603–610, 1996
13. Husain K, Somani SM: Effect of exercise training and chronic ethanol ingestion on cholinesterase activity and lipid peroxidation in blood and brain regions of rat. Prog Neuropsychopharmacol Biol Psychiatry 22: 411–423, 1998 14. Repka T, Hebbel RP: Hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents. Blood 78: 2753–2758, 1991 15. Paolini M, Pozzetti L, Pedulli GF, Marchesi E, Cantelli-Forti G: The nature of prooxidant activity of vitamin C. Life Sci 64: PL273–278, 1999 16. Kurtel H, Granger DN, Tso P, Grisham MB: Vulnerability of intestinal interstitial fluid to oxidant stress. Am J Physiol 263: G573–G578, 1992 17. Elmann GL: Tissue sulyphydryl groups. Arch Biochem Biophys 82: 70–77, 1959 18. Berger J, Shepard D, Morrow F, Taylor A: Relationship between dietary intake and tissue levels of reduced and total vitamin C in the nonscorbutic guinea pig. J Nutr 119: 734–740, 1989 19. Liu L, Yeo HC, Overvik-Douki E, Hagen T, Doniger SJ, Chyu WD, Brooks GA, Ames BN, Chu DW: Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants. J Appl Physiol 89: 21–28, 2000 20. Somani SM, Husain K: Interaction of exercise training and chronic ethanol ingestion on antioxidant system of rat brain regions. J Appl Toxicol 17: 329–336, 1997 21. Ozkaya YG, Agar A, Yargicoglu P, Hacioglu G, Bilmen-Sarikcioglu S, Ozen L, Aliciguzel Y: The effect of exercise on brain antioxidant status of diabetic rats. Diabetes Metab 28: 377–384, 2002 22. Radak Z, Kaneko T, Tahara S, Nakamoto H, Pucsok J, Sasvari M, Nyakas C, Goto S: Regular exercise improves cognitive function and decreases oxidative damage in rat xbrain, Neurochem Int 38: 17–23, 2001 23. Hara M, Iigo M, Ohtani-Kaneko R, Nakamura N, Suzuki T, Reiter RJ, Hirata K: Administration of melatonin and related indoles prevents exercise-induced cellular oxidative changes in rats. Biol Signals 6: 90– 100, 1997 24. Radak Z, Taylor AW, Ohno H, Goto S: Adaptation to exercise-induced oxidative stress: from muscle to brain. Exerc Immunol Rev 7: 90–107, 2001 25. Brahma B, Forman RE, Stewart EE, Nicholson C, Rice ME, Ascorbate inhibits edema in brain slices. J Neurochem 74: 1263–1270, 2000 26. Ashton T, Young LS, Peters JR, Jones E, Jackson SK, Davies B, Rowland CC: Electron spin resonance spectroscopy, exercise and oxidative stress: an ascorbic acid intervention study. J Appl Physiol 87: 2032–2036, 1999
