Many studies reported that intense exercise caused oxidative stress because of increased generation of ROS, whereas regular exercise is known as an important factor in preventing many diseases. Oxidative stress induced-cellular damage often appeared as changes in macromolecules such as proteins, lipids, and nucleic acids (DNA). In many studies, 8-OHdG was evaluated as a biomarker of oxidative DNA damage (6,29,40) because it represents 5% of the total oxidized bases in the DNA and is present in quantities that are sufficient to be readily detected (17). This 8-OHdG is a potentially important factor in carcinogenesis because it is prone to induce G-C to T-A transversion during DNA replication, which are frequently found in tumor-relevant genes. Thus, increases in the level of 8-OHdG can have important implications for mutagenesis and the induction of tumors (35).
In this study, urinary 8-OHdG excretion significantly increased immediately and 24 hours post RE in both groups before the Cr supplementation period, which is in accordance with study of Radak et al. (29) who reported that the 8-OHdG level was significantly increased in biopsy samples of the quadriceps at 24 hours after performing 200 eccentric contractions with knee extensors, but our findings are inconsistent with those of Bloomer et al. (6), who did not observe significant changes in plasma concentrations of 8-OHdG and MDA after 30-minute intermittent dumbbell squat with 70% of 1RM. The possible reasons for the inconsistency in our findings with those of the previous study can imply a difference in the RE protocol. In this study, whole-body RE protocol performed in flat pyramid loading pattern (FPLP), which includes 7 sets of 3–6 repetitions with 80–90% of 1RM. Flat pyramid loading pattern is the most effective method to gain maximal strength, and the physiological advantage of this method is that using a load of only 1 intensity level, the best neuromuscular adaptation for maximal strength is achieved without confusing the body with several intensities (7), which in comparison to the protocol used in the previous study (6) possess a higher intensity and volume. In this case, it has been previously reported that training intensity may be the main factor in producing free radicals (5,6,10).
In addition to oxidative DNA damage, free radicals may damage cellular compartments specially lipids and lead to initiation of chain reactions that are known as lipid peroxidation, the most important consequence of oxidative stress. In this study, before the Cr supplementation period, MDA, a biomarker of lipid peroxidation, is significantly increased immediately and 24 hours post RE in both groups, which is in accordance with the finding of Ramel et al. (32) who reported a significant increase in plasma MDA concentration after a circuit RE bout (18 minutes of RE with 75% of 1RM in 10 exercises) in trained and untrained subjects (3); an increase in plasma MDA concentration after 2 days of whole-body RE as well has been reported (25).
It has been shown that endogenous antioxidant substances could not completely prevent oxidative damage under physiological and pathological conditions, such as strenuous exercise and exercise at altitude, and many diseases. It is possible that these situations disturb endogenous antioxidant balance, which could not neutralize the oxidant effects. During these situations, dietary antioxidants have the most important roles owing to enhancement in the ability of body antioxidant systems. Recently, Cr has been introduced as an antioxidant (23). Therefore, in this study, the effect of 7-day Cr supplementation (4 doses of 5 g·d−1) on MDA and 8-OHdG, which are biomarkers of lipids peroxidation and oxidative DNA damage, is examined after a single bout of RE.
To our knowledge, this was the first investigation to compare Cr supplementation on biomarkers of DNA and lipid oxidation after acute RE. The primary findings of our study were that urinary 8-OHdG concentrations were significantly decreased in Cr group compared to in the PL group immediately and 24 hours after RE and that plasma MDA concentrations were significantly decreased immediately and 24 hours after RE in the Cr group compared to in the PL group. However, in both groups, urinary 8-OHdG and MDA concentrations were significantly higher post RE compared to pre RE. These results imply that oxidative stress and oxidative DNA damage are lesser in the Cr group than in the PL group after a single bout of RE. These changes are attributed to the antioxidant characteristics of Cr. Lawler et al. (23) for the first time showed in vitro that Cr had an ability to remove the superoxide anion radical, ABTS+ cation, and peroxynitrite radical (ONOO−).
Antioxidant properties of Cr may be attributed to the presence of arginine in its molecule. Arginine is also a substrate for the nitric oxide synthase family and can increase the production of nitric oxide, a free radical that modulates metabolism, contractility, and glucose uptake in skeletal muscle (21,34). Other amino acids such as glycine and methionine because of the presence of sulfhydryl groups may be especially susceptible to free-radical oxidation (13). Up to this time, there is no study regarding the effect of Cr supplementation on oxidative DNA damage after RE, in athletes. However, in 3 studies (22,36,45), indirect antioxidative properties of Cr were confirmed, which is in accordance with our findings. In a study, Vergnani et al. (45) reported the antioxidative role of arginine, the precursor of Cr, in the oxidative modifications of the low-density lipoprotein cholesterol in endothelial cells and aortal rings. Also, Santos et al. (36) examined the effect of Cr supplementation (4 dose of 5 g for 5 days) upon inflammatory and muscle soreness markers after a 30-km race. They reported that Cr supplementation reduced cell damage and inflammation after an exhaustive intense race. As previously mentioned, lower concentration of MDA in the Cr group compared to in the PL group immediately and 24 hours after the RE protocol are in accordance with the findings of Basta et al. (3), who reported that Cr supplementation led to the reinforcement of the antioxidative system (superoxide dismutase [SOD], GPx) in rowers' blood, confirmed by a significantly lower concentration of lipid peroxidation products upon a 24-hour recovery period and by a lower postexercise activity of glutathione peroxidase. As mentioned above, these changes may be because of the antioxidative properties of Cr. Also, reduction in glutathione peroxidase activity because of Cr supplementation could be the reason for the lower concentration of MDA, lipid peroxidation products, in the Cr group.
In addition, the attenuated oxidative DNA damage and lipid peroxidation responses are consistent with the findings of other studies using high antioxidant diets in athletes (2,26). For example, Arent et al. (2) found that, compared with PL, supplementing with Resurgex® decreased lipid peroxidation (8-isoprostane, lipid hydroperoxide) and creatine kinase (CK) in college soccer players. Also, our findings are consistent with those of the study using isolated Glisodin® supplementation that has reported a protective effect on DNA and reduced 8-isoprostane levels (26).
Although, our subjects were recreationally resistance trained, they were unaccustomed to the format and intensity of the RE protocol used in this study. The unfamiliarity of the subjects with FPLP most likely is the reason for increasing oxidative stress and oxidative DNA damage before Cr loading. In summary, acute the RE with FPLP method induced oxidative DNA damage and lipid peroxidation in trained subjects, but short-term Cr supplementation decrease these effects, which may be because of increasing the activity of antioxidant enzymes and reducing oxidant production.
A single bout of RE which is performed using the FPLP method increases oxidative DNA damage and lipid peroxidation in athletes. However, Cr supplementation for a short period decreased urinary 8-OHdG excretion and plasma MDA levels after acute RE with the FPLP method, which suggests a positive effect of the Cr supplementation as a strategy in reducing oxidative DNA damage and lipid peroxidation after a strenuous RE protocol. The exact mechanisms by which Cr supplementation exert its antioxidant actions remain to be elucidated, however, increased activity of antioxidant enzymes and ability to remove ROS and reactive oxygen and nitrogen species (RONS) have been implicated, as previously demonstrated (23,38). Also, 90% of the total Cr in the body is stored in skeletal muscles and mitochondria is one important source of ROS that includes H2O2, O2−, and possibly OH− and peroxynitrite, in skeletal muscles. Therefore, in this case, further studies on animal or human modals will be required to examine short-term Cr supplementation on mitochondrial antioxidant enzymes and components such as mitochondrial genome. The coach and athlete should consider that in current form and dosage, Cr supplementation has ergogenic benefit, so we recommended its use in resistance-trained men seeking to enhance performance and to reduce oxidative DNA damage and lipid peroxidation.
The authors are grateful to the subjects who participated in this study. The authors also thank Mr. Mehdi Malaki at the Fadai Laboratory of Endocrinology for technical assistance with ELISA procedures.
1. Ahmun, RP, Tong, RJ, and Grimshaw, PN. The effects of acute creatine supplementation on multiple sprint cycling and running performance in rugby players. J Strength Cond Res
19: 92–97, 2005.
2. Arent, SM, Pellegrino, JK, Williams, CA, DiFabio, DA, and Greenwood, JC. Nutritional supplementation, performance, and oxidative stress in college soccer players. J Strength Cond Res
24: 1117–1124, 2010.
3. Basta, P, Skarpańska-Stejnborn, A, and Pilaczyńska-Szcześniak, L. Creatine supplementation and parameters of exercise-induced oxidative stress after a standard rowing test. Stud Phys Cult Tour
13: 17–23, 2006.
4. Bloomer, RJ, Falvo, MJ, Fry, AC, Schilling, BK, Smith, WA, and Moore, CA. Oxidative stress response in trained men following repeated squats or sprints. Med Sci Sports Exerc
38: 1436–1442, 2006.
5. Bloomer, RJ and Goldfarb, AH. Anaerobic exercise and oxidative stress: A review. Can J Appl Physiol
29: 245–263, 2004.
6. Bloomer, RJ, Goldfarb, AH, Wideman, L, Mckenzie, MJ, and Consitt, LA. Effects of acute aerobic and anaerobic exercise on blood markers of oxidative stress. J Strength Cond Res
19: 276–285, 2005.
7. Bompa, TO. Periodization Training for Sports
. Champaign, IL: Human Kinetics, 1999.
8. Brenner, M, Walberg Rankin, MJ, and Sebolt, D. The effects of creatine supplementation during resistance training in women. J Strength Cond Res
14: 207–213, 2003.
9. Cheng, KC, Cahill, DS, Kasai, H, Nishimura, S, and Loeb, LA. 8-Hydroxy, an abundant form of oxidative DNA damage, causes G-T and A-C substitutions. J Biol Chem
267: 166–172, 1992.
10. Dixon, CB, Robertson, RJ, Goss, FL, Timmer, JM, Nagle, EF, and Evans, RW. The effect of acute resistance exercise on serum malondialdehyde
in resistance-trained and untrained collegiate men. J Strength Cond Res
20: 693–698, 2006.
11. Eckerson, JM, Stout, JR, Moore, GA, Stone, NJ, Nishimura, K, and Tamura, K. Effect of two and five days of creatine loading on anaerobic working capacity in women. J Strength Cond Res
18: 168–172, 2004.
12. Esterbauer, H, Schaur, RJ, and Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde
and related aldehydes. Free Rad Biol Med
11: 81–128, 1991.
13. Grune, T, Reinheckel, T, and Davies, KJA. Degradation of oxidized proteins in mammalian cells. FASEB J
11: 526–534, 1997.
14. Gutteridge, JM and Halliwell, B. Free radicals and antioxidants in the year: A historical look to the future. Ann NY Acad Sci
899: 136–147, 2000.
15. Halliwell, B. Free radicals and antioxidants: A personal view. Nutr Rev
52: 253–265, 1994.
16. Halliwell, B and Aruoma, OI. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett
281: 9–19, 1991.
17. Helbock, HJ, Beckman, KB, and Ames, BN. 8-Hydroxyguanosine as biomarker of oxidative DNA damage. Meth Enzymol
300: 156–165, 1999.
18. Ji, LL. Free radicals and antioxidants in exercise and sports. In: Exercise and Sport Science
. W.E. Garrett and D.T. Kirkendall, eds. Philadelphia, PA: Lippincott Williams and Wilkins, 2000. pp. 299–317.
19. Kasai, H, Tanooka, H, and Nashimura, S. Formation of 8-hydroxyguanine residues in DNA by X-irradiation. Gann
75: 1037–1039, 1984.
20. Konig, D, Wagner, KH, Elmadfa, I, and Berg, A. Exercise and oxidative stress: Significance of antioxidants with reference to inflammatory, muscular, and systemic stress. Exerc Immunol Rev
7: 108–133, 2001.
21. Kraemer, WJ and Volek, JS. Creatine supplementation. Its role in human performance. Clin Sports Med
18: 651–666, 1999.
22. Kreider, RB, Melton, C, Rasmussen, CJ, Greenwood, M, Lancaster, S, Cantler, EC, and Almada, AL. Long-term creatine supplementation does not significantly affect clinical markers of health in athletes. Mol Cell Biochem
244: 95–104, 2003.
23. Lawler, JM, Barnes, WS, Wu, G, and Song, W. Direct antioxidant properties of creatine. Biochem Biophys Res Commun
290: 47–52, 2002.
24. Lefavi, RG, McMillan, JL, Kahn, PJ, Crosby, JF, Digioacchino, RF, and Streater, AJ. Effects of creatine monohydrate on collegiate baseball and basketball players. J Strength Cond Res
12: 275, 1998.
25. McBride, JM, Kraemer, WJ, Triplett McBride, T, and Sebastianelli, W. Effect of resistance exercise on free radical production. Med Sci Sports Exerc
30: 67–72, 1998.
26. Muth, CM, Glenz, Y, Klaus, M, Radermacher, P, Speit, G, and Leverve, X. Influence of an orally effective SOD on hyperbaric oxygen-related cell damage. Free Radical Res
38: 927–932, 2004.
27. Naclerio, FJ, Jiménez, A, Alvar, BA, and Peterson, MD. Assessing strength and power in resistance training. J Hum Sport Exerc
4: 100–113, 2009.
28. Nakajima, S, Kamohara, S, Nakano, M, and Ohno, M. Antioxidant supplementation decrease the amount of urinary 8-OHdG excretion induced by a single bout of exercise. Jpn J Phys Fitness Sports Med
55: S251–S256, 2006.
29. Radak, Z, Pucsok, J, Meeseki, S, Csont, T, and Ferdinandy, P. Muscle soreness-induced reduction in force generation is accompanied by increased nitric oxide content and DNA damage in human skeletal muscle. Free Radic Biol Med
26: 1059–1063, 1999.
30. Rahimi, R, Boroujerdi, SS, Ghaeeni, S, and Noori, SR. The effect of different rest intervals between sets on the training volume of male athletes. Facta Univ Phys Educ Sport
5: 37–46, 2007.
31. Rahimi, R, Qaderi, M, Faraji, H, and Boroujerdi, SS. Effects of very short rest periods on hormonal responses to resistance exercise in men. J Strength Cond Res
24: 1851–1859, 2010.
32. Ramel, A, Wagner, KH, and Elmadfa, I. Plasma antioxidants and lipid oxidation after submaximal resistance exercise in men. Eur J Nutr
43: 2–6, 2004.
33. Rawson, ES and Persky, AM. Mechanisms of muscular adaptations to creatine supplementation. Int Sport Med J
8: 43–53, 2007.
34. Reid, MB. Redox modulation of skeletal muscle contraction: What we know and what we don't. J Appl Physiol
90: 724–731, 2001.
35. Remmen, HV, Hamilton, ML, and Richardson, A. Oxidative damage to DNA and aging. Exerc Sport Sci
31: 149–153, 2003.
36. Santos, RVT, Bassit, RA, Caperuto, EC, and Costa Rosa, LFBP. The effect of creatine supplementation upon inflammatory and muscle soreness markers after a 30 km race. Life Sci
75: 1917–1924, 2004.
37. Sen, CK, Rankinen, T, Vaisanen, S, and Rauramaa, R. Oxidative stress after human exercise: Effect of N
-acetylcysteine supplementation. J Appl Physiol
76: 2570–2577, 1994.
38. Sestili, P, Martinelli, C, Bravi, G, Piccoli, G, Curci, R, Battistelli, M, Falcieri, E, Agostini, D, Gioacchini, AM, and Stocchi, V. Creatine supplementation affords cytoprotection in oxidatively injured cultured mammalian cells via direct antioxidant activity. Free Radic Biol Med
40: 837–849, 2006.
39. Shephard, RJ and Shek, PN. Associations between physical activity and susceptibility to cancer: Possible mechanisms. Sports Med
26: 293–315, 1998.
40. Shi, M, Wang, X, Yamanaka, T, Ogita, F, Nakatani, K, and Takeuchi, T. Effects of anaerobic exercise and aerobic exercise on biomarkers of oxidative stress. Environ Health Prev Med
12: 202–208, 2007.
41. Shibutani, S, Takeshita, M, and Grollman, AP. Insertion of specific bases during DNA synthesis past the oxidation damaged 8-oxodG. Nature
349: 431–434, 1991.
42. Surmen-Gur, E, Ozturk, E, Gur, H, Punduk, Z, and Tuncel, P. Effect of vitamin E supplementation on post-exercise plasma lipid peroxidation and blood antioxidant status in smokers: With special reference to haemoconcentration effect. Eur J Appl Physiol Occup Physiol
79: 472–478, 1999.
43. Takeuchi, T, Nakajima, M, and Morimoto, K. Establishment of a human system that generates O2
and induces 8-hydroxydeoxyguanosine, typical of oxidative DNA damage, by a tumour promoter. Cancer Res
54: 5837–5840, 1994.
44. Takeuchi, T, Nakajima, M, Ohta, Y, Mure, K, Takeshita, T, and Morimoto, K. Evaluation of 8-hydroxydeoxyguanosine, a typical oxidative DNA damage, in human leukocytes. Carcinogenesis
15: 1519–1523, 1994.
45. Vergnani, L, Hatrick, S, Ricci, F, Passaro, A, Manzoli, N, Zuliani, G, and Brokovych, V. Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production: Key role of l-arginine availability. Circulation
101: 1261–1266, 2000.