Oxidative DNA damage in human peripheral leukocytes induced by massive aerobic exercise

Free Radical Biology & Medicine, Vol. 31, No. 11, pp. 1465–1472, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00729-8

Original Contribution OXIDATIVE DNA DAMAGE IN HUMAN PERIPHERAL LEUKOCYTES INDUCED BY MASSIVE AEROBIC EXERCISE KELVIN TSAI,*† TAI-GER HSU,‡ KUANG-MING HSU,‡ HU CHENG,‡ TSUNG-YUN LIU,*† CHEN-FU HSU,*† CHI-WOON KONG*†

and

*Oxidative Stress Clinical Research Group and Division of Critical Care, Department of Medicine, Veterans General Hospital, Taipei, Taiwan; †National Yang-Ming University School of Medicine, Taipei, Taiwan; and ‡Institute of Sports Science, Taipei Physical Education College, Taipei, Taiwan [Received 22 May 2001; Accepted 6 September 2001]

Abstract—Reactive oxygen species produced during vigorous exercise may permeate into cell nuclei and induce oxidative DNA damage, but the supporting evidence is still lacking. By using a 42 km marathon race as a model of massive aerobic exercise, we demonstrated a significant degree of unrepaired DNA base oxidation in peripheral immunocompetent cells, despite a concurrent increase in the urinary excretion of 8-hydroxy-2⬘-deoxyguanosine. Single cell gel electrophoresis with the incorporation of lesion-specific endonucleases further revealed that oxidized pyrimidines (endonuclease III-sensitive sites) contributed to most of the postexercise nucleotide oxidation. The oxidative DNA damage correlated significantly with plasma levels of creatinine kinase and lipid peroxidation metabolites, and lasted for more than 1 week following the race. This phenomenon may be one of the mechanisms behind the immune dysfunctions after exhaustive exercise. © 2001 Elsevier Science Inc. Keywords—Comet assay, Running, Oxidants, Humans, Free radicals

INTRODUCTION

response occurring in injury or infection [8]. Tissue injuries and phagocyte activation during exercise all contribute to increased reactive oxygen species (ROS) productions. These processes are expected to be more prominent in strenuous exercise such as running a marathon. Lipid and protein oxidation induced by high-intensity exercise have been clearly demonstrated previously [7,9]. However, evidence supporting increased free radical attack on DNA during exercise is still limited. Urinary excretion of 8-hydroxy-2⬘-deoxyguanosine (8OHdG), the most abundant product of oxidative DNA modifications, increased 1.3-fold above resting approximately 10 h following a marathon race [10]. However, urinary 8-OHdG remained unchanged after exercises of more moderate intensity or shorter duration [11,12]. Of note, the increase in urinary excretion of oxidized nucleotides may reflect either higher levels of DNA oxidation or augmented enzyme repairing ability, and does not reflect the steady-state unrepaired DNA damage level. Due to the inherent problem in the interpretation of urinary 8-OHdG excretion and large discrepancies among different assay results, it appears that definite conclusions could not be drawn from these findings.

Oxidative damage to DNA has been suggested to contribute to aging and various diseases including cancer and chronic inflammation [1,2]. It has been estimated that free radical-induced DNA damage in humans is at biologically relevant levels, with approximately 104 DNA bases being oxidatively modified per cell per day [3,4]. The magnitude of DNA base oxidation is proportional to the change in metabolic rate [5], and thus is expected to increase in situations with increased oxygen consumption such as smoking and exercise [4]. Exercise is hypothesized to induce oxidative stress by a number of biochemical events [6,7] During exercise, oxygen consumption can increase up to 10- to 15-fold above resting levels, thus temporarily increasing the rate of mitochondrial free radicals production. Exercise may also induce inflammatory reactions similar to the acute phase Address correspondence to: Dr. Chi-Woon Kong. Oxidative Stress Clinical Research Group, Division of Critical Care, Department of Medicine, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Rd., Taipei, 112, Taiwan; Tel: ⫹886 (2) 28330656; Fax: ⫹886 (2) 28740010; E-Mail: [email protected]. 1465

1466

K. TSAI et al.

Recent advances in the single cell gel electrophoresis (SCGE) allow the measurement of in situ DNA damage in lymphocytes or mononuclear cells [13]. Application of this method has disclosed increased leukocyte DNA single strand breaks (SSB) after intensive exercise modes [14,15]. The extent of DNA damage seemed to depend on the training status and the anaerobic thresholds of the study subjects [14,16], and was attenuated by vitamin E supplementation [17]. Nevertheless, the SCGE did not directly detect oxidative DNA damage, and whether the exercise-induced DNA strand breaks were due to base oxidation remained unclear. With the incorporation of an endonuclease that could specifically recognize oxidized purines in the SCGE, Hartmann et al. did not find an increased DNA migration of peripheral leukocytes directly after a short-distance triathlon [15]. However, this finding was insufficient in reaching a definite conclusion because only oxidized purines were detected and sequential postexercise changes were not gauged. Since exercise-induced oxidative stress and immune dysfunctions may vary with different exercise intensities [18], the above controversies can only be resolved by employing a massive exercise in higher intensity and longer duration, such as a 42 km marathon race, as an experimental model. In the present study, we assessed oxidative DNA damage in peripheral blood mononuclear cells (PBMC) from 12 subjects completing a 42 km marathon race. The standard alkaline SCGE assay was performed at sequential time points during a 1-week postrace observation period, and the presence of oxidized purines or pyrimidines was identified with the incorporation of lesion-specific endonucleases [19]. Urinary 8-OHdG excretions were simultaneously measured to provide an improved understanding of the homeostasis between the nucleotide damage and repairing capability during the observation period. MATERIALS AND METHODS

Subjects Fourteen male runners (median age 21, range 20 –24) who completed the 2000 Taiwan 42 km Marathon Race (median running time 3:01 [h:min], range 2:55–3:18) volunteered to participate in this study. Questionnaires were used to exclude the presence of acute or chronic infectious, inflammatory, or immune disorders during the study period. The participants did not take anti-inflammatory agents, steroid hormones, antioxidants, or vitamin supplements. Informed consents were obtained from all the subjects. The participants were also asked to refrain from any form of exercise during the postrun recovery period. Twenty sedentary, healthy males (median age 21, range 20 –22) donated their blood and urine

samples as reference materials. The research protocol was approved by the Committee for Research on Human Subjects of the Taipei Physical Education College, and conformed to the guidelines of the Helsinki Conference for research on human subjects. Reagents Lesion-specific endonucleases, including endonuclease III (for oxidized pyrimidines) and formamidopyrimidine glycosylase (FPG) (for oxidized purines), were obtained from Trevigen (Gaithersburg, MD, USA). SYBR Green I was obtained from Molecular Probes (Eugene, OR, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA). Blood and urine sampling Approximately 5 ml of EDTA-treated venous blood samples was collected from each subject before (Rest), immediately after (Day 0), and then 24 h (Day 1), 72 h (Day 3), 1 week (Day 7), and 2 weeks (Day 14) after the race. A 2 ml blood sample was mixed with an equal volume of PBS. Three milliliters of Histopaque-1119 (Sigma) was layered in 10 ml polypropylene conical tubes. The diluted blood was carefully layered over the gradient and then spun at 300 ⫻ g for 15 min at 4°C. The opaque layer containing PBMC was aspirated and transferred to separate siliconized glass tubes and washed with 5 ml PBS. The cell pellet was resuspended with PBS to give a final cell concentration of 6 ⫻ 104 cells/ml. The remaining 4 ml blood sample was spun at 300 ⫻ g and the plasma stored at ⫺70°C until subsequent analysis (see below). The plasma sample was additionally mixed with 0.1% butylated hydroxytoluene to prevent further oxidation during storage. In addition, simultaneously with blood sampling, an 8 h urine sample was obtained from each participant. Two 10 ml aliquot of urine samples were stored at ⫺70°C until further analysis. Measurement of DNA base oxidative damage The principle of the SCGE (comet assay) for the detection of DNA strand breaks has been described previously [13]. Briefly, microscopic slides were coated with melted 1% standard agarose and dried down on a warm heater. PBMC suspensions (30 ␮l) were mixed with 60 ␮l low melting point agarose (LMA) and added to the slides. The slides were covered with a coverslip and kept in a refrigerator for 5 min. Then the coverslip was removed and the slides were immersed in a jar containing lysing solution (2.5 M NaCl, 0.1 M EDTA, 10

Oxidative DNA damage and exercise

mM Tris, and 1% Triton X-100) at 4°C for 1 h. After lysis, the slides were gently placed in an electrophoresis tank in 0.3 M NaOH, 1 mM Na2EDTA for 40 min. Electrophoresis was then carried out at 25 V and 300 mA for 30 min. After electrophoresis, the slides were neutralized in 0.4 M Tris buffer (pH 7.5). The slides were rinsed in distilled water. After staining with approximately 50 ␮l of SYBR Green I, each slide was immediately viewed by fluorescence microscopy. In separate experiments, the presence of oxidative DNA base damage was determined using lesion-specific endonuclease, as previously described [19,20]. Microscopic slides were coated with the mixture of PBMC and LMA as described above. After cell lysis, slides were washed three times in a buffer containing 40 mM HEPES, 0.1 KCl, 0.5 mM EDTA, and 0.2 mg/ml bovine serum albumin (pH 8.0). Then 50 ␮l of either endonuclease III (1 mg/ml) or FPG (22 pg/␮l), or 100 ␮l of the mixture of both enzyme solutions was placed onto agarose (a total of three separate slides), and the gel was covered with a coverslip and incubated at 37°C for 45 min. All subsequent steps were as mentioned above. Comets in each gel were analyzed using a CCD camera and Komet 3.0 image analysis program (Kinetic Imaging Ltd, Liverpool, UK). The frequency of DNA single strand breaks was determined by measuring the percent of DNA in the tail portion (tail DNA) from a total of 50 mononuclear cells per sample (25 cells from each of the two replicate slides). The data was further standardized by using an internal standard, obtained from the K562 human erythroleukemia cell line, as described previously [21]. The amount of oxidized pyrimidines (endonuclease III-sensitive sites) or purines (FPG-sensitive sites) or both (endocuclease III- and FPG-sensitive sites, a measure of total extents of nucleotide oxidation) in DNA was measured by subtracting the tail DNA without enzyme treatments from the tail DNA with enzyme incubation [22]. Measurement of urinary 8-OHdG excretion The urine sample was thawed and adjusted to pH 6 –7 by the addition of 1 M HCL or NaOH. The urine was then centrifuged at 1500 ⫻ g for 5 min and the supernatant used for analysis. Urinary 8-OHdG was measured using an ELISA kit (BIOXYTECH 8-OHdG-EIA Kit, OXIS, Portland, OR, USA). Each sample was assayed in triplicate and the results were expressed as the 8-OHdG to creatinine ratio. Measurements of plasma oxidants and creatinine kinase EDTA-anticoagulated plasma samples were used for the following assays. Metabolic products of nitric oxide

1467

(NO), including nitrite (NO2⫺ and nitrate (NO3⫺), were measured by a modified Griess method (OXIS). Plasma lipid peroxidation (LPO) metabolites (including malondialdehyde and 4-hydroxyalkenal) were analyzed by a colorimetric assay kit (LPO assay kit, CalBiochem, San Diego, CA, USA). Total plasma creatinine kinase (CK) activity was estimated by a test kit (Sigma, St. Louis, MO, USA). All the results were adjusted according to plasma volume changes following the race [23]. Statistics All continuous data was expressed as mean ⫾ SEM. Group comparison was judged by Mann-Whitney U tests. Sequential changes were assessed by repeated measurement ANOVA. Pearson’s correlation analyses were used to examine relation between markers. A p ⬍ .01 was considered statistically significant. RESULTS

DNA single strand breaks DNA SSB of PBMC, as evaluated by the percent of DNA in the tail after SCGE, gradually increased after the marathon race (Fig. 1A). The increase reached a significant difference on Day 1. The high percentage of DNA strand breaks did not decline until 2 weeks after the race. The sequential changes were statistically significant (p ⬍ .001 by repeated measurement ANOVA). Oxidative DNA damages Sequential changes of the levels of FPG- and endonuclease III-sensitive sites on nuclei of PBMC, are shown in Fig. 1B and 1C. Before the race, the mean level of FPG-sensitive sites was low (less than 1% of additional DNA in the tail after FPG treatments). Higher levels of FPG sites appeared immediately (Day 0) and 24 h (Day 1) after the race (Fig. 1B), and the level gradually normalized thereafter. In contrast, the sequential changes in the levels of endonuclease III sites were more pronounced, showing biphasic patterns after the race (Fig. 1C). The first peak appeared immediately postexercise, with the second on Day 7. When the cell nuclei of PBMC were treated simultaneously with FPG and endonuclease III, the overall magnitude of DNA base oxidation was quantified, which showed biphasic patterns of alterations similar to that of endonuclease III-sensitive sites (Fig. 1D). All the time-sequence changes were statistically significant (p ⬍ .001 by repeat measurement ANOVA). Also worth noting was that the level of FPG- and endonuclease III-sensitive sites seemed to be additive, since their sum approximated the

1468

K. TSAI et al.

Fig. 1. Time-sequence changes of strand breaks (A), formamidopyrimidine glycosylase (FPG)-sensitive sites (B), endonuclease III-sensitive sites (C), and FPG and endonuclease III-sensitive sites (D) in the nuclei of peripheral blood mononuclear cells following the marathon race. Values are expressed as means ⫾ SEM. Mean value of healthy controls (■) is also shown. *p ⬍ .01 compared with control value; †p ⬍ .01 by repeated measurement ANOVA.

level of tail DNA after simultaneous treatments of both enzymes (r ⫽ 0.835, p ⬍ .001).

more prominent and prolonged (Fig. 3C). Peak LPO levels appeared at Day 1 (up to 4.5-fold), and the rise lasted until the end of the 2 week follow-up period.

Urinary 8-OHdG excretion Figure 2 shows sequential changes of urinary concentration of 8-OHdG before and after the marathon race (p ⬍ .001 by repeat measurement ANOVA). The level increased approximately 2-fold immediately postrace, remaining supranormal through the 1 week follow-up (Day 7). Plasma CK, NO metabolites, and LPO Figure 3 displays time-sequence changes of plasma levels of CK, nitrite/nitrate, and LPO before and after the marathon race. One day following the race, CK increased more than 2-fold, indicating a significant degree of skeletal muscle damage. CK levels did not normalize until 2 weeks after the race (Fig. 3A). Immediately after the race, plasma levels of nitrite/nitrate increased abruptly (1.6-fold), and declined thereafter (Fig. 3B). In contrast, the postexercise alterations in plasma LPO levels were

Fig. 2. Urinary 8-hydroxy-2⬘-deoxyguanosine (8-OHdG) excretions before and during various times after the marathon race. Values are expressed as means ⫾ SEM. Mean value of healthy controls (■) is shown. *p ⬍ .01 compared with control value; †p ⬍ .01 by repeated measurement ANOVA.

Oxidative DNA damage and exercise

1469

Table 1. Correlations of Oxidative DNA Damages with Associating Parameters FPG and Endonuclease endonuclease Urinary FPG sites III sites III sites 8-OHdG CK NO metabolites LPO

0.279 0.247 0.318*

0.489** 0.375* 0.336*

0.459** 0.332* 0.446**

0.368* 0.286 0.349*

Values are Pearson’s correlation coefficients. FPG ⫽ formamidopyrimidine glycosylase; 8-OHdG ⫽ 8-hydroxy-2⬘-deoxyguanosine; CK ⫽ cratinine kinase; NO ⫽ nitric oxice; LPO ⫽ lipid peroxidation products. * p ⬍ .05, ** p ⬍ .01.

plasma nitrite/nitrate levels showed significant correlation with endonuclease III sites and the total extent of DNA base oxidation (FPG and endonuclease III-sensitive sites), but not with FPG sites. Significant correlation existed between plasma LPO levels and all markers of DNA damage. When all time point data are considered together, there are no statistically significant correlations between urinary 8-OHdG and various comet parameters (SSB, FPG- and/or endonuclease III-sensitive sites, p ⬎ .05). The mutual correlation between FPG- and endonuclease III-sensitive sites is also insignificant (p ⬎ .05). DISCUSSION

Fig. 3. Time sequence changes of plasma levels of creatinine kinase (CK) (A), nitric oxide (NO) metabolites (B), and lipid peroxidation (LPO) metabolites (C) before and after the marathon race. Values are adjusted according to plasma volume changes following the race and are expressed as means ⫾ SEM. Mean value of healthy controls (■) is also shown. *p ⬍ .01 compared with control value; †p ⬍ .01 by repeated measurement ANOVA.

Correlations among markers Mutual correlations between peak DNA damage and associated plasma parameters immediately postexercise are shown in Table 1. Plasma CK levels were correlated significantly with endonuclease III-sensitive sites and urinary 8-OHdG, but not with FPG sites. Similarly,

Our results corroborate previous findings of the delayed increase in DNA strand breaks after a massive aerobic exercise. Although the exact pathomechanisms behind this phenomenon remains unclear, it seems that the duration and extent of DNA effects increase synchronously with the amount and intensity of exercise. After a single bout of exhaustive treadmill running, DNA strand breaks in PBMC reached its maximum at 24 h and subsided 72 h later [16]. When exercise of longer duration was performed (triathlons with a mean exercise duration of 2.5 h), the DNA damage peaked 3 d following the exercise and lasted for more than 5 d [15]. In the present study, the long-duration (more than 3 h) competitive marathon race produced even more prominent DNA strand breaks, which lasted for more than 1 week following the race. Of note, the magnitude of exerciseinduced DNA effects may also be affected by prior training status and the physical fitness of the subjects [14,16]. Regular training seemed to confer protection against further exercise-induced DNA damage, which had been attributed to adaptive responses involving upregulation of cellular proteolytic enzymes and repairing systems [24]. Although the athletes enrolled in the present study undertake regular running training, substantial increase in DNA strand breaks of PBMC still occurred. This finding suggests the potentially harmful

1470

K. TSAI et al.

effects of exhaustive exercise on the DNA of peripheral leukocytes [16]. The traditional SCGE assay does not detect in situ, unrepaired base damage. By incorporating FPG and endonuclease III in the analysis of DNA migration [19], our study further identified oxidative base damage following the marathon race. As shown in Fig. 1, the oxidative modification to nucleotides appeared immediately postexercise and persisted for more than 1 week. Oxidized pyrimidines, as determined by endonuclease III-sensitive sites, seemed to account for most of the oxidative DNA damage induced by a marathon race. It is possible that ROS produced during exhaustive exercise is more likely to cause oxidation of pyrimidines than purines. However, this notion contradicts the general belief that purines such as guanines are more easily oxidized, as 8-OHdG is the most abundant oxidatively modified DNA lesion [2]. Alternatively, the less marked increase in FPG-sensitive sites, coupled with a prominent postrace increase in the urinary excretion of 8-OHdG, the most abundant repair product of oxidative damages to purines, seemed to imply that oxidized purines were repaired more effectively that oxidized pyrimidines in PBMC. Previously, a short-distance triathlon did not lead to the appearance of in situ oxidized DNA bases in leukocytes or an increase in urinary 8-OHdG excretions [15]. However, it should be noted that only FPG-sensitive sites on DNA were evaluated in this study, and the exercise mode employed is not a competitive one. As shown in our results, the changes in endonuclease III-sensitive sites contributed to most of the postexercise oxidative DNA damage. FPG-sensitive sites on PBMC DNA only showed modest changes after the marathon race. Furthermore, exercise-induced leukocyte DNA damage may vary according to the mode, intensity, and duration of the exercise protocol and prior training status of the participants, which may lead to inconsistent results in relevant studies [14,16]. In the present study, the 42 km competitive marathon race was more vigorous than a treadmill run or a short-distance triathlon. It was conceivable that under the present long-duration and massive exercise mode, prior controversies in the exercise-related DNA effects can be resolved. The measurement of urinary 8-OHdG as a marker of oxidative DNA damages in the present study demands further discussions. First, urinary excretion of 8-OHdG reflects the average rate of oxidative DNA damages in all the cells in a body. Its increase postexercise may be the net result of exercise-induced muscle damages and increased cell turnover, and is not specific to PBMC. Second, the kinetics of the repair of oxidative DNA lesions in human cells have not been fully understood. It may take more than 4 h for human lymphocytes to remove their oxidized pyrimidines after the oxidative

damage [25]. Therefore, the 8 h urine samples might not contain the total repairing products of oxidized nucleotides after the marathon race. Third, the urinary excretion of 8-OHdG did not correlate significantly with various comet parameters. This may suggest that the repair products and the unrepaired lesions of oxidative DNA damages do not correspond with each other. They should be taken as independent parameters that provide supplementary data during the measurement of oxidative DNA damages in vivo. The mechanisms behind the leukocyte oxidative DNA damage following a marathon race may be multifold. First, exhaustive exercise may induce increased production of ROS in the vascular compartments due to myofibril injuries with inflammatory cell infiltrations and the activation of circulating phagocytes and, possibly, endothelial xanthine oxidase [6,7]. ROS produced by this manner may permeate into peripheral leukocyte and lead to modification of nucleic acid [26]. Aside from direct attack of leukocyte DNA by ROS, the various products of lipid peroxidation can also interact with DNA and cause oxidation of nucleotides [27]. This notion derived support from the positive correlations of plasma LPO levels with various markers of oxidative DNA damage. In addition, ROS may also be produced during the reparative process of tissue damage following exhaustive exercise [28,29]. This mechanism may help account for the delayed appearance of endonuclease III sites 1 week following the race. Second, as demonstrated by Mars et al., exhaustive exercise may produce a high percentage (up to 86%) of apoptosis in lymphocytes [30]. Although characteristic apoptotic cells with DNA fragmentations were not found under the present alkaline SCGE assay conditions [31], we could not exclude the possibility that some of the included cells were those undergoing early apoptic changes. If this was the case, the increased uncoupling of mitochondria electron transport during apoptotic events may lead to increased intracellular production of ROS, producing oxidative DNA damage [32]. Third, after the marathon race, metabolic products of NO in plasma increased up to 1.6-fold, probably due to increased inducible NO synthase expression and NO synthesis in peripheral leukocytes [26]. The increased amounts of NO may contribute to the leukocyte DNA damage due to its capability of damaging DNA and inhibiting DNA repair enzymes [33,34]. The significant correlation of plasma levels of NO metabolites with endonuclease III sites on DNA seemed to confer partial support to the presence of this mechanism. The clinical significance of exercise-induced leukocyte oxidative DNA damage is obscure. Conceptually, DNA strand breaks and modified bases may lead to miscoding, mutation, and decreased efficiency of DNA reproduction [2,35]. The accumulation of these base

Oxidative DNA damage and exercise

modifications may potentially contribute to carcinogenesis and cellular dysfunction. However, epidemiological data do not show that long-term strenuous competitive sports increase the risk of cancer [36 –38]. In fact, the increased DNA strand breaks of peripheral leukocytes after exercise were not coupled with significant chromosome changes such as micronuclei or sister chromatid exchanges, implicating an adequate repair of DNA lesions [15,16]. Another finding worth mentioning was the parallel changes of leukocyte oxidative DNA damage and depressed innate immunity after a marathon race. Massive and long duration exercise such as marathon running increases the risk of opportunistic infections, which may persist for more than 1 week following the race [39]. At the present stage of knowledge it is hard to discriminate whether the DNA oxidation of peripheral immunocompetent cells have casual relationships with postexercise immune disturbances or both are secondary to increased oxidative stress induced by exhaustive exercise. Caution must also be taken in the interpretation of the alterations of peripheral blood leukocyte parameters in the present clinical model. It was not due to the changes of the same cell population but instead due to postexercise leukocyte subpopulational changes [40]. The acute changes in the PBMC DNA could be related to the recruitment of young cells with immature ability to repair their DNA damages [41]. Further laboratory investigations in this regard are needed to further explore the basic mechanisms leading to the observed changes. In conclusion, a long duration, massive exercise such as a marathon race leads to oxidative DNA damage in PBMC. The damages are more prominent on pyrimidines and last long following the exercise. Whether the DNA oxidation has mutagenic effects and whether antioxidant supplementations may reverse this phenomenon remains to be investigated. Acknowledgements — This project was supported, in part, by grant VAC 90044 from Taipei Veterans General Hospital and by NSC 89-2320-B-154-004 from the National Science Council, Taiwan. We wish to express our appreciation to the elite athletes who participated in the study.

REFERENCES [1] Adelman, R.; Saul, R. L.; Ames, B. N. Oxidative damage to DNA: relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. USA 85:2706 –2708; 1988. [2] Loft, S.; Poulsen, H. E. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74:297–312; 1996. [3] Shigenaga, M. K.; Gimeno, C. J.; Ames, B. N. Urinary 8-hydroxy-2⬘-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. USA 86:9697–9701; 1989. [4] Loft, S.; Vistisen, K.; Ewertz, M.; Tjønneland, A.; Overvad, K.; Poulsen, H. E. Oxidative DNA-damage estimated by 8-hydroxydeoxyguanosine excretion in man: influence of smoking, gender and body mass index. Carcinogenesis 13:2241–2247; 1992.

1471

[5] Loft, S.; Astrup, A.; Buemann, B.; Poulsen, H. E. Oxidative DNA damage correlates with oxygen consumption in humans. FASEB J. 8:534 –537; 1994. [6] Sjo¨ din, B.; Westing, Y. H.; Apple, F. S. Biochemical mechanisms for oxygen free radical formation during exercise. Sports Med. 10:236 –254; 1990. [7] Alessio, H. M. Exercise-induced oxidative stress. Med. Sci. Sports Exerc. 25:218 –224; 1993. [8] Camus, G.; Deby-Dupont, G.; Duchateau, J.; Deby, C.; Pincemail, J.; Lamy, M. Are similar inflammatory factors involved in strenuous exercise and sepsis? Intensive Care. Med. 20:602– 610; 1994. [9] Ji, L. L. Oxidative stress during exercise: implication of antioxidant nutrients. Free Radic. Biol. Med. 18:1079 –1086; 1995. [10] Alessio, H. M.; Culter, R. G. Evidence that DNA damage-andrepair cycle activity increases following a marathon race. Med. Sci. Sports Exerc. 22:751; 1990. [11] Nielsen, H. B.; Hanel, B.; Loft, S.; Poulsen, H. E.; Pedersen, B. K.; Diamant, M.; Vistisen, K.; Secher, N. H. Restricted pulmonary diffusion capacity after exercise is not an ARDS-like injury. J. Sports Sci. 13:109 –113; 1995. [12] Witt, E. H.; Reznick, A. Z.; Viguie, C. A.; Starke-Reed, P.; Packer, L. Exercise, oxidative damage and effects of antioxidant manipulation. J. Nutr. 122:766 –773; 1992. [13] Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. A simple technique for quantification of low levels of DNA damage in individuals cells. Exp. Cell Res. 175:184 –191; 1988. [14] Niess, A. M.; Hartmann, A.; Guchs-Gru˝ nert-Fuchs, M.; Poch, B.; Speit, G. D. N. A.; damage after exhaustive treadmill running in trained and untrained men. Int. J. Sports Med. 17:397– 403; 1996. [15] Hartmann, A.; Pfuhler, S.; Dennog, C.; Germadnik, D.; Pilger, A.; Speit, G. Exercise-induced DNA effects in human leukocytes are not accompanied by increased formation of 8-hydroxy-2⬘-deoxyguanosine or induction of micronuclei. Free Radic. Biol. Med. 24:245–251; 1998. [16] Hartmann, A.; Plappert, U.; Raddatz, K.; Gru˝ nert-Fuchs, M.; Speit, G. Does physical activity induce DNA damage? Mutagenesis 9:269 –272; 1994. [17] Hartmann, A.; Nieß, A. M.; Gru˝ nert-Fuchs, M.; Poch, B.; Speit, G. Vitamin E prevents exercise-induced DNA damage. Mutat. Res. 346:195–202; 1995. [18] Woods, J. A.; Davis, J. M.; Smith, J. A.; Nieman, D. C. Exercise and cellular innate immune function. Med. Sci. Sports Exerc. 31:57– 66; 1999. [19] Collins, A. R.; Dusˇinska´ , M.; Gedik, C. M.; Sˇ te˘ tina, R. Oxidative damage to DNA: do we have a reliable biomarker? Environ. Health Perspect. 104(Suppl. 3):465– 469; 1996. [20] Piperakis, S. M.; Visvardis, E. E.; Tassiou, A. M. Comet assay for nuclear DNA damage. Methods Enzymol. 300:184 –194; 1999. [21] De Boeck, M.; Touil, N.; De Visscher, G.; Vande, P. A.; KirschVolders, M. Validation and implementation of an internal standard in comet assay analysis. Mutat. Res. 469:181–197; 2000. [22] Collins, A. R.; Rasˇlova, K.; Somorovska´ , M.; Petrovska´ , H.; Ondrusˇova´ , A.; Vohnout, B.; Fa´ bry, R.; Dusˇinska´ , M. DNA damage in diabetes: correlation with a clinical marker. Free Radic. Biol. Med. 25:373–377; 1998. [23] Costill, D. L.; Fink, W. J. Plasma volume changes following exercise and thermal dehydration. J. Appl. Physiol. 37:521–525; 1974. [24] Rada´ k, Z.; Kaneko, T.; Tahara, S.; Nakamoto, H.; Ohno, H.; Sasva´ ri, N.; Nyakas, C.; Goto, S. The effect of exercise training on oxidative damage of lipids, proteins, and DNA in rat skeletal muscle: evidence for beneficial outcomes. Free Radic. Biol. Med. 27:69 –74; 1999. [25] Collins, A. R.; Ai-guo, M.; Duthie, S. J. The kinetics of repair of oxidative DNA damage (strand breaks and oxidized pyrimidines) in human cells. Mutat. Res. 336:69 –77; 1995. [26] Niess, A. M.; Dickhuth, H.; Northoff, H.; Fehrenbach, E. Free radicals and oxidative stress in exercise—immunological aspects. Exerc. Immunol. Rev. 5:22–56; 1999. [27] Chaudhary, A. K.; Nokubo, M.; Reddy, G. R.; Yeola, S. N.;

1472

[28] [29] [30] [31] [32]

[33]

[35]

[36]

K. TSAI et al. Morrow, J. D.; Blair, I. A.; Marnett, L. J. Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265:1580 –1581; 1994 Sjo¨ din, B.; Westing, Y. H.; Apple, F. S. Biochemical mechanisms for oxygen free radical formation during exercise. Sports Med. 10:236 –254; 1990. Zarba, E.; Komorowski, T.; Faulkner, J. A. Free radical injury to skeletal muscles of young, adult, and old mice. Am. J. Physiol. 258:C429 –C435; 1990. Mars, M.; Govender, S.; Weston, A.; Naicker, W. V.; Chuturgoon, A. High intensity exercise, a cause of lymphocyte apoptosis? Biochem. Biophys. Res. Commun. 249:366 –370; 1998. Olive, P.; Frazer, G.; Banath, J. P. Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay. Radiat. Res. 136:130 –136; 1993. Zamzami, N.; Marchetti, P.; Castedo, M.; Decaudin, D.; Macho, A.; Hirsch, T.; Susin, S. A.; Petit, P. X.; Mignotte, B.; Kroemer, G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182:367–377; 1995. Delaney, C. A.; Green, I. C.; Lowe, J. E.; Cunningham, J. M.; Butler, A. R.; Renton, L.; D’Costa, I.; Green, M. H. Use of the comet assay to investigate possible interactions of nitric oxide and reactive insulin-secreting cell line. Mutat. Res. 375:137–146; 1997. [34] Wink, D. A.; Laval, J. The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and vivo. Carcinogenesis 15:2125–2129; 1994. Floyd, R. A.; Watson, J. J.; Harris, J.; West, M.; Wong, P. K. Formation of 8-hydroxydeoxyguanosine, hydroxyl free radical adduct of DNA in granulocytes exposed to the tumor promoter, tetradecanoylphobolacetate. Biochem.Biophys. Res. Commun. 137:841– 846; 1986. Thune, I.; Brenn, T.; Lund, E.; Gaard, M. Physical activity and the risk of breast cancer. N. Engl. J. Med. 336:1269 –1275; 1997.

[37] Thune, I.; Lund, E. The influence of physical activity on lung cancer risk: a prospective study of 81,516 men and women. Int. J. Cancer 70:57– 62; 1997. [38] VanSaase, J. L.; Noteboom, W. M.; Vandenbrouke, J. P. Longevity of men capable of vigorous physical activity exercise: a 32-year follow-up of 2259 participants in the Dutch Eleven Cities Ice Skating Tour. Br. Med. J. 301:1409 –1411; 1990. [39] Woods, J. A.; Davis, J. M.; Smith, J. A.; Nieman, D. C. Exercise and cellular innate immune function. Med. Sci. Sports Exerc. 31:57– 66; 1999. [40] Pyne, D. B. Regulation of neutrophils function during exercise. Sports Med. 17:245–258; 1994. [41] Inoue, T.; Mu, Z.; Sumikawa, K.; Adachi, K.; Okochi, T. Effect of physical exercise on the content of 8-hydroxydeoxyguanosine in nuclear DNA prepared from human lymphocytes. Jpn. J. Cancer Res. 84:720 –725; 1993.

ABBREVIATIONS

ROS—reactive oxygen species 8-OHdG— 8-hydroxy-2⬘-deoxyguanosine SCGE—single cell gel electrophoresis SSB—single strand break PBMC—peripheral blood mononuclear cell FPG—formamidopyrimidine glycosylase LMA—low melting point agarose NO—nitric oxide LPO—lipid peroxidation products CK— creatinine kinase