Objective: Physical exercise is capable of enhancing or suppressing the immune response depending on the intensity and duration of exercise. This study investigated how exercise intensity influences the lymphocyte antioxidant response and the induction of cellular oxidative damage.
Design: Eighteen voluntary male pre-professional soccer players participated in this study. Sportsmen played a 60 min training match, and were divided into three groups depending on the intensity degree during the match: low, medium and high intensities.
Measurements: Malondialdehyde (MDA), vitamins C and E and haem oxygenase-1 (HO-1) gene expression were measured in lymphocytes. Reactive oxygen species (ROS) production was determined in lymphocytes and neutrophils.
Results: Lymphocyte MDA levels and H2O2 production were significantly increased in the group which performed the most intense exercise. Neutrophil counts and ROS production increased progressively with the exercise intensity. Vitamin C significantly decreased after exercise in the highest-intensity group in comparison with initial values, whereas vitamin E levels significantly increased in the medium and high-intensity groups. HO-1 gene expression significantly increased in the medium and high-intensity groups.
Conclusions: Exercise intensity affects the lymphocyte and neutrophil oxidant/antioxidant balance, but only exercise of high intensity induces lymphocyte oxidative damage.
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The physiological response to physical exercise involves a number of changes in the oxidative balance and in the metabolism of some important biological molecules.1 Physical exercise is characterised by an increase in oxygen consumption by the whole body and by an increase in reactive oxygen species (ROS) production.2 The main sources of ROS during exercise are the mitochondrial respiratory chain, xanthine oxidase-catalysed reaction and neutrophil activation.3 ROS are known to cause oxidative modifications of lipids, proteins and nucleic acids leading to cell and tissue damage.4–7 Under physiological conditions, these deleterious species are mostly removed by the cellular antioxidant systems. Exercise-induced ROS are also thought to modulate acute-phase inflammatory responses8 and to have a role in cell signalling inducing specific cellular adaptations to exercise.9 10
Exhaustive exercise elicits a stress response similar to the acute phase immune response.8 Exercise affects lymphocytes as reflected in total blood cell counts and lymphocyte proliferative response.11 12 It is widely accepted that athletes undergoing intensive training and competition schedules are at increased risk of developing upper respiratory tract infections.13 Strenuous physical exercise, characterised by a remarkable increase in oxygen consumption with concomitant ROS production, could lead to an oxidative stress situation and cell damage.3 It has been evidenced that athletes undergoing regular and adequate training show improved intracellular antioxidant status14 15 and increased resistance to upper respiratory tract infections.16
Soccer is one of the most popular sports worldwide, but only a few studies have focused on the effect of soccer practice on the antioxidant status of players. Competitive soccer engages many of the body’s systems to a major extent, to a point where oxidative-derived damage can appear.17 Some studies have compared the oxidant and antioxidant status of soccer players and sedentary controls. Total antioxidant capacity, superoxide dismutase activity, uric acid, ascorbic acid and tocopherol plasma levels were all higher in soccer players, while MDA levels were lower.14 18 The levels of autoantibodies against oxidised LDL in professional soccer players are increased as result of intensive training-induced oxidative stress.19 Despite these studies, there are few data concerning the effects of a soccer match on the antioxidant status of players.
The aim of this study was to determine the differential effects of the exercise intensity on the antioxidant response and on the cellular oxidative damage in lymphocytes induced by a training soccer match. Neutrophil capability to produce ROS was also determined for comparison with lymphocytes.
MATERIALS AND METHODS
Subjects and protocol
Eighteen voluntary male pre-professional soccer players participated in this study after giving their written consent to participate. The protocol was in accordance with the Declaration of Helsinki for research on human subjects and was approved by the Ethical Committee of Clinical Investigation of CAR-Sant Cugat (Barcelona). All sportsmen participating in the study had a controlled diet and expended similar periods of training and competition.
The sportsmen played a training soccer match for 60 minutes. The exercise intensity was determined by a pulsometer, and sportsmen were divided into three groups (n = 6) depending on intensity degree. As the cardiac heart rate increases linearly with oxygen consumption,20 we can indirectly evaluate the work done during maximal and intervallic exercise through the heart rate.21 We categorise the subjects according to the perspective of the work performed during the training sessions and the competition in relation to the reference values of a progressive and maximal exercise test. Five metabolic zones are usually considered. From zone one (Z1) to zone five (Z5), the relation to maximal oxygen consumption is respectively <70%, 70–80%, 80–90%, 90–100% and 100% or higher. Groups were simplified to three (table 1), the low-intensity (more than 50% of match into zones Z1 plus Z2) the medium-intensity (more than 50% of match into zone Z3) and the high-intensity (more than 50% of the match into zones Z4 plus Z5).
The three groups had similar anthropometric values (table 2).
Venous blood samples were obtained from the antecubital vein with EDTA as anticoagulant. Samples were obtained in basal conditions and immediately after exercise finished. Blood samples were used to purify lymphocytes and neutrophils, and to obtain plasma. Lymphocyte and neutrophil counts were quantified in an automatic flow cytometer analyser Technicon H2 (Bayer) VCS system.
Lymphocyte, neutrophil and plasma purification
Blood cells were immediately purified from whole blood following an adaptation of the method of Boyum.22 Blood was introduced onto Ficoll and centrifuged at 900×g at 4°C for 30 min. The lymphocyte layer was carefully removed and washed twice with PBS and centrifuged for 10 min at 1000×g at 4°C. This method ensures that 95% (SD 5%) of cells in fraction are mononucleocytes with 95% (SD 5%) viability. The cellular precipitate of lymphocytes was lysed with distilled water. The precipitate obtained after centrifugation with Ficoll, containing erythrocytes and neutrophils, was incubated at 4°C with ammonium chloride 0.15 mol/l to haemolyse erythrocytes. The suspension was centrifuged at 750×g at 4°C for 15 min and the supernatant was discarded. The neutrophil phase at the bottom was washed first with ammonium chloride and then with PBS. Neutrophils were resuspended in Hank’s balanced salt solution (HBSS) for chemiluminescence assays.
Plasma was obtained after centrifugation for 15 min at 1000×g at 4°C of another blood sample and was stored at −80°C until use.
Lymphocyte MDA concentration
MDA as a marker of lipid peroxidation was analysed in lymphocytes using a colorimetric assay kit (Calbiochem, San Diego, CA, USA) by following the manufacturer’s instructions. This assay kit is specific for MDA, avoiding the poor reproducibility and the interference by several agents of the classical determination by the TBARs method.23
Lymphocyte vitamin C and vitamin E determination
Samples for vitamin C were deproteinised with 5% metaphosphoric acid and centrifuged for 5 min at 15 000×g at 4°C, and the supernatants recovered. The mobile phase consisted of 0.05 mol/l sodium phosphate, 0.05 mol/l sodium acetate, 189 μmol/l dodecyltrimethylammonium chloride and 36.6 mmol/l tetraoctylammonium bromide in 25/75 methanol/water (v/v), pH 4.8. The HPLC system was a Shimadzu with a Waters Inc. electrochemical detector and a Nova Pak, C18, 3.9×150 mm column. The potential of the chromatographic detection was set at 0.7 V versus an Ag/AgCl reference electrode. Vitamin C was quantified by using a standard curve of known concentration.
Vitamin E was extracted from lymphocyte lysates using n-hexane after deproteinisation with ethanol. Vitamin E concentration was determined by HPLC in the n-hexane extract after samples had been dried under nitrogen and dissolved in methanol. The mobile phase consisted of 550:370:80 acetonitrile:tetrahydrofuran:H2O, and the column was Nova Pak, C18, 3.9×150 mm. α-tocopherol isoform was determined at 290 nm and was quantified by comparison to a standard curve of known concentration.
Vitamin concentrations were calculated by taking into account the lymphocyte volume of 2.1×10−5 μl/lymphocyte.24
Lymphocyte hydrogen peroxide production
H2O2 production in lymphocytes was measured before and after stimulation with phorbol myristate acetate (PMA) using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator. DCFH-DA (30 μg/ml) in PBS was added to a 96-well microplate containing lymphocyte suspension. PMA (3 μmol/l) prepared in HBSS or HBSS alone was added to the wells and the fluorescence (Ex 480 nm; Em 530 nm) was recorded at 37°C for 1 h in a FLx800 Microplate Fluorescence Reader (Bio-tek Instruments, Inc., USA).
Neutrophil chemiluminescence assay
Opsonised zymosan (OZ) was used as neutrophil stimulant. Zymosan A (Sigma) was suspended in HBSS at a concentration of 1 mg/ml and incubated with 10% human serum at 37°C for 30 min, followed by centrifugation at 750×g for 10 min at 4°C. The precipitate was washed twice in HBSS and finally resuspended in HBSS at 1 mg/ml. OZ suspension (100 μl) was added to a 96-well microplate containing 50 μl neutrophil suspension and 50 μl luminol solution (2 mmol/l in PBS, pH 7.4). Chemiluminescence was measured at 37°C for 90 min in a FLx800 Microplate Fluorescence Reader.
Real-time reverse transcriptase–polymerase chain reaction
Lymphocyte mRNA was isolated by phenol–chloroform extraction. cDNA was synthesised from 1 μg total RNA using reverse transcriptase with oligo-dT primers. Quantitative PCR was performed using the LightCycler instrument (Roche Diagnostics) with DNA-master SYBR Green I. The primers used were: HO-1, forward: 5′-CCAGCG GGCCAGCAACAAAGTGC-3′, reverse: 5′-AAGCCTTCAGTGCCCACGGTAAGG-3′ and 18S, forward: 5′-ATGTGAAGTCACTGTGCCAG-3′, reverse: 5′-GTGTAATCCGTCTCCACAGA-3′. The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 0 s, 60°C for 5 s and 72°C for 10 s for HO-1 and for ribosomal 18S 40 cycles at 95°C for 10 s, 60°C for 7 s and 72°C for 12 s. The relative quantification was performed by standard calculations considering 2(-ΔΔCt). Basal mRNA levels of the low-intensity group were arbitrarily referred to as 1. The expression of target gene was normalised with respect to ribosomal 18S.
Statistical analysis was carried out using a statistical package (SPSS 12.0 for Windows). Results are expressed as mean (SEM) and p<0.05 was considered statistically significant. The statistical significance of the data was assessed by two-way analysis of variance (ANOVA). The statistical factors analysed were the exercise intensity (Fc) and the soccer match (E). When significant effects were found, a one-way ANOVA was used to determine the differences between the groups involved.
Lymphocyte number and haematocrit were similar in the three groups in basal conditions and did not change during the exercise in any of the three studied groups (data not shown).
No significant differences were observed between groups in the lymphocytes’ basal ROS production (fig 1). ROS production in non-activated lymphocytes significantly increased only in the most intense group after the match (24%). This increase is significantly different when compared with the post-exercise values obtained in the low-intensity group. In PMA-activated lymphocytes, ROS production maintained initial values in the groups which performed the lower and medium-intensity exercise. ROS production significantly increased in the high-intensity group after exercise (33%), the final values in this group being significantly higher than the final values measured in the low and medium-intensity groups.
Neutrophil number and ROS production were similar in the three groups in basal conditions (table 3).
Exercise induced an increase in the number of circulating neutrophils, and the increase degree depends on the exercise intensity. In the low-intensity group circulating neutrophils increased 36%, in the medium group 61% and in the high group 83%. ROS production in zymosan-stimulated neutrophils only increased significantly in the high-intensity group with respect to pre-exercise values. This increase is significantly different compared with post-exercise values of the low-intensity group.
Both exercise and exercise intensity influenced lymphocyte vitamin C and E levels (table 4).
Vitamin C significantly decreased after exercise in the highest-intensity group with respect to initial values (34%), whereas basal values were maintained in the other groups. This vitamin C decrease was significantly different when compared with the vitamin C levels obtained in the lower-intensity group, measured after the match. Vitamin E levels significantly increased in the medium (28%) and high (34%)-intensity groups after exercise. The after-match values in the most intense group were significantly higher than the ones obtained in the low-intensity group after the match. There were no significant differences in the basal values of MDA concentration between groups. MDA levels were significantly increased in the high-intensity group after the match (42%), whereas the other two groups maintained initial values. The increase in MDA levels in the high-intensity group after the match was not significantly different from the results obtained in the medium and low-intensity groups.
HO-1 expression was similar in the three groups in the basal point (Figure 2). After the training match, HO-1 expression significantly increased in the groups which performed the medium and high exercise intensities (57% and 86% respectively). However, the post-exercise values in the medium and high intensity groups were not significantly different respect the post-exercise values measured in the low intensity group.
Moderate exercise is a healthy practice; however, exhaustive exercise induces oxidative stress. Moderate exercise attenuates lymphocyte apoptosis induced by oxidative stress, possibly by improving intracellular antioxidative capacity.15 In previous studies, we reported that a mountain cycling stage induced a significant lymphopenia and high levels of oxidative stress in lymphocytes,25 whereas a cycling stage without mountainous terrain maintained lymphocyte counts.26 These results suggest that the exercise intensity could be responsible for the stress-induced changes. In fact, in the present study, MDA concentration increased only in the group that performed the most intense exercise. We also reported an inverse correlation between lymphocyte protein carbonyl derivatives and lymphocyte number.11
We studied the lymphocytes’ capability to produce ROS by using DCFH-DA before and after stimulation with PMA as a possible source of oxidative stress. However, DCFH can suffer an auto-oxidation that appears to form trace amounts of H2O2, but the rate of auto-oxidation should be of equal importance in each sample.27 The progressive increase in the capabilities of ROS production in the groups which performed the moderate (with no significant differences) and high-intensity exercise after the match indicates a relationship between exercise intensity and the appearance of oxidative stress. Exercise probably induces an increase in mitochondrial oxidant production as result of the increased oxygen availability. However, recent publications on the production of ROS show that a small increase in H2O2 is necessary for the activation of some intracellular signalling pathways, responsible for the development of an adaptive response to exercise-induced oxidative damage.28 29
Exercise induces an increase in the number of circulating neutrophils related to the intensity of the physical activity. In previous studies, we evidenced that an exhaustive exercise such as a duathlon competition or a cycling mountain stage increases the neutrophil counts about fourfold,30 31 while in another study, following a flat cycling stage, neutrophil number only increased twofold.32 Circulating neutrophils in the high-intensity group, but not in the low and medium-intensity groups, seem primed for an oxidative burst after exercise, as is evidenced by the progressive increase in the maximum luminol chemiluminescence. This response is similar to that obtained for lymphocytes. The increased preactivation state of neutrophils from the most intensely exercised group could contribute, at least in part, to the induction of oxidative stress.
Deficiency of antioxidant nutrients appears to hamper antioxidant systems and augment exercise-induced oxidative stress and tissue damage.3 33 Vitamin C prevents initiation of lipid peroxidation and spares other critical antioxidants including α-tocopherol and urate.34 Vitamin C is also an essential metabolite for cell and tissue metabolism, especially for collagen synthesis and for regulation of HIF-1α.35 36 Although vitamin C may interact with ‘free’ active metal ions contributing to oxidative damage,37 its relevance in vivo has been a matter of controversy. Lymphocyte vitamin C levels decreased significantly only in the group which performed the most intense exercise, probably because this group produced more ROS and consequently more vitamin C was consumed. Vitamin E levels were unchanged in the low-intensity group, but significantly increased in the other two groups. These results are in agreement with the increase observed in lymphocyte vitamin E just after a half-marathon or after a mountain cycling stage.28 38 It seems that lymphocyte vitamin E uptake is activated by oxidative stress in order to protect the cell from the action of ROS, according to the levels of oxidative stress.
When HO-1 gene expression was analysed, a significant increase was evidenced in the medium and higher-intensity groups. HO-1 is an antioxidant stress protein induced by stressful and inflammatory stimuli.39 HO-1 is particularly sensitive to acute exercise, being activated by the NF-kB pathway. Several authors have evidenced an increase in HO-1 after a half-marathon39 or after a treadmill test until exhaustion,40 but not after a short run or eccentric exercise.39 Our results suggest that HO-1 is activated depending on the degree of ROS production.
In conclusion, exercise affects the lymphocyte antioxidant system response and induces cellular oxidative damage depending on its intensity. Intense exercise enhances lymphocyte and neutrophil ROS production and produces lipid peroxidative damage and lymphocyte ascorbate consumption, activating the antioxidative response as evidenced by the increase in lymphocyte HO-1 expression. Intense exercise-induced oxidative stress is probably related to a disturbance in redox homeostasis. Thus, physical exercise provides an excellent model to study the relationship between pro-oxidants and the antioxidant defenses in healthy subjects.
What is already known on this topic
Data studying the effects of soccer practice on the antioxidant status of players are scarce. Several studies evidenced the deleterious effects of increased reactive species after exhaustive exercise resulting in oxidative stress. However, these reactive species also have a role in cell signalling, being important in the adaptive response to exercise.
What this study adds
The present manuscript shows that soccer induces oxidative stress. This study evidences the importance of intensity and duration on exercise-induced oxidative stress. The predominance of ROS’ deleterious effects over their signalling role depends on the intensity degree of exercise. Only very intense exercise results in evident signals of oxidative damage and induces an antioxidant response in lymphocytes.
This work was supported by a grant from the Spanish Ministry of Science and Education (Projects DEP2005-00238-CO4-01 and DEP2005-00238-CO4-02/EQUI) and the FEDER funding.
Competing interests: None.
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