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Competitive swimmers have marked oxidative stress at the airway and systemic levels but without concomitant inflammation.
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Almost half of healthy non-asthmatic competitive swimmers have bronchial hyperresponsiveness in the peak training season.
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However, we could not confirm that oxidative stress is associated with bronchial hyperresponsiveness (AHR).
Abstract
Background
The environment in swimming pools, which contain chlorine, might interact with the airway epithelium, resulting in oxidative stress and/or inflammation during high intensity training periods.
Methods
We evaluated pulmonary functional (metacholine challenge test, FEV1 and VC), cellular (eosinophils and neutrophils), inflammatory (FeNo, IL-5, IL-6, IL-8 and TNF-α), oxidative (8-isoprostanes) and angiogenesis factors (VEGF) in induced sputum and peripheral blood of 41 healthy non-asthmatic elite swimmers (median 16 years) during the period of high intensity training before a national championship. The second paired sampling was performed seven months later after training had been stopped for one month.
Results
There was a ten-fold increase (median 82–924 pg/ml; P < 0.001) in 8-isoprostanes in induced sputum and five-fold increase (median 82–924 pg/ml; P < 0.001) in sera during training in comparison to the period of rest. However, there was no difference in FEV1 (113 vs 116%), VC (119 vs 118%), FeNo (median 34 vs 38 ppb), eosinophils (2.7 vs 2.9% in sputum; 180 vs 165 cells/μl in blood), neutrophils, different cytokines or VEGF in induced sputum or sera. The only exception was TNF-α, which was moderately increased in sera (median 23 vs 40 pg/ml; P = 0.02) during the peak training period. Almost half (18 of 41) of swimmers showed bronchial hyperresponsiveness during the peak training period (PC20 cutoff was 4 mg/ml). There was no correlation between hyperresponsiveness and the markers of oxidative stress or inflammation.
Conclusions
High intensity training in healthy, non-asthmatic competitive swimmers results in marked oxidative stress at the airway and systemic levels, but does not lead to airway inflammation. However, we could not confirm that oxidative stress is associated with bronchial hyperresponsiveness (AHR), which is often observed during the peak exercise training period.
The mechanisms of airway disorders associated with competitive swimming are not fully understood. The high level of ventilation during exercise is believed to affect the airway mucosa through two mechanisms: dehydration and mechanical stress [
]. Chemical treatment with chlorine is used to disinfect water in indoor swimming pools, and the gaseous chlorine by-products above the water level, such as trichloramines or trihalometans, may be especially important. However, there are conflicting data concerning the impact of the chlorinated pool environment on the airways of competitive swimmers [
]. The chlorine hypothesis suggests that the interaction between chlorine and the airway epithelium results in oxidative stress and airway inflammation. When inhalation of chlorine by-products repeatedly occurs, such as in elite swimmers, it may result in an impairment of antioxidant activity and/or the pro-inflammatory response, contributing to increased airway responsiveness or asthma [
Bronchial challenges and respiratory symptoms in elite swimmers and winter sport athletes: airway hyperresponsivenes in asthma: its measurement and clinical significanca.
In previous studies of induced sputum in swimmers, there were no significant differences in neutrophil counts between swimmers and mild asthmatic subjects and controls. Eosinophils were increased in both swimmers and asthmatic subjects compared with controls [
]. Furthermore, no significant differences in inflammatory cell counts were observed between swimmers with a PC20 FEV1 of less than 4 mg/mL and those with a PC20 FEV1 of more than 4 mg/mL [
]. Regarding the cytokine response to exercise, swimmers, but not the controls, showed a decrease in the amount of some cytokines, including IL-6 and TNF-α [
Oxidative stress is defined as the presence of active oxygen species in excess of the available antioxidant buffering capacity. Major reactive oxygen species belong to free radicals, and reactive oxygen species may damage proteins, lipids, DNA and carbohydrates by changing their structure and function [
]. They are involved in non-enzymatic processes that involve the peroxidation of membrane phospholipids, resulting in the generation of isoprostanes. Isoprostanes are thus major oxidative stress markers that are generated from polyunsaturated fatty acids, mainly from arachidonic acid. Isoprostanes are stable compounds that are present in urine, plasma, cerebrovascular fluid and exhaled breath condensate. They are unaffected by the lipid content and diet [
]. Oxidative stress is known to be associated with many acute and chronic diseases, such as cancer and cardiovascular, neurodegenerative and lung diseases. There are several reports highlighting oxidative stress as a part of the pathogenesis of asthma [
]. Balanza and colleagues found elevated concentrations of isoprostane levels in exhaled breath condensate in children with moderate persistent asthma compared to healthy controls [
The aim of our study was to monitor pulmonary function, airway hyperresponsiveness (AHR), FeNO and respiratory symptoms in healthy non-asthmatic elite swimmers during the period of high intensity training before a national championship and then during the period of rest after training had stopped for one month.
We therefore aimed to characterise systemic and airway (induced sputum) oxidative stress and inflammation in competitive swimmers during two different swimming periods: the period of high intensity training just before a national championship and after training had stopped for one month. At both time points, we evaluated systemic and airway (induced sputum) inflammation (eosinophils, neutrophils, IL-5, IL-6, IL-8 and TNF cytokines), oxidative stress (8-isoprostane), chemokines (MCP1) and angiogenesis factors (VEGF).
To understand the environmental impact on these processes, we compared swimmers in two different swimming pools with different exposures to chlorine and its by-products.
2. Methods
2.1 Study subjects
We included 41 healthy competitive swimmers. The exclusion criteria included a previous diagnosis of asthma or any other chronic disease. All subjects were prospectively followed for 8 months: first during high intensity training before the beginning of the national championship in February 2015 and then after training had stopped for one month in September 2015. At the first time point in February, we performed pulmonary function tests, metacholin testing, induced sputum and peripheral blood sampling. At the second time point in September, we repeated all of the testing and sampling, except metacholin testing. Swimmers were trained in two different pools with different levels of ventilation: 23 swimmers were trained in pool 1 and 18 swimmers were trained in pool 2.
2.2 Spirometry, metacholin and FeNO
Spirometry was carried out according to American Thoracic Society Criteria [
Guidelines for methacholine and exercise challenge testing 1999. This official statement of American Thoracic society was adopted by ATS Board of Directors, July 1999.
Am. J. Respir. Crit. Care Med.2000 Jan; 161: 309-329
] on a spirometer (Vyntus CPX, CareFusion Germany 234 GmbH). Airway responsiveness to metacholine was measured during the high intensity training period by using the tidal breathing method according to the standards recommended by the American Thoracic Society [
Guidelines for methacholine and exercise challenge testing 1999. This official statement of American Thoracic society was adopted by ATS Board of Directors, July 1999.
Am. J. Respir. Crit. Care Med.2000 Jan; 161: 309-329
]. The PD20 FEV1 (provocation dose of inhaled metacholine causing a 20% fall in FEV1) was obtained by linear interpolation on the log concentration response curve. A cutoff of a maximum of 2 mg of metacholine was used to determine the PD20.
FeNO was measured with the online chemiluminescence FeNO analyser CLD 88 Series (ECOMEDICS, Duernten, Switzerland) according to published guidelines from the European Respiratory Society (ERS) and American Thoracic Society (ATS) [
Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and Children—1999.
Am. J. Respir. Crit. Care Med. [Internet].1999 Dec; 160 ([cited 2017 May 9] Available from:): 2104-2117
]. Briefly, subjects inhaled 4,5% hypertonic saline, nebulized via an ultrasonic nebulizer (PARI MASTER Type 84.0100, PARI GmbH, Starnberg, Germany), during three 5-min periods, and at least 2 ml of sputum was collected into a sterile container. The sputum was immediately processed and homogenized with 0,1% dithiothreitol (Sputolysin, Calbiochem, San Diego, CA, USA), and cell-free supernatants was frozen at −80 °C until subsequent analysis. The total number of nonsquamous cells (TNNC) per ml of sputum sample was assessed using a hemocytometer. Cytospins were stained according to the May-Grünwald-Giemsa and Papanicolaou methods. Differential cell counts were performed by one observer counting 200 nonepithelial cells. The quality of the induced sputum was assessed according to the recommendations of Pizzichini E et al. [
]. Briefly, IL-5, IL-6, IL-8, TNF, MCP1 and VEGF (subtypes 165 and 121), were measured with cytometric bead arrays (BD Biosciences, San Diego, CA, USA) containing microparticles dyed to different fluorescence intensities. The captured beads were incubated with standards (purified from human plasma) or test samples (serum or induced sputum supernatant), followed by a wash and incubation with phycoerythrin-conjugated detection antibodies to form sandwich complexes. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). Data were acquired and analysed using cytometric bead array software.
2.5 8-Isoprostanes
8-isoprostane in serum or induced sputum supernatant was measured with a competitive enzyme immunoassay for the quantification of 8-isoprostane (8-isoprostane EIA KIT, Cayman Chemical Company, MI, USA) according to the manufacturer's instructions. Immediately after the collection of the sample, we added 0.005% butylated hydroxytoluene stabilizer. The detection limit of this immunoassay is in the pg/ml range.
2.6 Chlorine and trihalometans
We determined the free chlorine and total chlorine in swimming pools by using a colorimetric method with N,N-diethyl-1,4-phenylenediamine (according EN ISO 7393:2000 standard). Trihalometans were determined with Electron Capture Detectors and HP-5 columns according to SIST EN ISO 10301:1998 standard (Agilent, Santa Clara, CA, USA) in the air 5 cm above the water level.
3. Results
3.1 Study subjects
There were 23 (56.1%) females and 18 (43.9%) males. The median age was 16 years (IQR 4 years). All subjects were healthy, without any previous or current diagnosis of asthma, and they were not taking any inhaled or systemic therapies. Twenty-seven of the 41 swimmers had a history of mild and occasional respiratory symptoms during the high training period.
3.2 Pulmonary function
There was no difference in FEV1 (median 116% in high training vs. 113% in rest; P = 0.248) or VC (median 118% in high training vs. 119% in rest) (Table 1). However, almost half (18 of 41) of the swimmers showed bronchial hyperresponsiveness during the peak training period (PD20 cutoff was 4 mg/mL, median 0.59 mg/mL). There was no difference in FeNO measurements (median 39 ppb in high training vs 34 ppb in rest).
Table 1Pulmonary function test results. Data are presented as the median with interquartile ranges (IQR).
There was a higher total cell number in induced sputum during the high intensity period in comparison to the rest period (median 2880 vs.1170 cells/ml) (Table 2). However, we could not find any difference in the percentages of eosinophils (median 2.9% vs 2.7%) and neutrophils (median 48% vs 48.5%) in the induced sputum between the training and rest period. There was only a slight difference in the absolute number of eosinophils in peripheral blood at high intensity vs rest (training median 165 cells/μl vs rest median 180 cells/μl). There was no significant difference in the induced sputum concentrations of eosinophils (p = 0.898) and neutrophils (p = 0.323) between swimmers with negative and positive AHR.
Table 2Cell counts in the induced sputum in the high intensity training period and in the rest period. Data are presented as the median with interquartile ranges (IQR).
There was a ten-fold increase (median 82–924 pg/ml) in the 8-isoprostane concentrations in induced sputum and five-fold increase (median 82–924 pg/ml) in sera during the training period in comparison to the rest period (Fig. 1A–B; Table 3). There was no difference in the 8-isoprostane measurements between swimmers with and without AHR.
Fig. 1A–B. 8-Isoprostane concentration in A induced sputum and B serum during high the intensity training period compared to the non-training period. The bars indicate the median value with interquartile ranges for each group.
Table 3Concentration of isoprostanes in serum and induced sputum in the high intensity training period and in the rest period. Data are presented as the median with interquartile ranges (IQR).
There were no differences in cytokines (IL-5, IL-6, IL-8) or VEGF (vascular endothelial growth factor) in induced sputum or sera between the training and rest period (Table 4). We found only a moderate increase in TNF in the sera during the peak training period (median 27.9 vs 17.9 pg/ml; P = 0.02), but this difference was not evident in the induced sputum. There was also a difference in the concentrations of the chemokine MCP1 in serum (median 115.4 in high intensity vs. 147.2 pg/ml in rest; P < 0.001) and induced sputum (median 3.07 in high intensity vs. 1.37 pg/ml in rest; P = 0.002).
Table 4Concentrations of cytokines, MCP1 and VEGF in the high intensity training period and in the non-training period. Data are presented as the median with interquartile ranges (IQR).
During the intensive training period, the concentration of free chlorine in water was 0.5–0.6 mg/L (normative 0.3–0.6 mg/L) and the concentration of total chlorine was 0.1–0.2 mg/L (normative 0.3 mg/L in both swimming pools). The concentration of trihalometans in the water was 0.005–0.007 mg/L (normative 0.050 mg/L), and at 5 cm above the water surface, the concentration was 9.8 mg/m3 in swimming pool 1 and 20.1 mg/m3 in swimming pool 2. Overall, there were 18 swimmers in pool 1 and 23 in pool 2. However, despite training in environments with different concentrations of trihalometans, we did not find any differences in pulmonary function tests or the measurements of 8-isoprostanes, eosinophils, neutrophils, cytokines or VEGF during the high intensity period and rest.
4. Discussion
In the present study we showed that high intensity training in healthy, non-asthmatic competitive swimmers results in marked oxidative stress at the airway level. Chlorine and its by-products are strong oxidants, which may irritate the airways and are considered increasingly responsible for the occurrence of respiratory disorders in swimmers. There is a recent report on isoprostanes in swimmers [
], where researchers found increased levels of 8-isoprostane in exhaled breath condensate 10 min after the training session, highlighting the impact of exercise-induced hyperpnoea on oxidative stress. We corroborated those data and showed marked airway and systemic oxidative stress during the high training period, indicating that the problem of airway oxidative stress is not only an » acute « airway problem, linked to hyperpnoea. The impact of air quality on oxidative stress in the lung has been studied during the Beijing Olympics in young healthy non-athlete subjects [
]. Findings from this study suggest that air pollution adversely affected the airway, measured through 8-isoprostanes in exhaled air condensate.
We have shown marked oxidative stress at the systemic level in swimmers during the high intensity training period. There are also other reports of systemic oxidative stress and induction of muscle damage markers by high intensity interval training in competitive swimmers [
According to our results, exercise-induced oxidative stress in competitive swimmers was not followed by airway or systemic inflammation. Previously, studies on bronchial biopsies of competing swimmers have shown inflammatory and remodelling changes, similar to patients with mild asthma [
], suggesting that swim training in chlorinated pools affects the structure of the airway in elite swimmers. Researchers found increased eosinophils in swimmers and asthmatics, but no significant differences in neutrophil counts. According to the results of our study, a high training indoor pool environment does not result in significant changes at the level of airway inflammation, FeNO, eosinophils, neutrophils or different cytokines. The explanation for these differences might due to different methodologies (biopsy vs induced sputum) and the age of swimmers. The median age of our swimmers was 5 years less, and we can speculate that the occurrence of eosinophilia may be dependent on many years of intensive training, and therefore it is too early to find eosinophilia in our younger group of swimmers. Our results are more comparable to the results of previous studies on induced sputum [
], which found minimal or no airway inflammation in elite swimmers, except for those with AHR, who had a significant increase in airway eosinophil count.
In our group, almost half of swimmers have AHR to metacholin. How ewer, there was no correlation between hyperresponsiveness and the markers of oxidative stress or inflammation. Screening in competitive swimmers has demonstrated that up to 76% have symptomatic or asymptomatic airway hyperresponsiveness (AHR) and/or exercise-induced bronchoconstriction (EIB) [
Bronchial challenges and respiratory symptoms in elite swimmers and winter sport athletes: airway hyperresponsiveness in asthma: its measurement and clinical significance.
Chest [Internet].2010 Aug; 138 ([cited 2017 May 29] Available from:): 31S-37S
]. In some athletes, provocation tests with direct stimuli, such as methacholine, may be negative, while EIB can be confirmed by indirect tests such as eucapnic voluntary hyperpnoea (EVH) [
Airway hyperresponsiveness to methacholine, adenosine 5-monophosphate, mannitol, eucapnic voluntary hyperpnoea and field exercise challenge in elite cross-country skiers.
] reported that the airways of elite skiers clearly react in a heterogeneous manner in the training season, being more responsive to metacholine than to indirect provocation. They also reported that AHR to metacholine was more prevalent in those not reporting asthma-like symptoms. Furthermore, asthmatic airway inflammation was not a prerequisite for AHR to metacholine. We now highlight a similar point in competitive swimmers, where we did not find a correlation between airway inflammation and AHR to metacholin. The limitation of our study is that we did not repeat the metacholin testing in the rest period to document possible decreasing AHR. Previous study suggest that training may contribute to the development of AHR in elite swimmers, but this seems reversible in many athletes after training cessation for at least 2 weeks [
In conclusion, high intensity training in healthy, non-asthmatic elite swimmers results in marked oxidative stress at the airway and systemic levels, but does not lead to airway inflammation. Further studies are needed to confirm the clinical relevance of oxidative stress in swimmers and the possible risk in cold-air athletes.
Conflicts of interest
I declare no conflict of interest.
Acknowledgements
We thank Prof. Ratko Djukanović, NIHR Respiratory Biomedical Research Unit, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK, for comments and revision of the manuscript.
References
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Exercise-induced asthma: is it the right diagnosis in elite athletes?.
Bronchial challenges and respiratory symptoms in elite swimmers and winter sport athletes: airway hyperresponsivenes in asthma: its measurement and clinical significanca.
Guidelines for methacholine and exercise challenge testing 1999. This official statement of American Thoracic society was adopted by ATS Board of Directors, July 1999.
Am. J. Respir. Crit. Care Med.2000 Jan; 161: 309-329
Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and Children—1999.
Am. J. Respir. Crit. Care Med. [Internet].1999 Dec; 160 ([cited 2017 May 9] Available from:): 2104-2117
Bronchial challenges and respiratory symptoms in elite swimmers and winter sport athletes: airway hyperresponsiveness in asthma: its measurement and clinical significance.
Chest [Internet].2010 Aug; 138 ([cited 2017 May 29] Available from:): 31S-37S
Airway hyperresponsiveness to methacholine, adenosine 5-monophosphate, mannitol, eucapnic voluntary hyperpnoea and field exercise challenge in elite cross-country skiers.
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