Effect of exposure to disinfection by-products during swimming exercise on asthma-related immune responses

Recently, due to the increasing interest in leisure and health, the number of indoor swimming pool users is increasing along with the increase in the social sport population (Jo et al. 2023). Swimming, a representative exercise in water, consumes more energy than aerobic exercise performed on land because it uses the four characteristics of water: buoyancy, water temperature, water pressure, and resistance. Compared to exercise on land, it reduces the burden of body load and puts less strain on joints. Therefore, it is a full-body exercise where patients can experience the effects of exercise without much difficulty (Park et al. 2021). Furthermore, it is evident that swimming exerts a positive influence not only on diseases but also on immune function (Lee et al. 2019a, 2019b). Specifically, it has been reported to reduce cough perception, enhance respiratory muscle strength, and decrease the likelihood of exercise-induced asthma (Chandratilleke et al. 2012). Swimming exercise has been documented as a safe and primarily recommended rehabilitation method for patients with asthma (Beggs et al. 2013).

However, recent reports on the potential adverse effects of disinfection by-products (DBPs) generated during the disinfection process, which can harm the health of swimming pool users through skin contact or respiratory exposure, have sparked increased interest in the quality of swimming pool water (Jo et al. 2015). Just as with drinking water, chlorine disinfectants, used essentially for the prevention of infectious diseases, are the most commonly employed oxidizing and disinfecting agents in swimming pools due to their cost-effectiveness, efficient bacterial eradication capabilities, and convenient application (Kim et al. 2017; Sun et al. 2019). However, the use of disinfectants generates DBPs, such as total trihalomethanes, haloacetic acids, haloacetonitriles, and chloral hydrate, and the highly oxidizing sodium hypochlorite, produced during the disinfectant manufacturing process, may also be introduced (You 2020). In particular, DBPs that have been shown to have a higher risk of exposure in swimming pool water than in drinking water are also found to be significantly involved in the risk of association with respiratory symptoms such as allergies and asthma and in causing inflammation (Villanueva et al. 2004; Caro & Gallego 2007; Kaydos-Daniels et al. 2008; Florentin et al. 2011; Del Giacco et al. 2015).

Some studies showed that an increase in the number of patients with asthma had been reported to be associated with exposure to chlorinated irritants in swimming pools (Varraso et al. 2002; Kohlhammer et al. 2006), and the association between the duration spent in indoor swimming pools and adverse health outcomes, including asthma, has also been reported (Zheng et al. 2020, 2021). However, these reports have limited data to detect harmful associations (Del Giacco et al. 2015), and scientific evidence regarding inhalation of DBPs and changes in the immune system has not been clearly established to date (Lee 2023).

In this study, we sought to determine how factors such as DBPs, asthma, and inhalation time of DBPs to swimming exercise affect the immune system.

Six-week-old male C57BL/6 mice (Dongnam Institute of Radiological and Medical Sciences Animal Inc., Busan, Korea) were used after a 1-week quarantine and acclimatization period. The mice were housed in a room maintained at 23 ± 2 °C, with a relative humidity of 50 ± 5%. The lighting schedule consisted of artificial light from 08:00 to 20:00; the air within the room underwent 13–18 air changes per hour. The mice were provided a standard laboratory diet and had unlimited access to water. All experimental procedures adhered to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Dongnam Institute of Radiological and Medical Sciences (Permit Number: DI-2023-010). Animal welfare was ensured in compliance with the regulations stipulated by the National Animal Welfare Law of Korea.

We experimented with inhalation of DBPs and the indoor swimming environment by applying the indoor swimming pool environment model created in a previous paper (Lee 2023). The experimental method was the same as in the previous paper. Briefly, the mice were exposed to the DBP gas using the previous setup. To induce the generation of DBPs, sodium hypochlorite and the synthesized bodily fluid mimicry solution were reacted in a constant-temperature stirrer and a reaction tank. Chlorine concentration following chlorination was measured in the reactor tank using a residual chlorine analyzer, and an initial residual chlorine concentration of 5 mg/mL or higher was ensured. After the reaction, the final residual chlorine concentration was maintained at 1 mg/mL or less. The synthesized solution was loaded into a syringe for usage.

Aquatic exercise was performed in a constant-temperature water bath (width × length × height; 90 × 30 × 30 cm) filled with water to a height of 13 cm and temperature at 29 °C for 30 or 60 min. All mice were made to swim for 4 s at a speed of 10 cm/s with 4-s intervals between swims, and the backs of the mice were tapped with a soft brush at every 4 s to maintain the swimming intervals. This process was repeated five times a week for a total duration of 4 weeks (Lee et al. 2019a, 2019b).

The mice were euthanized 48 h after the final challenge through intraperitoneal injection of Alfaxan (0.5 mg/kg; Australia), and a tracheostomy was performed. To obtain the bronchoalveolar lavage fluid (BALF), ice-cold phosphate buffered saline (PBS) (0.5 mL) was infused into the lungs thrice and withdrawn each time through tracheal cannulation, resulting in a total volume of 1.5 mL. The total number of inflammatory cells was determined by counting the cells in at least five squares of a hemocytometer after excluding dead cells using trypan blue staining. Differential cell counts in BALF were determined using Systemex ADVIA 2120 (Siemens Healthcare Diagnostic Inc., IL, USA) following the manufacturer's instructions. The numbers of macrophages, neutrophils, and lymphocytes were calculated by multiplying the percentages obtained from the total yield. The slides were imaged using a digital camera attached to a microscope (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan).

The amounts of interleukin (IL)-2, IL-4, IL-6, IL-10, and interferon (IFN)-γ secreted in the serum of the controlled mice were quantitatively measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BD OptEIA™ Mouse IL-2 (#555148), IL-4 (#555232), IL-6 (#555240), IL-10 (#555252), and IFN-γ (#555138) ELISA KitII), according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA).

The isolated cells were resuspended in 100 μL of 1% fetal bovine serum (FBS) solution in PBS and incubated with anti-CD3 (PE-Cy7-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), anti-CD4 (PE-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), anti-CD8 (FITC-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), and anti-CD25 (APC-conjugated, BD MS CD25 APC, #558643) antibodies. The cell pellets were resuspended in 400 μL of 1% FBS solution in PBS and analyzed using flow cytometry (FACS) (FACSAria II cell sorter, BD Biosciences, USA).

Statistical differences between groups were analyzed using Student's two-tailed t-test for comparisons between two groups and one-way analysis of variance for comparisons between more than two groups. All calculations were performed using GraphPad Prism software (version 5.0). Significance was set at p < 0.05.

Swimming exercise is an important recreational activity for many people around the world and is often recommended by doctors for improving lung function. However, many studies have raised concerns about potential adverse health effects from exposure to chemically disinfected swimming pool water (Zwiener et al. 2007; Chowdhury et al. 2014; Valeriani et al. 2017; Lee 2023); in recent years, the association between the frequency of exposure in swimming pools and asthma has not been proven, and the results are often conflicting (Weisel et al. 2009; Font-Ribera et al. 2014; Voisin et al. 2014). Therefore, because the use of disinfectants in swimming pools has an important impact on public health in any country around the world (Voisin et al. 2010; Dehghani et al. 2018), this study examined the inhalation of DBPs in indoor swimming pools and swimming exercise. We would like to discuss the results of confirming the relationship and progression of asthma and analyzing changes in immune response. Asthma is a chronic inflammatory disease of the respiratory system that releases various chemical mediators that are closely related to inflammatory cells (Lee et al. 2019a, 2019b).

Studies looking at the effects of these mediators on asthma have found no significant changes. According to a study that compared and analyzed the frequency of DBP inhalation according to swimming pool attendance in 5,738 people, lung function and risk of asthma symptoms were not significantly related to the frequency of DBP inhalation (Font-Ribera et al. 2011), and there was no significant association between respiratory disease and the frequency of environmental exposure during swimming pool exercise among 3,223 participants (Font-Ribera et al. 2009). These reports supported our results that DBPs had a minimal effect on the expression of asthma-related factors. In other words, it could be seen that inhalation of DBPs in the swimming pool during swimming exercise had no or minimal effect on respiratory diseases, and the causal relationship was uncertain (Goodman & Hays 2008; Weisel et al. 2009; Villanueva & Font-Ribera 2012; Font-Ribera et al. 2014; Voisin et al. 2014).

Exercise is an important physical stress factor, and it has been reported that inflammation is induced and differentiated changes in T-cell distribution occur depending on the type and intensity of exercise. It means that exercise affects various immunological processes by affecting the concentration of catecholamines, a stress hormone, in blood and lymphocyte population (Krüger et al. 2008). That is, exercise promotes an intensity-dependent increase in sympathetic activity as well as activation of the hypothalamic–pituitary axis, leading to systemic secretion of catecholamines. In this study, the inhalation of DBPs did not statistically significantly show changes in T cells and cytokines, but changes in T cells were observed in all mice that underwent swimming exercise. These results are supported by studies showing that continuous swimming exercise has a positive effect on the body's immunity due to an increase in the number of T cells (Liu et al. 2011; Kang et al. 2021) and that swimming exercise is an important non-pharmacological intervention for chronic inflammation or T cells (Xie et al. 2019). Therefore, it can be seen that the change in lymphocytes through swimming exercise is safe for the efficiency of the immune system, and the increase in T cells through exercise is also effective in promoting the secretion of cytokines (Lee 2023).

Cytokines, which are signaling molecules secreted by immune cells, play an important role in maintaining homeostasis between cell-mediated and humoral immune responses and regulating inflammatory processes (Zuo et al. 2014; Couto et al. 2021). In particular, Th2 cytokines are considered the main cause for asthma because they promote the infiltration of eosinophils (Krüger & Mooren 2014). IFN-γ, IL-2, IL-4, IL-6, and IL-10 cytokines are said to be affected by physical activities such as swimming (Lee et al. 2019a, 2019b; Lee 2023). Many researchers have reported that although adequate exercise clearly promotes immune parameters, the effect is closely related to training parameters such as the type, intensity, and duration of exercise (Hillman et al. 2008; Font-Ribera et al. 2009; Radak et al. 2010; Walsh et al. 2011; Krüger & Mooren 2014). Therefore, long-term vigorous exercise may actually cause a higher incidence of upper respiratory tract infections, and immunological loss cannot be ruled out. Therefore, the positive effects of swimming and the suppression and improvement of asthma triggers are dependent on the individual's physical fitness level. Exercise should be encouraged.

This study analyzed how the inhalation of DBPs during swimming exercise affects asthma-related immune responses and drew the following conclusions. Factors such as inhalation of DBPs, asthma, and exposure time to swimming exercise did not appear to affect the immune system related to respiratory diseases. Therefore, considering that swimming is a recommended sport for individuals with asthma and respiratory diseases and that the immunological benefits gained from regular swimming exercise are greater, swimming is considered to be a safe and beneficial exercise for respiratory diseases. However, swimming pools are aggressive environments with high levels of DBPs in water and air due to constant disinfection and organic load from swimmers, so special attention to immune management is always required.

We thank Yonghae Son and Koanhoi Kim (Pusan National University) for their help in editing this manuscript.

B-A. Lee did all the work in this study. The author has read and agreed to the published version of the manuscript.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A1A01059491).

The animal study protocol was approved by the Institutional Review Board of the Institutional Animal Care and Use Committee of the Dongnam Institute of Radiological and Medical Sciences (Permit Number: DI-2023-010), and all experimental procedures adhered to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Animal welfare was ensured in compliance with the regulations stipulated by the National Animal Welfare Law of Korea.

All relevant data are included in the paper or its Supplementary Information.

The author declares there is no conflict.