**5. Lung inflammation and air pollution**

**Antioxidant Solubility Concentration in human serum (μM)**

Melatonin Hydrophilic and lipophilic Varies throughout the day

Most antioxidants undergo redox cycling. This means that once oxidized they can be reduced to their former state and act again as an antioxidant. Nevertheless, this redox cycling allows the antioxidant to act as pro-oxidant promoting free radical formation. This would happen if there is an unbalance in the antioxidant network system. Vitamin C and vitamin E are examples of antioxidants with this characteristic, while melatonin is considered a terminal antioxidant

Oxidant air pollutants such as ozone, particulate matter and nitrogen dioxide have been shown to induce lung inflammation through stimulation of the oxidative stress process. Little is known, however, about their effects as oxidant compounds in the lungs or about the role and the effectiveness of the antioxidants present in the respiratory tract lining fluid (RTLF) in scavenging and protecting against their harmful effects. A great variety of antioxidants can be found in human RTLF. Their concentration and distribution throughout the airways, however, is not homogeneous, with high levels of GSH in the alveolar epithelial regions and uric acid

Uric acid Hydrophilic 200-400

Glutathione Hydrophilic 4 Lipoic acid Hydrophilic 0.1-0.7

Ubiquinol (coenzyme Q) Lipophilic 5

Vitamin A (retinol) Lipophilic 1-3 Vitamin C (ascorbic acid) Hydrophilic 50-60 Vitamin E (α-tocopherol) Lipophilic 10-40 β-carotene Lipophilic 0.5-1

**Table 3.** Important antioxidants

6 Lung Inflammation

because it cannot be recycled.

**4.3. Lung oxidative stress**

**Figure 2.** Redox cycling of vitamin E (vit E) by vitamin C (Vit. C)

predominating in the upper airways [33, 34].

Airway inflammation and any other inflamed tissue can be characterized by an increase in inflammatory cells, such as neutrophils and macrophages, as well as inflammatory mediators: interleukin-6 (IL-6), interleukin-8 (IL-8), and prostaglandins. An increase in neutrophil numbers and percentage is a good indicator of the beginning of an inflammatory response because these cells account for 50-60% of the total white blood cells in the circulation and are the first cell type to migrate to sites of injury and inflammation. When a tissue is inflamed, an increase in the expression of adhesion molecules of the selectin family (E-and P-selectin molecules) occurs in the local endothelium. This process is mediated by cytokines and other inflammatory mediators. Neutrophils present in the blood recognise the site of inflammation because of these adhesion molecules which bind to the other molecules (mucin-like celladhesion molecules, CAM) on the neutrophil surface. This step is referred to as rolling: the first step to the attachment of neutrophils onto the endothelium. In order for them to adhere firmly and be able to migrate through the endothelial to the inflamed site, the neutrophils are activated by various chemoattractants derived from epithelial cells exposed to a foreign body, IL-8 being an important one. Once the adhesion processes is successful the neutrophils can initiate their transendothelium migration. Upon arrival at the inflamed tissue, neutrophils release a number of chemoattractants to amplify the inflammatory response by recruiting other cells.

not trapped in the mucus, though they can exacerbate its production and destroy the cilia making the airways more susceptible to the invasion of other foreign agents. Another conse‐ quence of airway inflammation is lung epithelial injury which leads to an open interface between the lung and the blood. This facilitates the dispersion of microbes to the rest of the body, initiating a systemic inflammatory response. If the lung epithelial injury is chronic and the tissue is recurrently going through a repairing process, this can lead to fibrosis with

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Injury and toxicity involving the respiratory epithelium can be assessed by a simple and noninvasive way by measuring the concentration of Clara cell protein, also known as CC16, CC10 orUteroglobin.Thisprotein is secreted, as the name indicates, by Clara cells.The function of these cells is mainly the protection of the respiratory tract. They present a high content of xenobiotic metabolizing enzymes which protect our system against inhaled particles includ‐ ing pollutants [46, 47]. CC16 is a small protein with an important role in decreasing the inflammation of the respiratory tract and protecting it against the harmful effects of oxidative stress [48]. This protein can be measured by the methods used to assess the respiratory airways, includingtheNLprocedure.Inaddition,CC16 canalsobe foundintheblood,where itisderived almost exclusivelyfromtheairways [46].Innormalhealthyindividuals,the serumlevelofCC16 ranges, on average, from 10 to 15 ug⋅l-1 [49]. Yet, the concentration of this protein in the blood has been shown to rise as a result of pulmonary inflammation and increases in the permeabili‐ ty of the lung epithelial barrier. The lung–blood barrier offers some resistance to the bidirectional movement of large proteins such as albumin. Nevertheless, the high concentration of CC16 in the respiratory tract secretions and its small size permit its diffusion into the blood

consequential decrease in lung function, chest discomfort, fatigue and weakness.

[50, 51] where it can easily be detected by conventional enzyme immunoassays [52].

The bi-directional exchange of proteins between lung and blood is regulated by several factors, such as the size of the proteins, the epithelium permeability and the driving force of the transepithelial concentration gradient. The concentration gradient allows the movement of proteins from an area of high concentration to an area of low concentration. In the case of CC16, this if from the lung to the blood; but albumin, for example, moves in the opposite direction. The large difference between the concentration gradients can be related to the difference in the compartment sizes in which the proteins are diluted. The concentration gradient is also influenced by the removal of the protein from the compartment into which it is leakingproteins that enter the lung interstitium are rapidly cleared by lymphatic drainage [51].

The changes that occur in serum concentrations of CC16 may result from three different mechanisms. The first mechanism would result from the increase in the permeability of the lung epithelial barrier, and this has as a consequence a higher diffusion of CC16 into the blood. This can happen following exposure to ozone, which causes epithelial lung injury, or more specifically, damage to the tight junctions of the cells (fig. 4) [51, 53]. A second possibility is the decrease or increase in the production or secretion of CC16 from the Clara cells present in the respiratory tract. A reduction in the number of Clara cells has been shown to occur following chronic exposure to lung toxicants such as silica particles [54]. The third mechanism that would lead to an enhancement in the levels of serum CC16 would result from a reduction in the clearance of this protein by the kidney. Serum CC16 has a half-life of approximately 2-3

The cytokine IL-8 is a mediator of the immune function and helps regulate the immune response. It is secreted by a variety of cells, including neutrophils, macrophages and endo‐ thelial cells, and is a chemotactic for cells such as neutrophils and T cells. In addition, it has been linked to a wide variety of pathologic conditions characterized by an increase in neutro‐ phil count. Thus, an increase in IL-8 levels is linked to an increase in neutrophils [39]. IL-6 is another important mediator in the development of an inflammatory process. It is produced mainly by T-cells and macrophages; and, together with IL-1 and TNF-α, stimulate both local and systemic changes of an inflammatory response. This cytokine – IL-6 – has been thoroughly studied in immunological responses to exercise [40-41].

Inhalation of air pollution has been shown to stimulate airway inflammation due to its oxidative nature. Airway inflammation can be detected by both a local increase in inflamma‐ tory cells as well as inflammatory mediators. The depletion of antioxidants found in the airways, characterizes the oxidative process that triggers an inflammatory response due to epithelial damage [42, 43]. Both the inflammatory mediators and the antioxidants can be measured using different techniques, each with its own advantages and disadvantages. Bronchial biopsies and brochoalveolar lavage (BAL) are quite invasive procedures that require local anesthesia and need to be performed in a medical environment [44]. These two techniques have the advantages of sampling the more distal regions of the airway and the biopsies retrieves tissue samples which can give further information about the local inflammatory process. The sputum induction procedure and the nasal lavage (NL) procedure, on the other hand, are less invasive and less technically difficult procedures than the previously-mentioned bronchial techniques and can be repeated at multiple time points [37, 45]. Sputum induction and NL are techniques that sample the upper respiratory airways.

There has been a variety of studies analysing airway inflammation of individuals exposed to O3 pollution. Due to the similar oxidative nature of O3 in relation to other pollutants, the results of these studies can be, in a certain way, generalized to include them too. Nevertheless, when analysing the literature, it is always essential to take into account the total volume of pollutants inspired by the participants, as well as the techniques used to sample the airway compart‐ ments. Ideally it would also be relevant to take into account the antioxidant concentrations in the RTLF, which has been shown to vary between individuals. In order to increase the amount of air – and consequently air pollution – inspired in a shorter amount of time, most studies use exercise protocols in association with the exposure.

Airway inflammation can lead to the destruction of the cilia of the epithelial cells that line the respiratory tract. The cilia have an important immune function because they constantly move the mucus up from the lungs to the back of the throat where it is eliminated or swallowed and digested. The mucus serves as a "trap" to infectious agents and small particles, such as pollutants and allergens, preventing them to enter deep into the airways. Gas pollutants are not trapped in the mucus, though they can exacerbate its production and destroy the cilia making the airways more susceptible to the invasion of other foreign agents. Another conse‐ quence of airway inflammation is lung epithelial injury which leads to an open interface between the lung and the blood. This facilitates the dispersion of microbes to the rest of the body, initiating a systemic inflammatory response. If the lung epithelial injury is chronic and the tissue is recurrently going through a repairing process, this can lead to fibrosis with consequential decrease in lung function, chest discomfort, fatigue and weakness.

activated by various chemoattractants derived from epithelial cells exposed to a foreign body, IL-8 being an important one. Once the adhesion processes is successful the neutrophils can initiate their transendothelium migration. Upon arrival at the inflamed tissue, neutrophils release a number of chemoattractants to amplify the inflammatory response by recruiting other

The cytokine IL-8 is a mediator of the immune function and helps regulate the immune response. It is secreted by a variety of cells, including neutrophils, macrophages and endo‐ thelial cells, and is a chemotactic for cells such as neutrophils and T cells. In addition, it has been linked to a wide variety of pathologic conditions characterized by an increase in neutro‐ phil count. Thus, an increase in IL-8 levels is linked to an increase in neutrophils [39]. IL-6 is another important mediator in the development of an inflammatory process. It is produced mainly by T-cells and macrophages; and, together with IL-1 and TNF-α, stimulate both local and systemic changes of an inflammatory response. This cytokine – IL-6 – has been thoroughly

Inhalation of air pollution has been shown to stimulate airway inflammation due to its oxidative nature. Airway inflammation can be detected by both a local increase in inflamma‐ tory cells as well as inflammatory mediators. The depletion of antioxidants found in the airways, characterizes the oxidative process that triggers an inflammatory response due to epithelial damage [42, 43]. Both the inflammatory mediators and the antioxidants can be measured using different techniques, each with its own advantages and disadvantages. Bronchial biopsies and brochoalveolar lavage (BAL) are quite invasive procedures that require local anesthesia and need to be performed in a medical environment [44]. These two techniques have the advantages of sampling the more distal regions of the airway and the biopsies retrieves tissue samples which can give further information about the local inflammatory process. The sputum induction procedure and the nasal lavage (NL) procedure, on the other hand, are less invasive and less technically difficult procedures than the previously-mentioned bronchial techniques and can be repeated at multiple time points [37, 45]. Sputum induction

There has been a variety of studies analysing airway inflammation of individuals exposed to O3 pollution. Due to the similar oxidative nature of O3 in relation to other pollutants, the results of these studies can be, in a certain way, generalized to include them too. Nevertheless, when analysing the literature, it is always essential to take into account the total volume of pollutants inspired by the participants, as well as the techniques used to sample the airway compart‐ ments. Ideally it would also be relevant to take into account the antioxidant concentrations in the RTLF, which has been shown to vary between individuals. In order to increase the amount of air – and consequently air pollution – inspired in a shorter amount of time, most studies use

Airway inflammation can lead to the destruction of the cilia of the epithelial cells that line the respiratory tract. The cilia have an important immune function because they constantly move the mucus up from the lungs to the back of the throat where it is eliminated or swallowed and digested. The mucus serves as a "trap" to infectious agents and small particles, such as pollutants and allergens, preventing them to enter deep into the airways. Gas pollutants are

studied in immunological responses to exercise [40-41].

and NL are techniques that sample the upper respiratory airways.

exercise protocols in association with the exposure.

cells.

8 Lung Inflammation

Injury and toxicity involving the respiratory epithelium can be assessed by a simple and noninvasive way by measuring the concentration of Clara cell protein, also known as CC16, CC10 orUteroglobin.Thisprotein is secreted, as the name indicates, by Clara cells.The function of these cells is mainly the protection of the respiratory tract. They present a high content of xenobiotic metabolizing enzymes which protect our system against inhaled particles includ‐ ing pollutants [46, 47]. CC16 is a small protein with an important role in decreasing the inflammation of the respiratory tract and protecting it against the harmful effects of oxidative stress [48]. This protein can be measured by the methods used to assess the respiratory airways, includingtheNLprocedure.Inaddition,CC16 canalsobe foundintheblood,where itisderived almost exclusivelyfromtheairways [46].Innormalhealthyindividuals,the serumlevelofCC16 ranges, on average, from 10 to 15 ug⋅l-1 [49]. Yet, the concentration of this protein in the blood has been shown to rise as a result of pulmonary inflammation and increases in the permeabili‐ ty of the lung epithelial barrier. The lung–blood barrier offers some resistance to the bidirectional movement of large proteins such as albumin. Nevertheless, the high concentration of CC16 in the respiratory tract secretions and its small size permit its diffusion into the blood [50, 51] where it can easily be detected by conventional enzyme immunoassays [52].

The bi-directional exchange of proteins between lung and blood is regulated by several factors, such as the size of the proteins, the epithelium permeability and the driving force of the transepithelial concentration gradient. The concentration gradient allows the movement of proteins from an area of high concentration to an area of low concentration. In the case of CC16, this if from the lung to the blood; but albumin, for example, moves in the opposite direction. The large difference between the concentration gradients can be related to the difference in the compartment sizes in which the proteins are diluted. The concentration gradient is also influenced by the removal of the protein from the compartment into which it is leakingproteins that enter the lung interstitium are rapidly cleared by lymphatic drainage [51].

The changes that occur in serum concentrations of CC16 may result from three different mechanisms. The first mechanism would result from the increase in the permeability of the lung epithelial barrier, and this has as a consequence a higher diffusion of CC16 into the blood. This can happen following exposure to ozone, which causes epithelial lung injury, or more specifically, damage to the tight junctions of the cells (fig. 4) [51, 53]. A second possibility is the decrease or increase in the production or secretion of CC16 from the Clara cells present in the respiratory tract. A reduction in the number of Clara cells has been shown to occur following chronic exposure to lung toxicants such as silica particles [54]. The third mechanism that would lead to an enhancement in the levels of serum CC16 would result from a reduction in the clearance of this protein by the kidney. Serum CC16 has a half-life of approximately 2-3

**Figure 4.** Movement of CC16 from the airways to the blood. (A) Under normal conditions, (B) after exposure to ozone. The thickness of the arrows is used to illustrate the relative permeability of the different barriers and the increase in the CC16 flux after ozone exposure. Abbreviations: Ep – epithelium; In-interstitium; En – endothelium (adapted from

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Both acute and chronic exposure to toxicants has been shown to elicit changes in serum CC16 levels. This supports the theory that this protein is a sensitive and suitable biomarker of lung injury [46, 54]. A study conducted with firefighters [55] showed that acute smoke inhalation significantly increased serum CC16 levels. In addition to smoke inhalation, the firefighters also had to perform physically demanding tasks. Serum CC16 concentration was measured immediately after exposure and was three times higher than that of control participants. The change in serum CC16 concentration was attributed to a transient increase in the lung epithelial permeability, but with no sign of lung function impairment. Ten days after exposure, the CC16

When two groups of individuals were compared, a chronic toxic effect on Clara cells was observed in workers inhaling silica-rich dust for an average of 15 months [54]. One group was composed of workers exposed to silica and the other was a control group, matched for age, body mass index and proportion of smokers. After 15 months, the mineworkers showed a significant reduction in serum CC16, even though they did not present any lung function impairment or abnormalities in their chest X-ray. The decrease was reported for both the nonsmokers and the smokers, but an additional and significant effect of tobacco smoking was found. The authors associated this decrease with a reduction in the release of CC16 from the Clara cells probably due to their damage from the toxic action of silica. Pertaining literature suggests that the toxic metabolites of tobacco smoke not only increase the permeability of the

**5.1. Studies investigating the effect of air pollution on performance, lung inflammation and**

Exercise leads to various physiological changes that can aggravate the effects of air pollutants. At resting conditions our breathing is predominantly nasal. This has various advantages which

lung epithelium, but also cause a progressive destruction of Clara cells [47].

Broeckaert *et al.* 2000b).

**injury**

concentration had returned to baseline levels.

**Figure 3.** Respiratory bronchiole before (A) and after (B) air pollution exposure.

h due to its rapid clearance through the kidney [46]. Hence, the variation in CC16 serum levels can only be used as a specific biomarker of the airway epithelium integrity if the individual does not present renal dysfunction.

**Figure 4.** Movement of CC16 from the airways to the blood. (A) Under normal conditions, (B) after exposure to ozone. The thickness of the arrows is used to illustrate the relative permeability of the different barriers and the increase in the CC16 flux after ozone exposure. Abbreviations: Ep – epithelium; In-interstitium; En – endothelium (adapted from Broeckaert *et al.* 2000b).

Both acute and chronic exposure to toxicants has been shown to elicit changes in serum CC16 levels. This supports the theory that this protein is a sensitive and suitable biomarker of lung injury [46, 54]. A study conducted with firefighters [55] showed that acute smoke inhalation significantly increased serum CC16 levels. In addition to smoke inhalation, the firefighters also had to perform physically demanding tasks. Serum CC16 concentration was measured immediately after exposure and was three times higher than that of control participants. The change in serum CC16 concentration was attributed to a transient increase in the lung epithelial permeability, but with no sign of lung function impairment. Ten days after exposure, the CC16 concentration had returned to baseline levels.

When two groups of individuals were compared, a chronic toxic effect on Clara cells was observed in workers inhaling silica-rich dust for an average of 15 months [54]. One group was composed of workers exposed to silica and the other was a control group, matched for age, body mass index and proportion of smokers. After 15 months, the mineworkers showed a significant reduction in serum CC16, even though they did not present any lung function impairment or abnormalities in their chest X-ray. The decrease was reported for both the nonsmokers and the smokers, but an additional and significant effect of tobacco smoking was found. The authors associated this decrease with a reduction in the release of CC16 from the Clara cells probably due to their damage from the toxic action of silica. Pertaining literature suggests that the toxic metabolites of tobacco smoke not only increase the permeability of the lung epithelium, but also cause a progressive destruction of Clara cells [47].

#### **5.1. Studies investigating the effect of air pollution on performance, lung inflammation and injury**

h due to its rapid clearance through the kidney [46]. Hence, the variation in CC16 serum levels can only be used as a specific biomarker of the airway epithelium integrity if the individual

does not present renal dysfunction.

10 Lung Inflammation

**Figure 3.** Respiratory bronchiole before (A) and after (B) air pollution exposure.

Exercise leads to various physiological changes that can aggravate the effects of air pollutants. At resting conditions our breathing is predominantly nasal. This has various advantages which includes not only humidifying and heating the inspired air but also filtering it. Once exercise starts becoming more intense, individuals automatically switch the nasal breathing to oral breathing in an attempt to increase the amount inhaled. Nevertheless avoiding the nasal filtration system potentially enhances the pollutant concentration that reaches the lungs.

With the beginning of exercise, the ventilatory exchange rate (VE) starts to increase and, depending on the exercise intensity and the size of the individual, the VE can be higher than 160 l/min which also leads to an increase in air pollution inhaled. For example, it has been shown that, with the start of an exercise bout, there is an increase in the proportion of ultrafine particles (nanosized particulate matter) inhaled and deposited in the airways but not elimi‐ nated [56, 57]. This could be due to impaired nasal mucociliary clearance and reduced cilia beat frequency which can occur with strenuous exercise [58, 59]. This impairment of respira‐ tory defences together with a higher VE and deeper breathing makes the active and athletic population of large cities more vulnerable to the harmful effect of air pollution on health and on performance, especially with long-duration high-intensity exercise. Most of the major sports events (e.g. Summer Olympic Games, Football World Cup) take place within or near large cities, e.g. polluted Olympic Games venues Barcelona 1992; Atlanta 1996; Athens 2004; Beijing 2008; London 2012 [60, 61]. Rio de Janeiro, Brazil's second biggest city (population density of 5346 hab/km2 ) and host of the 2016 Olympic Games, also presents high levels of air pollution [5].

Studies that have investigated the deleterious effects of air pollution on performance do indeed report that athletes have an impaired performance [62-64]. This can be further exacerbated depending on other environmental conditions, such as elevated temperature and humidity [65]. This impairment can be attributed not only to an increase in lung inflammation, which can decrease its function, but also to the increase in respiratory symptoms that the athletes experience, including cough, nausea, pain on deep inspiration and wheezing, amongst others. This would lead to a decrease in maximal inspiration volume via neural stimulation of sensory fibers present in the lungs, affecting. In more reactive individuals, the ozone could activate "irritant" receptors leading to contraction of alveolar smooth muscles and as a result changes in respiratory muscle force and mechanic properties of the lungs would occur [66, 67]. Endurance exercise alone has also been shown to decrease lung function because hyperven‐ tilation affects the airway smooth muscles [68].

Table 4 presents a summary of studies that have investigated the effect of O3 air pollution on lung inflammation, injury and function. It is interesting to see the different markers that were analysed, as well as the protocols used. More details of some studies are described below.

Devlin *et al.* [72] analyzed the concentration of a broad range of inflammatory mediators in BAL fluid 1 h after ozone exposure. In this study, volunteers performed intermittent heavy treadmill exercise (66 l min-1) for 2 h in a chamber where the ozone concentration was 0.4 ppm. An increase was observed in mediators of inflammation such as neutrophils, IL-6 and lactate dehydrogenase (LDH), which is an indicator of cell damage. Similarly Holz and colleagues [39] observed a significant increase in neutrophil count and percentage in induced sputum 1 h after participants completed 3 h of light intermittent exercise-14 l min-1 m-2 of body surface

area-exposed to 0.25 ppm of O3. Nevertheless, when the participants performed the same exercise bout exposed to a lower O3 concentration (0.12 ppm), no changes in neutrophils were observed. Furthermore, sputum IL-8 concentration was reported to be elevated only after the

**Study Subjects Exercise andOzone levels Results**

Field study with cyclists

Healthy male individuals

nonasthmatics

Field study, trained

Children (10-11 years

**Table 4.** Studies investigating the effect of O3 after exercise

cyclists

of age)

Endurance runners 1 h training or competition simulation 0; 0.2; 0.35 ppm

Cyclists 1 h 1 h competitive cycling

simulation protocol 0; 0.12; 0.18; and 0.24ppm

0.04 – 0.1 ppm + 17,9 ºC

3 h intermittent cycling

Average 35 km cycling Average 0.076 ppm

2 h of outdoor exercise

0.1ppm 31ºC + 70% rh

0.059 ppm

↑ Increase ↓decrease. \*Non-smokers, male and female, the study does not report their physical fitness level.

2 h heavy intermittent cycling

75 min cycling

0.4 ppm

0.12 ppm

0.12 ppm 0.25 ppm

\*Healthy individuals 2 h intermittent cycling

\*Healthy individuals 2 h intermittent cycling 0.2 ppm

Healthy individuals 2 h intermittent cycling 0.2 ppm

Elite runners 8 km time trial

↓ lung funtion at 0.2 and 0.35 ppm ↑ Respiratory symptoms with higher O<sup>3</sup> concentration, impairment in performance

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↓ lung funtion at 0.18 and 0.24ppm ↑ Respiratory symptoms with higher O<sup>3</sup> concentration, impairment in performance

Correlation between the O3 exposure and the impairement in lung function

1 h post-exposure = ↑ neutrophil, IL-6 and

6 h post-exposure = ↑ neutrophil, IL-8 in BAL

1.5 h post-exposure: no effect on neutrophil count or percentage in BAL, but ↑ in

↑ neutrophil percentage and count in

Post-exercise = ↑ in serum CC16

2 h and 4 h post-exposure = ↑ in serum CC16

No effect on serum CC16 or lung function

Immediately post-exercise: ↑ CC16 in NL and no difference on neutrophil counts No effect on lung function Decrease in performance

Small ↓ in lung function

No effect on lung function

inflammatory mediators ↓ Lung function

sputum (0.25 ppm O3) ↑IL-8 in sputum (0.25 ppm O3) No effect on lung function

LDH in BAL

Adams & Schelegle,

Schelegle & Adams,

Brunekreef *et al*., 1994 [71]

Devlin *et al*., 1996

Krishna *et al*., 1998

Blomberg *et al.,* 1999

Broeckaert *et al.,* 2000 [51]

Blomberg *et al*., 2003

Lagerkvist *et al.* 2004

Gomes *et al.* 2010 [64] and 2011 [65]

Holz *et al*., 1999 [39] \*Mild asthmatics +

[72]

[73]

[38]

[76]

[77]

1983 [69]

1986 [70]

Contrasting some of the previous findings, Blomberg *et al.* [38] were unable to find either mucosal and airway neutrophilia or LDH increase at 1.5 h after a 2 h exposure to 0.2 ppm O3

0.25 ppm exposure.


↑ Increase ↓decrease. \*Non-smokers, male and female, the study does not report their physical fitness level.

**Table 4.** Studies investigating the effect of O3 after exercise

includes not only humidifying and heating the inspired air but also filtering it. Once exercise starts becoming more intense, individuals automatically switch the nasal breathing to oral breathing in an attempt to increase the amount inhaled. Nevertheless avoiding the nasal filtration system potentially enhances the pollutant concentration that reaches the lungs.

With the beginning of exercise, the ventilatory exchange rate (VE) starts to increase and, depending on the exercise intensity and the size of the individual, the VE can be higher than 160 l/min which also leads to an increase in air pollution inhaled. For example, it has been shown that, with the start of an exercise bout, there is an increase in the proportion of ultrafine particles (nanosized particulate matter) inhaled and deposited in the airways but not elimi‐ nated [56, 57]. This could be due to impaired nasal mucociliary clearance and reduced cilia beat frequency which can occur with strenuous exercise [58, 59]. This impairment of respira‐ tory defences together with a higher VE and deeper breathing makes the active and athletic population of large cities more vulnerable to the harmful effect of air pollution on health and on performance, especially with long-duration high-intensity exercise. Most of the major sports events (e.g. Summer Olympic Games, Football World Cup) take place within or near large cities, e.g. polluted Olympic Games venues Barcelona 1992; Atlanta 1996; Athens 2004; Beijing 2008; London 2012 [60, 61]. Rio de Janeiro, Brazil's second biggest city (population

Studies that have investigated the deleterious effects of air pollution on performance do indeed report that athletes have an impaired performance [62-64]. This can be further exacerbated depending on other environmental conditions, such as elevated temperature and humidity [65]. This impairment can be attributed not only to an increase in lung inflammation, which can decrease its function, but also to the increase in respiratory symptoms that the athletes experience, including cough, nausea, pain on deep inspiration and wheezing, amongst others. This would lead to a decrease in maximal inspiration volume via neural stimulation of sensory fibers present in the lungs, affecting. In more reactive individuals, the ozone could activate "irritant" receptors leading to contraction of alveolar smooth muscles and as a result changes in respiratory muscle force and mechanic properties of the lungs would occur [66, 67]. Endurance exercise alone has also been shown to decrease lung function because hyperven‐

Table 4 presents a summary of studies that have investigated the effect of O3 air pollution on lung inflammation, injury and function. It is interesting to see the different markers that were analysed, as well as the protocols used. More details of some studies are described below.

Devlin *et al.* [72] analyzed the concentration of a broad range of inflammatory mediators in BAL fluid 1 h after ozone exposure. In this study, volunteers performed intermittent heavy treadmill exercise (66 l min-1) for 2 h in a chamber where the ozone concentration was 0.4 ppm. An increase was observed in mediators of inflammation such as neutrophils, IL-6 and lactate dehydrogenase (LDH), which is an indicator of cell damage. Similarly Holz and colleagues [39] observed a significant increase in neutrophil count and percentage in induced sputum 1 h after participants completed 3 h of light intermittent exercise-14 l min-1 m-2 of body surface

) and host of the 2016 Olympic Games, also presents high levels of air

density of 5346 hab/km2

tilation affects the airway smooth muscles [68].

pollution [5].

12 Lung Inflammation

area-exposed to 0.25 ppm of O3. Nevertheless, when the participants performed the same exercise bout exposed to a lower O3 concentration (0.12 ppm), no changes in neutrophils were observed. Furthermore, sputum IL-8 concentration was reported to be elevated only after the 0.25 ppm exposure.

Contrasting some of the previous findings, Blomberg *et al.* [38] were unable to find either mucosal and airway neutrophilia or LDH increase at 1.5 h after a 2 h exposure to 0.2 ppm O3  in subjects performing intermittent moderate cycling exercise producing an average minute ventilation of 20 l min-1 m-2 of body surface area. This difference might be explained by the lower exercise and O3 levels in the latter study which would have as consequence the lowering of the inhaled O3 dose. Nevertheless, in tissue obtained from bronchial mucosal biopsies, Blomberg *et al.* [38] were able to detect increases in the expression of vascular endothelium Pselectin and ICAM-1 after the ozone exposure. These molecules mediate adhesion and rolling of leukocytes on the vessel walls. Hence, it was suggested that although there was an increase in the expression of vascular adhesion molecules in the vascular endothelium, this had not yet resulted in an increase in neutrophil numbers at the analyzed sites. Stenfors *et al.* [74] using the same study design as the previously-mentioned researchers, demonstrated a significant increase in BAL neutrophil number and percentage 6 h after the exercise trial. In addition, vascular endothelium P-selectin and ICAM-1 were also elevated. This reinforces the impor‐ tance of these adhesion molecules in the inflammatory response since they recruit inflamma‐ tory cells into the airways of healthy individuals.

Blomberg *et al*. [76] conducted a lab study where 22 subjects performed 2 h of moderate intermittent exercise. They were exposed to two different environment conditions: 0.2 ppm of O3 and filtered air. The participants' lung function was assessed and peripheral blood samples were obtained 2 h pre, immediately pre, immediately post, 2 and 4 h post-exercise. Significant decreases in the lung function parameters, FEV1 and FVC, were observed immediately post O3 exposure. However, at 2 and 4 h post-exercise this decrease was no longer observed. Moreover, a significant increase in CC16 serum levels was seen around 2 and 4 h post O3 exposure. No relationship was noted between CC16 and lung function at any of the analyzed points. Serum CC16 concentrations were shown to have returned to baseline 18 h postexposure. Other epithelial permeability markers, albumin and total protein concentration, which were also assessed, did not show a significant increase. The data from this study supports the theory that serum CC16 is a more sensitive marker of altered lung epithelial

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Contrary to the above-mentioned studies, Lagerkvist *et al.* [77] did not find any significant changes in serum levels of CC16 in children (10-11 years of age). They performed 2 h of outdoor exercise, where the maximal O3 value reached 0.059 ppm. Blood samples and lung function performance were analyzed pre and post-exercise. Yet no decrease in lung function or changes in CC16 were observed. In relation to CC16 concentration, the authors reported that children who regularly visited chlorinated indoor swimming pools presented significantly lower levels of serum CC16 both before and after the outdoor exercise when compared to the nonswimming children. In this study, it is important to observe that the maximum level of O3 reported is lower than in the previous studies discussed above. Furthermore, the authors mentioned that the children performed light exercise; though, they did not report the type of exercise nor how the exercise intensity was controlled. More investigation is needed to establish the effect of ozone and exercise on airway permeability of different populations, as

there are still contradictions in the literature using CC16 as a marker of lung injury.

animal studies, but needs further investigation in humans [53].

Acute exposure to ozone, as investigated in the studies above, is very relevant and can also be related to some real-life situations when cities experience intense pollution episodes or when individuals travel to more polluted areas. Nevertheless, chronic exposure to air pollution also needs further investigation. Unfortunately, several challenges are present for the development of research on chronic exposure to air pollution on a human exercising population. Christian *et al.* [53] showed an attenuation of the inflammatory response in BAL after four consecutive days of exposure to ozone. Nevertheless, it seems that, although neutrophil recruitment and IL-6 concentration in the respiratory tract is attenuated with multiday short-term exposures, airway epithelial injury may continue to occur. The data from Jörres *et al.* [44] support the previous finding, with them additionally reporting that, after four consecutive exposures, an increase in airway mucosa inflammation as well as the neutrophil count was observed in bronchial mucosal biopsies. These data, thus, demonstrate that airway inflammation persists despite the attenuation of some inflammatory markers in BAL. It is important to point out that such persistent injury could lead to airway remodeling, which has been observed in several

permeability when compared to traditional markers.

Gomes and colleagues [64, 65] investigated well trained runners performing an intense exercise bout (8 km time trial) in an environment that, in addition to ozone pollution, was warm and humid. This kind of environment is relevant because the formation of ozone is intensified during the summer when there is a high incidence of sun light. Even though they did not report any changes in lung function, there were signs of lung inflammation and lung injury, the latter observed by an increase in NL CC16 concentration. In addition, there was a positive correlation between lung antioxidant concentrations and performance, that is, the athletes who presented lower concentrations of lung antioxidants were the ones who had a higher impairment in their performance in that extreme environment.

Other studies have also looked into changes in CC16 with exercise associated with ozone pollution. Broeckaert *et al.* [75] investigated 24 non-smoking cyclists, performing a 2 h of moderate intensity cycling during episodes of photochemical smog. The average concentration of ozone was 0.076 ppm. Immediately before and after each ride, the participants provided blood samples and also performed lung function tests. Significant correlations were found between the O3 concentration and the cyclists' serum levels of CC16 both pre and post-exercise. By contrast, when comparing pre and post rides, no decrease on lung function parameters were found-these are usually impaired by O3 exposure. Thus, this study showed that shortterm exposures to ambient-levels of O3 induced an early increase in serum CC16 which took place before other manifestations of lung toxicity. However, there was no control group to verify if this increase was due to the exercise, the ozone or a combination of both. The authors suggested that the increase seen in the serum CC16 was due to an increase in the pulmonary epithelium permeability and not to an increase in the production of this protein. As this study was conducted in the field, it is difficult to attribute these results directly to O3 exposure, as there may have been other pollutants that could also have influenced the epithelial leakage. In addition, the levels of serum CC16 pre-ride were also correlated with the levels of ambient O3, indicating that the cyclists initiated the exercise with an increased lung epithelium permeability.

Blomberg *et al*. [76] conducted a lab study where 22 subjects performed 2 h of moderate intermittent exercise. They were exposed to two different environment conditions: 0.2 ppm of O3 and filtered air. The participants' lung function was assessed and peripheral blood samples were obtained 2 h pre, immediately pre, immediately post, 2 and 4 h post-exercise. Significant decreases in the lung function parameters, FEV1 and FVC, were observed immediately post O3 exposure. However, at 2 and 4 h post-exercise this decrease was no longer observed. Moreover, a significant increase in CC16 serum levels was seen around 2 and 4 h post O3 exposure. No relationship was noted between CC16 and lung function at any of the analyzed points. Serum CC16 concentrations were shown to have returned to baseline 18 h postexposure. Other epithelial permeability markers, albumin and total protein concentration, which were also assessed, did not show a significant increase. The data from this study supports the theory that serum CC16 is a more sensitive marker of altered lung epithelial permeability when compared to traditional markers.

 in subjects performing intermittent moderate cycling exercise producing an average minute ventilation of 20 l min-1 m-2 of body surface area. This difference might be explained by the lower exercise and O3 levels in the latter study which would have as consequence the lowering of the inhaled O3 dose. Nevertheless, in tissue obtained from bronchial mucosal biopsies, Blomberg *et al.* [38] were able to detect increases in the expression of vascular endothelium Pselectin and ICAM-1 after the ozone exposure. These molecules mediate adhesion and rolling of leukocytes on the vessel walls. Hence, it was suggested that although there was an increase in the expression of vascular adhesion molecules in the vascular endothelium, this had not yet resulted in an increase in neutrophil numbers at the analyzed sites. Stenfors *et al.* [74] using the same study design as the previously-mentioned researchers, demonstrated a significant increase in BAL neutrophil number and percentage 6 h after the exercise trial. In addition, vascular endothelium P-selectin and ICAM-1 were also elevated. This reinforces the impor‐ tance of these adhesion molecules in the inflammatory response since they recruit inflamma‐

Gomes and colleagues [64, 65] investigated well trained runners performing an intense exercise bout (8 km time trial) in an environment that, in addition to ozone pollution, was warm and humid. This kind of environment is relevant because the formation of ozone is intensified during the summer when there is a high incidence of sun light. Even though they did not report any changes in lung function, there were signs of lung inflammation and lung injury, the latter observed by an increase in NL CC16 concentration. In addition, there was a positive correlation between lung antioxidant concentrations and performance, that is, the athletes who presented lower concentrations of lung antioxidants were the ones who had a higher impairment in their

Other studies have also looked into changes in CC16 with exercise associated with ozone pollution. Broeckaert *et al.* [75] investigated 24 non-smoking cyclists, performing a 2 h of moderate intensity cycling during episodes of photochemical smog. The average concentration of ozone was 0.076 ppm. Immediately before and after each ride, the participants provided blood samples and also performed lung function tests. Significant correlations were found between the O3 concentration and the cyclists' serum levels of CC16 both pre and post-exercise. By contrast, when comparing pre and post rides, no decrease on lung function parameters were found-these are usually impaired by O3 exposure. Thus, this study showed that shortterm exposures to ambient-levels of O3 induced an early increase in serum CC16 which took place before other manifestations of lung toxicity. However, there was no control group to verify if this increase was due to the exercise, the ozone or a combination of both. The authors suggested that the increase seen in the serum CC16 was due to an increase in the pulmonary epithelium permeability and not to an increase in the production of this protein. As this study was conducted in the field, it is difficult to attribute these results directly to O3 exposure, as there may have been other pollutants that could also have influenced the epithelial leakage. In addition, the levels of serum CC16 pre-ride were also correlated with the levels of ambient O3, indicating that the cyclists initiated the exercise with an increased lung epithelium

tory cells into the airways of healthy individuals.

14 Lung Inflammation

performance in that extreme environment.

permeability.

Contrary to the above-mentioned studies, Lagerkvist *et al.* [77] did not find any significant changes in serum levels of CC16 in children (10-11 years of age). They performed 2 h of outdoor exercise, where the maximal O3 value reached 0.059 ppm. Blood samples and lung function performance were analyzed pre and post-exercise. Yet no decrease in lung function or changes in CC16 were observed. In relation to CC16 concentration, the authors reported that children who regularly visited chlorinated indoor swimming pools presented significantly lower levels of serum CC16 both before and after the outdoor exercise when compared to the nonswimming children. In this study, it is important to observe that the maximum level of O3 reported is lower than in the previous studies discussed above. Furthermore, the authors mentioned that the children performed light exercise; though, they did not report the type of exercise nor how the exercise intensity was controlled. More investigation is needed to establish the effect of ozone and exercise on airway permeability of different populations, as there are still contradictions in the literature using CC16 as a marker of lung injury.

Acute exposure to ozone, as investigated in the studies above, is very relevant and can also be related to some real-life situations when cities experience intense pollution episodes or when individuals travel to more polluted areas. Nevertheless, chronic exposure to air pollution also needs further investigation. Unfortunately, several challenges are present for the development of research on chronic exposure to air pollution on a human exercising population. Christian *et al.* [53] showed an attenuation of the inflammatory response in BAL after four consecutive days of exposure to ozone. Nevertheless, it seems that, although neutrophil recruitment and IL-6 concentration in the respiratory tract is attenuated with multiday short-term exposures, airway epithelial injury may continue to occur. The data from Jörres *et al.* [44] support the previous finding, with them additionally reporting that, after four consecutive exposures, an increase in airway mucosa inflammation as well as the neutrophil count was observed in bronchial mucosal biopsies. These data, thus, demonstrate that airway inflammation persists despite the attenuation of some inflammatory markers in BAL. It is important to point out that such persistent injury could lead to airway remodeling, which has been observed in several animal studies, but needs further investigation in humans [53].

Using a different technique, sputum induction, to assess airway inflammation, Ratto *et al.* [45] found an increase in the percentage of neutrophils after 4 h of 0.2 ppm O3 exposure during 4 consecutive days. This finding is in contrast to the afore-mentioned studies using BAL where it was observed an attenuation of the inflammatory response. Nevertheless, the increased recruitment of neutrophils to proximal airway tissue, showed by the analysis of induced sputum, was consistent with the examination of endobronchial biopsies samples taken by Jörres *et al.* [44]. Once more, it shows that different techniques sample different airway compartments producing differing results. In addition, the exposure and exercise protocol is also essential for the outcome of the inflammatory process. An important additional factor that appears to affect results in these studies is individual responsiveness due to differences in ozone sensitivity of individuals [39, 78].

34 workers (shoe-cleaners), who were constantly exposed to pollution (Mexico City), partici‐ pated in a double blind supplementation/placebo crossover design study. The supplementa‐ tion consisted of a mix of different antioxidants (650 mg vitamin C+100 IU vitamin E+15 mg b-carotene) ingested during a 10-week period. The washout period was 2 weeks. The average daily ozone concentration was 0.07 ppm, and on 55% of the days the concentration exceeded the Mexican standard of 0.11 ppm. As reported, antioxidant supplementation resulted in a protective effect on the lung function against the ozone exposure. The authors, however, do mention that individuals who consumed antioxidants first presented less lung function impairment after consuming placebo than subjects that initially ingested placebo. The authors attributed this result to the short washout period, especially in relation to vitamin E, which accumulates in the tissues; but they did not investigate this issue. Field studies, such as this one, have some limitations, as for instance, the interference of other pollutants and the change in concentration of the analyzed pollutant throughout the long period in which the study was conducted. In addition, the participants of this study were not exercising while being exposed

Lung Inflammation, Oxidative Stress and Air Pollution

http://dx.doi.org/10.5772/58252

17

Two subsequent field studies, conducted with amateur and recreational cyclists [86, 87], reached similar results in relation to antioxidant supplementation providing some protection against the acute effects of ozone on lung function. Grievink *et al.* [86] observed two groups of cyclists, during a 3-month period. One group was supplemented with vitamin C (650 mg day-1), vitamin E (100 IU day-1) and β-carotene (15 mg day-1); while the other group ingested placebo. This study was conducted during the summer months, and the lung function of the cyclists was measured before and after training or competition on 4 to 14 occasions. Of note, the supplementation started 1 week before the first measurement and was maintained during the study period, and this could have influenced the result as the participants were not all tested on the same occasions. The mean temperature throughout the study period was 23°C and the ozone concentration averaged 0.05 ppm. In the subsequent study by these researchers [87], the same protocol was followed in relation to the exercise and measurements during the summer; however, the supplementation protocol varied slightly (3 months of daily vitamin C 500 mg and vitamin E 150 IU). In this later study, even though it was reported that the supplementation was able to partly counteract the decreases of lung function, the authors also mentioned that, when participants who had not complied fully with the supplementation were excluded from the analysis, the effects of ozone on lung function were no longer observed. The average temperature and ozone concentration in this study were lower than in the previous study: 17°C and 0.04 ppm respectively. Both studies presented some disadvantages. Firstly, they analysed two different groups of individuals, making comparison challenging as the effects of ozone have been shown to vary a lot from one individual to another. Secondly, the placebo group and the vitamin group were not balanced in relation to individuals presenting respiratory allergies or asthma. In addition, in the latter study [87], the placebo group, acting as the control, was not taking any pills, therefore, it was not blinded. Thirdly, some individuals in the supplemented group were already taking antioxidants prior to the start of the study. Lastly, it was reported that the adjustment for environmental temperature as a possible confounder was difficult due to the high correlation with ozone. All in all, it is important to

to the pollution.

#### **6. Air pollution, exercise and antioxidant supplementation**

The respiratory tract lining fluid is the first barrier encountered by inspired gases and, therefore, it has a network of antioxidants, such as ascorbic acid, GSH, α-tocopherol and uric acid to provide protection against oxidative stress [42, 78, 79]. For this reason, antioxidant supplementation has been suggested as a benefit for people exercising in an air-polluted environment. The rationale behind such hypothesis is that increasing the availability of antioxidants in the respiratory-tract lining fluid will provide additional sacrificial substrates for the oxidant-be it PM, O3 or any other oxidant gas. These additional sacrificial substrates will, in turn, decrease oxidation reactions occurring within this fluid and within the under‐ lining epithelial cells. In addition, an excess in antioxidants concentration might also confer protection by neutralizing free radical species, derived from these initial reactions or inflam‐ matory cells [26, 78]. As a result, the toxicity of the pollutant would be decreased limiting the inflammation response of the cells from the epithelium tract [80], consequently, lung injury would be minimized and lung function would be maintained.

Some studies have investigated this proposed benefit of increasing antioxidant availability in individuals exposed to ozone-pollution by supplementing the participants with a mix of antioxidants, mostly vitamins C and E. These two antioxidants are present in the RTLF, they both have strong antioxidant properties and together have been shown to present a synergistic effect in the protection against oxidative stress [78, 81]. Vitamin E is the major lipophilic antioxidant in human tissues, whether in the airways or otherwise; and vitamin C has been linked to maintenance of lung health, as for example, by improving lung function, having a positive effect on exercise-induced bronchoconstriction in asthmatic individuals and decreas‐ ing the adverse respiratory symptoms experienced during exercise [82-84]. As will be further elucidated, supplementation with these two vitamins has been shown to provide some protection in humans exposed to ozone-pollution [35, 85-88]. Little is known in relation to the benefits of antioxidants when it comes to exposure to other kinds of air pollutants other than O3. Nevertheless, the benefits would be expected to be similar for all the oxidant air pollutants.

Romieu *et al.* [85] have shown the existence of some protective effect when participants are supplemented with a mix of antioxidants. These researchers conducted a field study in which 34 workers (shoe-cleaners), who were constantly exposed to pollution (Mexico City), partici‐ pated in a double blind supplementation/placebo crossover design study. The supplementa‐ tion consisted of a mix of different antioxidants (650 mg vitamin C+100 IU vitamin E+15 mg b-carotene) ingested during a 10-week period. The washout period was 2 weeks. The average daily ozone concentration was 0.07 ppm, and on 55% of the days the concentration exceeded the Mexican standard of 0.11 ppm. As reported, antioxidant supplementation resulted in a protective effect on the lung function against the ozone exposure. The authors, however, do mention that individuals who consumed antioxidants first presented less lung function impairment after consuming placebo than subjects that initially ingested placebo. The authors attributed this result to the short washout period, especially in relation to vitamin E, which accumulates in the tissues; but they did not investigate this issue. Field studies, such as this one, have some limitations, as for instance, the interference of other pollutants and the change in concentration of the analyzed pollutant throughout the long period in which the study was conducted. In addition, the participants of this study were not exercising while being exposed to the pollution.

Using a different technique, sputum induction, to assess airway inflammation, Ratto *et al.* [45] found an increase in the percentage of neutrophils after 4 h of 0.2 ppm O3 exposure during 4 consecutive days. This finding is in contrast to the afore-mentioned studies using BAL where it was observed an attenuation of the inflammatory response. Nevertheless, the increased recruitment of neutrophils to proximal airway tissue, showed by the analysis of induced sputum, was consistent with the examination of endobronchial biopsies samples taken by Jörres *et al.* [44]. Once more, it shows that different techniques sample different airway compartments producing differing results. In addition, the exposure and exercise protocol is also essential for the outcome of the inflammatory process. An important additional factor that appears to affect results in these studies is individual responsiveness due to differences in

The respiratory tract lining fluid is the first barrier encountered by inspired gases and, therefore, it has a network of antioxidants, such as ascorbic acid, GSH, α-tocopherol and uric acid to provide protection against oxidative stress [42, 78, 79]. For this reason, antioxidant supplementation has been suggested as a benefit for people exercising in an air-polluted environment. The rationale behind such hypothesis is that increasing the availability of antioxidants in the respiratory-tract lining fluid will provide additional sacrificial substrates for the oxidant-be it PM, O3 or any other oxidant gas. These additional sacrificial substrates will, in turn, decrease oxidation reactions occurring within this fluid and within the under‐ lining epithelial cells. In addition, an excess in antioxidants concentration might also confer protection by neutralizing free radical species, derived from these initial reactions or inflam‐ matory cells [26, 78]. As a result, the toxicity of the pollutant would be decreased limiting the inflammation response of the cells from the epithelium tract [80], consequently, lung injury

Some studies have investigated this proposed benefit of increasing antioxidant availability in individuals exposed to ozone-pollution by supplementing the participants with a mix of antioxidants, mostly vitamins C and E. These two antioxidants are present in the RTLF, they both have strong antioxidant properties and together have been shown to present a synergistic effect in the protection against oxidative stress [78, 81]. Vitamin E is the major lipophilic antioxidant in human tissues, whether in the airways or otherwise; and vitamin C has been linked to maintenance of lung health, as for example, by improving lung function, having a positive effect on exercise-induced bronchoconstriction in asthmatic individuals and decreas‐ ing the adverse respiratory symptoms experienced during exercise [82-84]. As will be further elucidated, supplementation with these two vitamins has been shown to provide some protection in humans exposed to ozone-pollution [35, 85-88]. Little is known in relation to the benefits of antioxidants when it comes to exposure to other kinds of air pollutants other than O3. Nevertheless, the benefits would be expected to be similar for all the oxidant air pollutants. Romieu *et al.* [85] have shown the existence of some protective effect when participants are supplemented with a mix of antioxidants. These researchers conducted a field study in which

**6. Air pollution, exercise and antioxidant supplementation**

would be minimized and lung function would be maintained.

ozone sensitivity of individuals [39, 78].

16 Lung Inflammation

Two subsequent field studies, conducted with amateur and recreational cyclists [86, 87], reached similar results in relation to antioxidant supplementation providing some protection against the acute effects of ozone on lung function. Grievink *et al.* [86] observed two groups of cyclists, during a 3-month period. One group was supplemented with vitamin C (650 mg day-1), vitamin E (100 IU day-1) and β-carotene (15 mg day-1); while the other group ingested placebo. This study was conducted during the summer months, and the lung function of the cyclists was measured before and after training or competition on 4 to 14 occasions. Of note, the supplementation started 1 week before the first measurement and was maintained during the study period, and this could have influenced the result as the participants were not all tested on the same occasions. The mean temperature throughout the study period was 23°C and the ozone concentration averaged 0.05 ppm. In the subsequent study by these researchers [87], the same protocol was followed in relation to the exercise and measurements during the summer; however, the supplementation protocol varied slightly (3 months of daily vitamin C 500 mg and vitamin E 150 IU). In this later study, even though it was reported that the supplementation was able to partly counteract the decreases of lung function, the authors also mentioned that, when participants who had not complied fully with the supplementation were excluded from the analysis, the effects of ozone on lung function were no longer observed. The average temperature and ozone concentration in this study were lower than in the previous study: 17°C and 0.04 ppm respectively. Both studies presented some disadvantages. Firstly, they analysed two different groups of individuals, making comparison challenging as the effects of ozone have been shown to vary a lot from one individual to another. Secondly, the placebo group and the vitamin group were not balanced in relation to individuals presenting respiratory allergies or asthma. In addition, in the latter study [87], the placebo group, acting as the control, was not taking any pills, therefore, it was not blinded. Thirdly, some individuals in the supplemented group were already taking antioxidants prior to the start of the study. Lastly, it was reported that the adjustment for environmental temperature as a possible confounder was difficult due to the high correlation with ozone. All in all, it is important to view these results with caution because this array of uncontrolled variables could have influenced the outcome.

In the study of Samet *et al.* [35], participants were divided into two groups and all underwent a 1-week period of vitamin C-restricted diet. After this, one of the groups received supple‐ mentation (250 mg vitamin C+50 IU vitamin E+12 oz of carrot and tomato juice), while the other group received placebo and continued on the restricted diet. The supplementation period consisted of a 2-week period after which the subjects underwent a 2 h low-intensity exercise protocol in a high ozone-polluted chamber (0.4 ppm). After exposure, the subjects completed a respiratory symptom questionnaire, performed lung function tests and underwent a bronchoalveolar lavage. There were no differences between the supplemented group and the placebo group in respect to answers given in the respiratory symptom questionnaire. This suggests that dietary antioxidants do not minimize the perceived harmful effects of ozone. In addition, there were no differences in neutrophil counts or other inflammatory markers in the bronchoalveolar lavage fluid. Nevertheless, the authors did report attenuation in lung function impairment in relation to the subjects who ingested the antioxidant mixture.

Contrary to this finding, Mudway *et al.* (89) did not report any changes in lung function when they conducted a double-blind crossover study. The supplementation (500 mg vitamin C+150 IU vitamin E daily) period in this study was smaller than most supplementation protocols: just 1 week, with a 2-week washout period. The exposure protocol consisted of 2 h of intermittent cycling in a chamber with 0.2 ppm of ozone. Besides the lack of changes in lung function, there were no differences in airway inflammation, which was assessed 6 h post-exposure. It is important to point out that, after the supplementation protocol, the subjects did present an increased concentration of plasma ascorbic acid and α-tocopherol. This increased concentra‐ tion, however, was not observed in the respiratory airways when it was accessed 6 h after the ozone exposure. Nevertheless, the authors did report movement of α-tocopherol from the plasma into the RTLF following the ozone challenge.

In a 2011 study, Gomes et al [90] reported that there was a positive effect of a 2-week supple‐ mentation period of vitamin E and vitamin C on the pre-exercise levels of the total antioxidant concentration in the RTLF. In addition, after the 8 km time trial run the participants, when on the vitamins, presented decreased lung injury (higher CC16 levels in both the plasma and NL) compared to when they took the placebos. Participants also ran on average 49sec faster when taking the vitamins. The environment where the exercise took place had, besides the ozone pollution (0.1ppm), also heat and humidity. This study was also conducted in a double-blinded randomized and crossover way, which minimizes biases. A summary of the studies presented above is provided in Table 5.

The inconclusiveness of the literature can possibly be attributed to divergences in the meth‐ odologies used in the research, such as different supplementation protocols, exercise modes and participants' fitness levels. Additionally, only one study looked at the effect of antioxidant supplementation on performance in a polluted environment, with more information being necessary in relation to the antioxidant and inflammatory response. Due to the high antioxi‐ dant consumption by the physically active community and by a large portion of the general

population, this is an important topic for research, particularly when coupled with the fact that large urbanized areas might provide an additional reason, in the form of air pollutants,

**Study Subjects Design Supplement Exercise and**

10 wks: vit C 650 mg + vit E 100 IU + b-carotene + 15

Started 1 wk before 1st measurement, total of 3 months: vit C 650mg + vit E 100 IU and b-carotene

Started 1 wk before first measurement, total of 3 months: vit C 500 mg + vit E

Placebo: 3 wks vitamin

Supplemented: 1 wk vitamin restriction + 2 weeks of 250 mg vitamin C + vitamin E 50 IU + 12 oz of carrot and tomato juice

1 week: vit C 500mg + vit E

2 weeks: vit C 500mg + vit E

mg daily

15mg daily

150 IU daily

restriction

daily

150 IU daily

100 IU daily

Shoe-cleaners Field study

Amateur and recreational cyclists

Amateur cyclists

Male and female, physical fitness not specified

Male and female, physical fitness not specified

Male elite runners

Crossover (n=34)

Field study Placebo (n =18) Supplemented (n=20)

Field study Placebo (n =9) Supplemented (n=11)

Placebo (n =16) Supplemented (n=15)

Crossover (n=14)

Crossover (n=10)

**Table 5.** Studies investigating vitamin C and E supplementation in ozone exposure

Romieu *et al*., 1998

Grievink *et al.,* 1998 [86]

Grievink *et al.,* 1999 [87]

Samet *et al.,* 2001

Mudway *et al.,* 2006 [89]

Gomes et al. 2011

[90]

[35]

[85]

**ozone levels**

Lung Inflammation, Oxidative Stress and Air Pollution

Daily work Average of 0.07 ppm O3

Training sessions Average of 0.05 ppm O3

Training sessions and competitive races

2 h lowintensity intermittent exercise on treadmill or cycling 0.4 ppm O3

Average of 0.04 ppm O3

2 h intermittent cycling 0.2 ppm O3

8km time trial

run

**Results**

http://dx.doi.org/10.5772/58252

Attenuation of lung function impairment with supplementation 19

Supplementation provided some protection on lung

No effect on lung function

Attenuation of lung function decrements with supplementation No differences in respiratory symptoms or lung inflammation

No effect on lung function No effect on lung inflammation

Attenuation of lung injury with supplementation 49 sec improvement in performance

function

to increase the antioxidant intake.


**Table 5.** Studies investigating vitamin C and E supplementation in ozone exposure

view these results with caution because this array of uncontrolled variables could have

In the study of Samet *et al.* [35], participants were divided into two groups and all underwent a 1-week period of vitamin C-restricted diet. After this, one of the groups received supple‐ mentation (250 mg vitamin C+50 IU vitamin E+12 oz of carrot and tomato juice), while the other group received placebo and continued on the restricted diet. The supplementation period consisted of a 2-week period after which the subjects underwent a 2 h low-intensity exercise protocol in a high ozone-polluted chamber (0.4 ppm). After exposure, the subjects completed a respiratory symptom questionnaire, performed lung function tests and underwent a bronchoalveolar lavage. There were no differences between the supplemented group and the placebo group in respect to answers given in the respiratory symptom questionnaire. This suggests that dietary antioxidants do not minimize the perceived harmful effects of ozone. In addition, there were no differences in neutrophil counts or other inflammatory markers in the bronchoalveolar lavage fluid. Nevertheless, the authors did report attenuation in lung function

Contrary to this finding, Mudway *et al.* (89) did not report any changes in lung function when they conducted a double-blind crossover study. The supplementation (500 mg vitamin C+150 IU vitamin E daily) period in this study was smaller than most supplementation protocols: just 1 week, with a 2-week washout period. The exposure protocol consisted of 2 h of intermittent cycling in a chamber with 0.2 ppm of ozone. Besides the lack of changes in lung function, there were no differences in airway inflammation, which was assessed 6 h post-exposure. It is important to point out that, after the supplementation protocol, the subjects did present an increased concentration of plasma ascorbic acid and α-tocopherol. This increased concentra‐ tion, however, was not observed in the respiratory airways when it was accessed 6 h after the ozone exposure. Nevertheless, the authors did report movement of α-tocopherol from the

In a 2011 study, Gomes et al [90] reported that there was a positive effect of a 2-week supple‐ mentation period of vitamin E and vitamin C on the pre-exercise levels of the total antioxidant concentration in the RTLF. In addition, after the 8 km time trial run the participants, when on the vitamins, presented decreased lung injury (higher CC16 levels in both the plasma and NL) compared to when they took the placebos. Participants also ran on average 49sec faster when taking the vitamins. The environment where the exercise took place had, besides the ozone pollution (0.1ppm), also heat and humidity. This study was also conducted in a double-blinded randomized and crossover way, which minimizes biases. A summary of the studies presented

The inconclusiveness of the literature can possibly be attributed to divergences in the meth‐ odologies used in the research, such as different supplementation protocols, exercise modes and participants' fitness levels. Additionally, only one study looked at the effect of antioxidant supplementation on performance in a polluted environment, with more information being necessary in relation to the antioxidant and inflammatory response. Due to the high antioxi‐ dant consumption by the physically active community and by a large portion of the general

impairment in relation to the subjects who ingested the antioxidant mixture.

plasma into the RTLF following the ozone challenge.

above is provided in Table 5.

influenced the outcome.

18 Lung Inflammation

population, this is an important topic for research, particularly when coupled with the fact that large urbanized areas might provide an additional reason, in the form of air pollutants, to increase the antioxidant intake.
